2008 Polar Targeting and Endocytic Recycling in Auxin-Dependent Plant Development

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绿色荧光蛋白研究的三个里程碑——2008年诺贝尔化学奖简介

生理活动；二把荧光化合物特别是标记了荧光化合物第的蛋白注射到细胞内，要特别的设备和专业训练—— 需微注射；三即使荧光化合物无毒，有微注射的条件，第也
每研究一种蛋白，必须先分离蛋白，后找到标记荧就然
突出成就而获得２００８年度诺贝尔化学奖。笔者拟对绿色荧光蛋白的研究历程以及应用领域做一些粗浅介绍。２００８年１０月８日，典皇家科学院诺贝尔奖委员瑞
会将２００８年度诺贝尔化学奖授予１美国科学家下村３裔修（ａｈｍｏｒ）美国科学家马丁・查尔非ＯｓｍｕＳｉｍｕａ、（ｒｉｈｌｉ）以及美国华裔科学家钱永健（ｇｒＭａｔＣａｆｅ，ｎＲｏｅ瑞舍一气之下，开了科学界，ＧＦ离将Ｐ的ｃＤＮＡ送给了几个实验室。很多人尝试用ＧＦＰ的基因来表达蛋白，
水母上分离出来的ＧＦＰ基因可以在其他物种上表达，正确地折叠，出荧光。这些研究成果为ＧＦ发Ｐ在生物
学研究上的使用，奠定了基础。当时如果要研究活体生物中的生命活动，以采用可
的编码区，将它克隆到表达载体中，过ｕＶ或蓝光激通
发，大肠杆菌和线虫细胞内均产生了很美妙的绿色荧在光。这才是ＧＦＰ作为荧光指示剂的真正突破，章发文表在Ｓｉｎｅ杂志上。这个实验的重要意义是证明了从ｃｅｃ

Regulation of DNA repair throughout the cell cycle

The genome is under constant threat of damage from exogenous agents that damage DNA. Endogenous processes can also induce DNA damage; for example, hydrolysis leads to spontaneous DNA depurination; reactive oxygen species (ROS) induce base oxidation and DNA breaks; replication defects can cause mis-matches; and replication fork collapse can result in strand breaks 1. The resulting DNA lesions must be repaired to prevent loss or incorrect transmission of genetic information, as errors can cause developmental abnormalities and tumorigenesis. However, the choice of which repair system to use depends both on the type of lesion and on the cell-cycle phase of the cell (FIG. 1). For example, a DNA double-strand break (DSB) in S and G2 phases is readily repaired by homologous recombination (HR) using the intact sister chromatid (BOX 1). However, as cells progress into G2–M, the chro-mosomes are condensed in a highly ordered chromatin structure that makes homology search difficult. The coordination of the DNA-repair pathway and the cell cycle is controlled through different cell-cycle activi-ties, such as cyclin-dependent kinases (CDKs). CDKs regulate cell-cycle transitions by inducing degradation of cell-cycle inhibitory proteins (BOX 2) and are peri-odically activated by their regulatory cyclin subunits, which are differentially expressed during the different cell-cycle phases 2. Cells integrate DNA-repair proc-esses with transcription and apoptosis in a network that is known as the DNA-damage response (DDR), which is orchestrated by checkpoint proteins. Understanding how DNA repair is modulated according to the cell-cycle phase has important applications for medicine and cancer.Over the past decade, tremendous progress has been made in the elucidation of the mechanistic intricacies of different repair pathways, and of the spatio–temporal orchestration of DNA repair. Post-translational modi-fications, such as checkpoint- and CDK-dependent phos-phorylation, ubiquitylation and sumoylation, were shown to be crucial for the regulation of the stability and activity of important components of the checkpoint machinery, thereby regulating important cell-cycle events. These post-translational modifications may affect the recruit-ment of repair proteins to damaged DNA or tune the efficiency or the specificity of the repair machinery towards a certain type of lesion, often to facilitate repair in a specific cell-cycle phase.In this review, we describe the most important DDR pathways that operate in a eukaryotic cell and discuss their activity through the cell cycle. We then discuss the main regulatory mechanisms that affect the choice of DNA-repair pathways through the cell cycle. Last, we provide examples of how the function of different repair factors are modulated through post-transla-tional modifications and discuss crucial questions that need to be addressed. The connections between DNA repair and other chromosome-metabolism processes, such as replication, transcription, cohesion and condensation, segregation, and DNA topology, have been discussed in other reviews 3–6.The checkpoint-activation networkCheckpoints are cellular surveillance and signalling pathways that coordinate DNA repair with chromo-some metabolism and cell-cycle transitions 7,8. The checkpoint proteins are often recruited to DNA lesionsFIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy;and Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy. e‑mails:dana.branzei@ifom‑ieo‑campus.it ;marco.foiani@ifom‑ieo‑campus.itdoi:10.1038/nrm2351Published online 20 February 2008Replication forkThe branch-point structure that forms during DNA replication between the two template DNA strands where nascent DNA synthesis is ongoing.Cyclin-dependent kinasesA group of serine/threonine protein kinases that are activated at specific points during the cell cycle, together with their regulatory cyclin subunits. They regulate cell-cycle transitions by inducing degradation of cell-cycle inhibitory proteins.ApoptosisA form of programmed cell death that is well defined in multicellular organisms.Regulation of DNA repair throughout the cell cycleDana Branzei and Marco FoianiAbstract | The repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity. DNA lesions activate checkpoint pathways that regulate specific DNA-repair mechanisms in the different phases of the cell cycle. Checkpoint-arrested cells resume cell-cycle progression once damage has been repaired, whereas cells with unrepairable DNA lesions undergo permanent cell-cycle arrest or apoptosis. Recent studies have provided insights into the mechanisms that contribute to DNA repair in specific cell-cycle phases and have highlighted the mechanisms that ensure cell-cycle progression or arrest in normal and cancerous cells.NATURE REvIEWS | molecular cell biologyvOLUME 9 | APRIL 2008 | 297© 2008Nature Publishing GroupNature Reviews |Molecular Cell BiologyStalled forkA replication fork at which progress is blocked. Progress may be blocked by the presence of bulky lesions, aberrant DNA structures, protein–DNA complexes or depletion of dNTP pools.by repair complexes that generate the intermediateDNA structures that function as signals to activate thecheckpoint response. For example, nucleotide-excisionrepair (NER) factors are required to process lesions inbudding yeast G1 cells that have been irradiated withultraviolet (Uv). By contrast, DSBs are first processedby the Mre11–Rad50–Xrs2 (MRX) complex in yeast(the MRE11–RAD50–NBS1 (MRN) complex in mam-mals) to generate long single-stranded (ss)DNA regionsthat activate the checkpoint response9–13(FIGS 1,2).During replication, stalled forks expose ssDNA, andthe collapsed forks that are deprived of replisome4 areprocessed by the exonuclease Exo1 in budding yeastto expose ssDNA14; when coated with replicationprotein A (RPA), ssDNA becomes an activating signalfor the checkpoint15(FIGS 1,2). Following activation,the checkpoint transducers transmit and amplify thecheckpoint signal to downstream targets such as theDNA-repair apparatus and the cell-cycle machinery7.The transmission of the signal or activation of thesetargets is often achieved by different phosphorylationevents that affect the transcription level or activity ofrepair genes, and modulate cell-cycle transitions byinfluencing the stability or activity of other proteins thatare implicated in checkpoint maintenance or cell-cycleprogression (FIG. 2;BOX 2).Figure 1 | cell-cycle-specific DNa structures and lesions and the checkpoint kinases that respond to them. During the G1 phase, double-strand breaks (DSBs) lead to activation of the phosphoinositide 3-kinase related kinases DNA-PK and ATM, whereas other types of damage, such as ultraviolet-induced pyrimidine dimers, are processed by nucleotide-excision repair enzymes and lead to ATR activation. DSBs or nicks that are not repaired during G1 result in collapse of replication forks, which activates ATM (following DSBs resection, ATR is also activated). S phase DNA damage, such as stalled forks or gaps that are generated during replication, activate ATR. In pathological conditions — for example, when cells contains mutations in genes of the ATM–ATR pathway — accumulation of reversed forks155,156 is processed by nucleases14 that lead to extensive gaps or DSBs. Topological problems during replications can form catenanes, which can result in nicks or, if unresolved, can lead to DSBs during chromosome segregation.298 | APRIL 2008 | vOLUME 9 /reviews/molcellbio©2008Nature Publishing GroupCollapsed forks Disjunction of the two partially replicated sister duplexes at the replication fork that is usually associated with the dissociation of the replisome from the replication fork. ReplisomeProtein machinery that is required to replicate DNA.T ranslesion-synthesis polymerasesLow-fidelity and non-processive polymerases that can be used to bypass DNA lesions at the replication fork, often in an error-prone way.T emplate switch(TS). A process that repairs gaps in newly replicated DNA. TS can occur, for example, when a replicative polymerase encounters a lesion on the parental strand. TS uses the information on the newly synthesized sister chromatid as a template to fill in the gaps.T opoisomerasesEnzymes that remove torsional stress from double-stranded DNA by breaking and rejoining one or two of the DNA strands.Central components of the checkpoint machineryare the phosphoinositide 3-kinase related kinasesATM, ATR and DNA-PK. ATM and DNA-PK respondmainly to DSBs, whereas ATR is activated by ssDNAand stalled replication forks8(FIGS 1,2). Activation andrecruitment of these kinases to DNA lesions occursthrough direct interactions with the specificity fac-tors NBS1 (for ATM), ATRIP (for ATR) and Ku80 (forDNA-PK)16,17. DSB resection, which is less efficient inG1 and is restricted by CDK activity12,13,18, also leadsto ATR activation13(FIG. 2). When ATM and ATR arerecruited to sites of damage, they target many sub-strates, including checkpoint kinase-2 (CHK2) andCHK1, respectively (FIG. 2; BOX 2). A comprehensivecatalogue of ATM and ATR substrates that might beinvolved in the damage response has recently beenpresented19. These signalling modules probably con-tribute to coordinate the checkpoint responses withDNA repair (BOX 2; REFS 8,19).Cell-cycle specificity of the DDREukaryotic chromosomes experience rounds of DNAreplication and segregation, and cycles of condensationand decondensation. Each of these events is coupledwith topological transitions that are mediated bytopoisomerases6. Chromatin structure and compactionis also regulated throughout the cell cycle, and can beinfluenced by checkpoints and other post-translationalmodifications20–22. Here, we discuss the main types oflesions that occur in different cell-cycle phases andthe repair pathways that are likely to operate in thesephases (FIG. 1). We also try to address, wherever possible(BOX 1; FIG. 2), how these repair pathways are connectedwith the checkpoint-activation network and how theyare integrated into the overall DDR.Repair during G1 phase. Cells in G1 phase need to repairaccidental damage, such as damage that is generated byendogenous ROS species or by chemical agents, Uv orionizing radiation (IR). This damage has to be repairedpreferentially before the onset of replication, when theprimary DNA lesions can stall replication or can be con-verted into other types of DNA damage, with hazardousconsequences for the cell (FIG. 1). For example, oxidationof guanine generates oxoG. OxoG is highly mutagenic asit can base-pair with adenine during replication and causea G:C to T:A transversion23 — one of the most commonmutations in human cancers24. OxoG can be removedby DNA glycosylases by the base-excision repair (BER)pathway25.The NER pathway is mainly responsible for repairingpyrimidine dimers, which are caused by Uv and can blockthe function of DNA polymerases1. Although NER has animportant role during G1, its activity is not restricted tothis phase of the cell cycle (BOX 1). NER is divided intoglobal repair and transcription-coupled repair (TCR).TCR repairs bulky lesions of transcribed genes, whereasglobal NER repairs lesions irrespective of genome locationand cell-cycle phase1. It has also been postulated that NERsupports other repair systems and shares componentswith other repair pathways. NER proteins together withHR proteins promote repair of the DSBs that are gener-ated by crosslinks during replication, and NER proteinscan function together with the error-prone polymerasesduring crosslink repair1,26.IR causes DSBs, which are perhaps the most harm-ful damage to DNA. DSBs can be repaired by differentpathways, the most important of which are HR and non-homologous end-joining (NHEJ)27–29(BOX 1; FIG. 3). TheHR pathway uses the information that is contained ingenetically identical, or almost identical, DNA molecules(usually the sister chromatid) to repair damaged DNA.In NHEJ, the Ku heterodimer, which consists of Ku70and Ku80, binds to the two ends of a DSB and recruitsDNA-PK catalytic subunit and Ligase4–XRCC4 to ligatethe termini and complete NHEJ (FIG. 3). In NHEJ, ligationoccurs regardless of whether the ends come from thesame chromosome, and so loss of genetic informationand translocation can occur1.The high compaction of chromatin and the absence ofsister chromatids are important factors that make NHEJthe predominant DSB repair (DSBR) pathway during G1(BOX 1). The cell-cycle phase is a decisive factor in the con-trol of the DSBR pathway on the basis of the phase-specificsensitivity profile to IR of mutants that are defective ineither HR or NHEJ30,31. In chicken DT40 cells, a RAD54-knockout mutant (HR defective) is sensitive to IR onlyduring the late S and G2 phases, whereas KU70-knockoutmutants are extremely sensitive to IR during the G1phase30. Sensitivity of NHEJ-defective (severe combinedimmunodeficiency) mouse cells to IR-induced DSBs iselevated only during G1 and early S phases31. In buddingyeast, diploid cells downregulate NHEJ in favour of HR,but haploid cells in the G1 phase of the cell cycle rely onNHEJ32,33. The choice of the DSBR pathway is also coordin-ated by the CDK activity, which is low until the S phaseand influences HR initiation12,13,18(BOX 1) (see below).NATURE REvIEWS |molecular cell biology vOLUME 9 | APRIL 2008 |299©2008Nature Publishing GroupSupercoilsContortions in DNA that are important for DNA packaging and DNA–RNA synthesis.T opoisomerases sense supercoiling and can either generate or dissipate it by changing DNA topology. PrecatenanesCruciform junctions that are formed by the intertwining of the sister duplexes in the replicated portion of a replicone.DNA-repair mechanisms that function in S phase.DNA synthesis is frequently associated with nucleotidemisincorporation, accumulation of nicks and gaps,slippage at repetitive sequences, fork collapse at DNAbreaks and aberrant transitions at collapsed forks thatcause reversed and/or resected forks4(FIG. 1). Theseaberrant replication-fork transitions can endanger thestability of the chromosomes if they are not promptlyrepaired. Moreover, the torsional stress that is generatedwhen the replication fork advances, when two repliconsfuse together at termination or when the forks encoun-ter transcription bubbles causes topological modifica-tions that lead to the accumulation of supercoils and/orprecatenanes6. The topoisomerase-mediated resolutionof these topological constraints allows the completionof S phase, chromosome condensation and segregationduring the G2 and M phases (FIG. 1).Base–base mismatches and small insertion and/ordeletion loops that are generated by faulty replicationare corrected by the mismatch repair (MMR) path-way, which functions mainly during S phase34(BOX 1).This pathway recognizes and removes the flawedstretch of DNA, and then novel DNA synthesis fillsin the gap. Chemical alterations of nucleotide basesare often removed by BER, as in G1 phase. BER is alsoinvolved in removing misincorporated uracils duringS phase1(FIG. 1).Single-strand gaps or nicks often occur during replica-tion (FIG. 1) and seem to be the main source of HR in mitoticcells35,36. However, they can also be dealt with by damagetolerance or bypass-replication mechanisms that operateduring S phase (BOX 1; FIG. 2). Cells have evolved twomechanisms that promote damage tolerance in S phase.The first mechanism is mediated by translesion synthesis(TLS) polymerases, which replicate across lesion, often inan error-prone manner. Template switch (TS) is an error-free mechanism that fills in gaps in the DNA template byrepriming events downstream of the lesion37,38(BOX 1). TheTS pathway uses the undamaged information of the sisterduplex, and the mechanism seems to share similaritieswith HR. In budding yeast, both pathways depend on theRAD6–RAD18 post-replication repair (PRR) pathwayand are largely controlled through covalent modificationsof proliferating cell nuclear antigen (PCNA; an essentialprocessivity clamp for DNA polymerases) by ubiquitin37,39(BOX 2; FIG. 4).DSBs can often occur during S phase as a result ofreplication-fork collapse (FIG. 1). Studies in buddingyeast have established that HR is carried out by theproducts of the conserved RAD52epistasis group ofgenes28,40. HR requires 5′–3′ resection of DSBs12,18, aprocess that depends largely on the activities of Exo1and the MRX complex12,41,42. The MRX (or MRN inmammals) complex has DNA-binding, endonucleaseand 3′–5′ exonuclease activity, and it is thought to func-tion together with a 5′–3′ exonuclease to resect DSBsand create 3′-ended ssDNA that is required to initiatestrand invasion (FIGS 2,3). Recently, both the fission yeastprotein Ctp1 and its mammalian orthologue CtIP wereshown to operate cooperatively with the MRN complexexclusively during the S and G2 phases of the cell cycle topromote DSB resection and HR43,44. Studies in buddingyeast have shown that the CDK activity facilitates theresection stage of the HR reaction and prevents NHEJ12,45(FIGS 2,3). The Ku heterodimer can still bind to DSBs,with even faster kinetics than HR factors46; therefore, itis possible that a competition might exist between NHEJand HR even during the S phase, which suggests thatadditional factors might suppress the binding of Ku infavour of HR proteins. Recent studies in chicken DT40cells indicated that both RAD18 and poly(ADP-ribose)polymerase (PARP) function to decrease the affinity of Kuto DSBs and to favour HR47,48. The mechanism throughwhich CDK facilitates HR in S phase is just beginning tobe elucidated, but it might involve the MRE11-associated300 | APRIL 2008 | vOLUME 9 /reviews/molcellbio©2008Nature Publishing GroupNature Reviews |Molecular Cell Biology Damage toleranceA post-replicative repairpathway in which the lesionsare not repaired, but bypassed(tolerated) during replication.Bypass can be achieved byeither using specializedpolymerases, or by using thenewly synthesized sisterchromatid strand as a template.EpistasisA group of genes that function in the same biological pathway, usually defined by genetic analysis of double mutants. Poly(ADP-ribose) polymeraseA polymerase that attaches ADP–ribose moieties to target proteins by means of covalent bonds, which is one of the earliest cellular responses to strand breaks. Differentiated cellsCells that are specialized for a particular function (such as neurons and muscle cells) and that cannot proliferate. Senescent cellsMitotic cells that cannot divide, but remain metabolically active. Senescence is often caused by stimuli that can cause cancer.factor CtIP43,44, which is phosphorylated by CDKs49.Remarkably, the protein levels of both fission yeast Ctp1and mammalian CtIP are very low during G1 and highduring S and G2 phases of the cell cycle43,50; this may alsoaccount for S–G2-specific HR (BOX 1; FIG. 2).DNA repair during G2 and M phases. Gaps and DSBsthat occur during replication, if left unrepaired by the endof the S phase, need to be repaired before mitosis. For HRto occur during S and G2 phases using the sister chroma-tid as a template, it is important that the sister chromatidsare in proximity to one another. This is probably estab-lished by cohesion, which provides a physical linkage thatconnects the sister chromatids from S phase until theirseparation during anaphase. Cohesion depends largely oncohesin, a protein complex that contains two structuralmaintenance of chromosomes (SMC) proteins, SMC1and SMC3, held together by sister-chromatid cohesion-1(SCC1) and SCC3 (REF. 51). Cohesion must be establishedduring S phase52, and this process requires additional pro-teins such as Eco1 in budding yeast53. However, DSBs cantrigger cohesion after DNA replication is complete, andthis event is required for sister-chromatid repair in cellsin G2 phase54,55(FIG. 2). Not surprisingly, mutations thataffect the cohesin complex, its loading, or factors thatare required to establish cohesion, are severely defectivein DSBR56–58.The topological problems that arise when two repli-cons fuse together at termination also need to be resolvedduring S–G2 in order to prevent chromosome breakageduring segregation6,59. When the DSBs occur during chro-mosome segregation — during which time chromosomesare already highly compact and the search for homologyis difficult — repair is likely to occur by NHEJ in the sub-sequent G1 phase if checkpoints or caretaker genes hadnot caused cell-cycle arrest during G2 and M phases60–62.DNA repair also occurs in non-dividing cells. Most DDRsare associated with replication, and it is therefore likelythat cells that do not divide (differentiated or senescentcells) may have dedicated repair mechanisms that repairendogenous damage when most DNA-repair pathwaysthat function in dividing cells are attenuated63. It hasbeen proposed that accumulating damage in the DNA ofthe human brain has a crucial role in ageing and in thepathogenesis of many neurological disorders, includingAlzheimer’s, Parkinson’s and amylotrophic lateral sclerosis(ALS)64,65. The most predominant type of damage in pathways. Ku and the MRE11–RAD50–NBS1 (MRN) complex bind to double-strand breaks (DSBs) and activate the kinases DNA-PK and ATM, respectively. Ku and DNA-PK promote non-homologous end joining (NHEJ) repair of DSBs. During S and G2 phases, DSBs are resected to expose single stranded (ss)DNA and activate ATR12,13,44. Activated ATM induces chromatin changes around the DSB site through phosphorylation of the histone H2AX, which leads to the recruitment of many checkpoint and repair factors, such as MDC1, MRN and the ubiquitin ligases RNF8 and UBC13. These factors promote the recruitment of 53BP1, BRCA1 (REFS 98–100,102) and ATM itself to facilitate the spreading of the damage signal through the nucleus8. ATM-mediated phosphorylation of KAP1 induces chromatin relaxation, whereas other modifications promote repair (such as modification of SMC1 or MRN), or further spreading of the damage signal (such as modification of MRN or checkpoint kinase-2 (CHK2)). ATM- and ATR-mediated phosphorylation of CHK2 and CHK1 promote cell-cycle arrest and DNA repair and reduce cyclin-dependent kinase (CDK) activity. Several targets of ATM and ATR are required for homologous recombination (HR), including SMC1, FANCD2 and FANCI. CDK-mediated phosphorylation of BRCA2 inhibits HR by impairing the interaction of BRCA2 with the HR protein RAD51. In fission yeast, the DNA-damage-checkpoint complex Rad9–Rad1–Hus1 (911) promotes translesion synthesis (TLS) and template switch (TS) damage-bypass mechanisms. Arrows indicate direct phosphorylation events. Dashed arrows indicate indirect events.NATURE REvIEWS |molecular cell biology vOLUME 9 | APRIL 2008 |301©2008Nature Publishing Group3′CDKDouble Holliday junction A central intermediate to homologous recombination. IschaemiaA restriction in blood supply, generally due to factors in the blood vessels, that causes tissue damage or dysfunction.neuronal cells is probably oxidative DNA damage, whicharises during normal cellular metabolism65. Oxidative basedamages are primarily removed by BER65, whereas DNAadducts are repaired mostly by NER64. NER is also impor-tant for survival and proper function in neuronal cells andis thought to be essential for repairing endogenous DNAdamage, as attested by the severe neurological defects thatare observed in patients with Xeroderma pigmentosum(XP) and Cockayne’s syndrome (CS). XP and CS arecharacterized by defects in NER and TCR, respectively66.MMR, HR and NHEJ have minor roles in neuronal cells(see REF. 65 and references therein), especially in condi-tions that occur following ischaemia or apoptotic stimuli.A major clinical effect of cancer treatments is neurocogni-tive dysfunction and neuropathy, and so much researchfocuses on understanding the repair pathways that areresponsible for the repair of neuronal DNA followingchemotherapy and IR64,65.Regulation of DNA repair by kinasesRegulation of DNA-repair pathways is important forgenome integrity. This can occur by modulating thechoice of the repair pathway when a lesion is a potentialsubstrate for two or several repair pathways, by regu-lating the stability or activity of a repair factor, or byregulating the period of time during which repair cantake place. Here we discuss several DNA regulatory path-ways that are either activated in a cell-cycle-dependentmanner or that crosstalk with the checkpoint machineryto induce cell-cycle arrest and DNA repair.The checkpoint kinases mediate arrest and allow repair.The checkpoint kinases promote the viability of cellsfollowing DNA damage through their ability to mediatecell-cycle arrest, which allows cells to repair DNA dam-age (BOX 2). Although ATM and ATR respond to differenttypes of lesions (BOX 2), to which they are recruited bydifferent factors (MRN recruits ATM; ATRIP and RPA-coated ssDNA recruit ATR) (FIG. 2), recent evidence sug-gests that ATR is also activated by IR-induced DSBs in acell-cycle regulated manner13. ATR activation by DSBsrequires ATM and MRN–CtIP, it occurs only duringS and G2 phases, and it is abolished by CDK inhibition13,44(FIG. 2). These results are in agreement with previousfindings showing that formation of IR-induced foci thatcontained the HR protein RAD51 in both yeast andhuman cells is restricted to S and G2 phases and dependson checkpoint activity67–69. The relevant targets of theCDK in this pathway, besides the likely CtIP44,49, remainto be identified.In certain occasions, CDK-mediated phosphorylationof checkpoint proteins is required to activate their DNA-repair mediator function. In fission yeast, the checkpointprotein Crb2 is phosphorylated by Cdk1. This modifica-tion is important to mediate later steps of HR-mediatedDSBR that implicate the RecQ helicase Rqh1 and thetopoisomerase Top3 (REF. 70).Numerous examples have shown that checkpoint-dependent phosphorylation of targets affects theirfunction in repair events that occur in a cell-cycle-specific manner. CHK1 phosphorylation of RAD51 isrequired for mammalian HR71; CHK1 also influences thereplacement of RPA on ssDNA with RAD51 and RAD52in a process that leads to RAD51 presynaptic filamentformation and initiation of HR-mediated DSBR72(FIG. 3). However, the replication-checkpoint-depend-ent phosphorylation of fission yeast protein Mus81, aconserved endonuclease that has been proposed to actat stalled replication forks, inhibits recombination73. TheATR-dependent phosphorylation of the NBS1 subunitrecombination (HR) during S and G2 phases of the cell cycle (see text and REF. 27).Binding of the Ku heterodimer to DSBs triggers the recruitment of DNA-PK catalyticsubunit and sealing of the DSBs by NHEJ. By contrast, DSBs that occur during S andG2 phases preferentially activate ATM, through the MRE11–RAD50–NBS1 (MRN;Mre11–Rad50–Xrs2 (MRX) in yeast) complex8. The higher cyclin-dependent kinase(CDK) activity that is specific for S and G2 phases of the cell cycle promotes DSBresection12, exposing 3′ overhangs of single stranded (ss)DNA. When the ssDNA of3′ overhangs is coated with replication protein A (RPA), it activates ATR; RPA can beremoved and replaced by RAD51 with the help of mediator proteins such as RAD52.This leads to the formation of RAD51 presynaptic filaments, which initiate HR byinvading the homologous region in the duplex to form a DNA joint called a D-loop,which can be further extended by DNA synthesis. Strand displacement of thisintermediate by a DNA helicase channels the reaction towards synthesis-dependentstrand annealing (SDSA). Alternatively, the second DSB end can be captured, giving riseto a double Holliday junction intermediate, which can be resolved by endonucleases ordissolved by the combined action of a helicase (BLM) and a topoisomerase (TOP3)40,134.302 | APRIL 2008 | vOLUME 9 /reviews/molcellbio©2008Nature Publishing Group。

RIC1在拟南芥根的生长发育过程中正调控生长素信号负调控ABA信号

Arabidopsis ROP-interactive CRIB motif-containing protein 1(RIC1)positively regulates auxin signalling and negatively regulates abscisic acid (ABA)signalling during root developmentYUNJUNG CHOI 1,YUREE LEE 1,SOO YOUNG KIM 3,YOUNGSOOK LEE 1,2&JAE-UNG HWANG 11POSTECH-UZH Global Research Laboratory,Division of Molecular Life Sciences,Pohang University of Science andTechnology (POSTECH),Pohang 790-784,Korea,2Division of Integrative Bioscience and Biotechnology,POSTECH,Pohang 790-784,Korea and 3Department of Molecular Biotechnology &Kumho Life Science Laboratory,College of Agriculture and Life Sciences,Chonnam National University,Gwangju 500-757,KoreaABSTRACTAuxin and abscisic acid (ABA)modulate numerous aspects of plant development together,mostly in opposite directions,suggesting that extensive crosstalk occurs between the signal-ling pathways of the two hormones.However,little is known about the nature of this crosstalk.We demonstrate that ROP-interactive CRIB motif-containing protein 1(RIC1)is involved in the interaction between auxin-and ABA-regulated root growth and lateral root formation.RIC1expression is highly induced by both hormones,and expressed in the roots of young seedlings.Whereas auxin-responsive gene induction and the effect of auxin on root growth and lateral root formation were suppressed in the ric1knockout,ABA-responsive gene induction and the effect of ABA on seed germination,root growth and lateral root for-mation were potentiated.Thus,RIC1positively regulates auxin responses,but negatively regulates ABA responses.Together,our results suggest that RIC1is a component of the intricate signalling network that underlies auxin and ABA crosstalk.Key-words :hormone crosstalk;lateral root;RIC protein;root growth;ROP GTPase.INTRODUCTIONAuxin and abscisic acid (ABA)are two major plant growth regulators.In general,auxin promotes the growth of vegeta-tive tissues,whereas ABA suppresses proliferation and confers stress resistance.For example,auxin promotes lateral root initiation,whereas ABA inhibits it.Auxin opens stomata,whereas ABA closes them.Such antagonistic effects of two hormones have been reported to regulate numerous stress responses and developmental and physiological pro-cesses in the plant (Gehring,Irving &Parish 1990;Casimiro et al .2003;Tanaka et al .2006).The interaction between auxin and ABA seems to be more complex during early seedlingdevelopment and primary root elongation than later on.Although both auxin and ABA are necessary for early seed-ling development,exogenously applied ABA,which presum-ably is applied at a signiﬁcantly greater concentration than the endogenous hormone,inhibits growth.Primary root elon-gation is promoted by nanomolar amounts of both auxin and ABA (Gaither,Lutz &Forrence 1975;Mulkey,Kuzmanoff &Evans 1982),but is inhibited by higher concentrations of these hormones (Pilet &Chanson 1981;Mulkey et al .1982;Eliasson,Bertell &Bolander 1989).The observation that the two hormones function together to regulate many responses indicates that the signalling pathways that transduce the primary hormonal signals to downstream responses may intersect at speciﬁc points and/or involve common players.Indeed,the expression of ABA INSENSITIVE 3(ABI3)is activated by both auxin and ABA,and ABI3functions as a positive regulator of ABA-mediated inhibition of seed ger-mination and as a negative regulator of auxin-mediated lateral root formation and ABA-mediated inhibition of primary root growth (Brady et al .2003;Zhang,Garreton &Chua 2005).Given the vast array of responses of plants to auxin and ABA,one would expect that many such points of crosstalk exist;however,this aspect of auxin and ABA signal transduction remains largely unexplored.Rho family GTPases act as molecular switches that mediate diverse cellular responses to multiple extracellular signal including hormones (Bos 2000).ROP (Rho of plants;also called RAC)GTPases represent the sole Rho family of Ras-related G proteins in plants (Yang 2002),and the model plant Arabidopsis contains 11ROP GTPases in its genome (Bischoff et al .1999;Winge et al .2000;Zheng &Yang 2000).Several studies have reported that ROP GTPases play impor-tant roles in auxin-and ABA-related responses (Lemichez et al .2001;Tao,Cheung &Wu 2002;Zheng et al .2002;Bloch et al .2005;Tao et al .2005).Auxin treatment increases the amount of activated ROP GTPase in tobacco (Tao et al .2002)and Arabidopsis (Xu et al .2010;Lin et al .2012)seed-lings.Overexpression of wild-type or constitutively active forms of ROP GTPases stimulates auxin-related phenotypes and auxin-responsive gene expression in Arabidopsis and tobacco (Li et al .2001;Tao et al .2002,2005).Activated ROP GTPases promote the 26S proteasome-dependentCorrespondence:Y.Lee.Fax:+82542792199;e-mail:ylee@postech.ac.kr;J-U.Hwang.Fax:+82542792199;e-mail:thecute@postech.ac.krY.L.and J-U.H.contributed equally to the manuscript.Plant,Cell and Environment (2013)36,945–955doi:10.1111/pce.12028©2012Blackwell Publishing Ltd945degradation of auxin/indole-3-acetic acid(AUX/IAA)pro-teins in tobacco and Arabidopsis(Tao et al.2005).ROP GTPase mutations cause defects in auxin-dependent cell expansion(Fu et al.2005,2009;Xu et al.2010).In contrast to the positive role of ROP GTPases in the auxin response, ROP GTPases appear to be negative regulators of ABA responses.ABA treatment reduces the amount of activated ROP GTPase in Arabidopsis suspension cells and seedlings (Lemichez et al.2001).Expression of constitutively active forms of Arabidopsis ROP2and ROP6reduces sensitivity to ABA during seed germination(Li et al.2001)and stomatal closing(Lemichez et al.2001;Hwang et al.2011).The obser-vation that an Arabidopsis mutant that lacks ROP10expres-sion is hypersensitive to ABA,and that ROP10expression is suppressed by ABA,suggests the existence of an interesting feedback regulation loop in the ABA signalling pathway (Zheng et al.2002).RICs(ROP-interactive CRIB motif-containing proteins) are a unique group of interacting partners of activated ROP GTPases.RIC proteins interact with multiple ROP GTPases via their conserved CRIB motif,and link ROP proteins to diverse target molecules that bind to their variable domains (Yang2002).The11RIC genes present in Arabidopsis are categorized into four phylogenetic groups(Wu et al.2001;Gu et al.2005).However,knowledge on RIC functions is limited; RIC3and RIC4have been shown to regulate[Ca2+]cyt and F-actin dynamics during the polar growth of pollen tubes (Wu et al.2001;Gu et al.2005).RIC7is reported to interact with active ROP2in stomatal guard cells and to suppress light-induced stomatal opening(Jeon et al.2008).In epider-mal cells of the leaf and hypocotyl,RIC1suppresses aniso-tropic cell expansion by regulating microtubule(MT) dynamics(Fu et al.2005,2009;Xu et al.2010).RIC1is expressed in a broad range of tissues(Wu et al. 2001).However,the function of RIC1has been analysed mostly in the development of leaf pavement cells(Fu et al. 2005).In this cell type,RIC1is associated with MTs and regulates their assembly.In the lobe-forming regions of pave-ment cells,RIC1is inactivated by active ROP2,which sup-presses MT assembly,but promotesﬁne F-actin assembly and thereby induces outgrowth of the region.In contrast,in the neck-forming regions of pavement cells,RIC1is activated by active ROP6and then promotes the assembly of MTs,which limits the expansion of the region and results in the forma-tion of a narrow neck.The cortical MTs in the leaf pavement cells of ric1mutants are randomly organized,resulting in pavement cells with wider necks.This ROP6-RIC1-MT sig-nalling pathway seems to function in both hypocotyl elonga-tion and leaf epidermal cell development(Fu et al.2005, 2009).In pollen tubes,however,RIC1is localized to the apical plasma membrane,where MTs are absent,and over-expression of RIC1suppresses the depolarized tube growth induced by ROP1overexpression(Wu et al.2001).Given its broad expression pattern,RIC1may mediate diverse pro-cesses in the growth and development of plants,which have yet to be elucidated.In this work,we established that RIC1positively regulates the auxin effect and negatively regulates the ABA effect during root growth and lateral root development.These results will advance our current limited understanding on the mode of action of RIC1protein during regulation of plant development by auxin and ABA.MATERIALS AND METHODSPlant materials and growth conditionsSeeds of wild-type,ric1,and ric1/RIC1p:GFP:RIC1Arabi-dopsis thaliana plants(ecotype Ws)were surface sterilized, placed at4°C in the dark for2d,and then sown in half-strength Murashige and Skoog(MS)agar medium.Arabi-dopsis seedlings were grown in a growth chamber with a16h light/8h dark cycle at22°C.Isolation of the RIC1knockout mutantsSeeds of T-DNA insertion mutant for RIC1(ric1; FLAG_075E05)were obtained from Institut National de la Recherche Agronomique(INRA)-Versailles Genomic Resource Center(http://www-ijpb.versailles.inra.fr/en/cra/ cra_accueil.htm).Reverse transcriptase(RT)-PCR analysis using gene-speciﬁc primers conﬁrmed that this is a null mutant.Primer information used for RT-PCR is available in Supporting Information Table S1.Complementation of ric1with RIC1p:GFP:RIC1For the ric1complementation assay,the RIC1promoter region(~2kb)and the RIC1open reading frame were indi-vidually obtained by PCR ampliﬁcation.These genomic DNA fragments and GFP coding sequence were sequentially cloned into a pCR®8/GW/TOPO®vector(Invitrogen,Carls-bad,CA,USA),and then transferred into a pMDC100 gateway vector(Curtis&Grossniklaus2003).The RIC1p:GFP:RIC1construct was transformed into ric1plants by the Agrobacterium-mediatedﬂoral dipping method (Clough&Bent1998).The phenotypes of the T3seedlings of homozygous ric1/RIC1p:GFP:RIC1lines were observed. RIC1p:GUS expression assayThe genomic DNA fragment containing the promoter region (~2kb)andﬁrst exon of RIC1was ampliﬁed by PCR and fused to the GUS-coding region of the pMDC164vector (RIC1p:GUS).The RIC1p:GUS construct was transformed into wild-type Arabidopsis plants using the Agrobacterium-mediatedﬂoral dipping method(Clough&Bent1998). RIC1p:GUS expression was observed in T3plants from six independently transformed lines.Brieﬂy,the seedlings of RIC1p:GUS were incubated in GUS staining buffer[100m m Na2HPO4(pH7.2),3m m potassium ferricyanide,3m m potas-sium ferrocyanide,10m m ethylenediaminetetraacetic acid (EDTA),0.1%Triton X-100,and2m m5-bromo-4-chloro-3-indolyl-b-D-glucuronide(Duchefa,Haarlem,The Nether-lands)]at37°C for12h.Chlorophyll was extracted in70% ethanol solution.946Y.Choi et al.©2012Blackwell Publishing Ltd,Plant,Cell and Environment,36,945–955Observation of the cellular localizationof GFP:RIC1Wild-type Arabidopsis plants were stably transformed with a GFP:RIC1construct under the control of CaMV35S pro-moter.In multiple independently transformed lines of Arabidopsis,the subcellular localization of GFP:RIC1was observed by using a Zeiss LSM510Meta Laser scanning microscope(Zeiss,/).To investigate the effects of auxin and ABA on the cellular localization of RIC1,7-day-old seedlings that stably express GFP:RIC1 were incubated in half-strength MS medium containing1m m auxin[naphthalene-1-acetic acid(NAA)]or10m m ABA for1h.Quantiﬁcation of RIC and ABA-orauxin-responsive gene transcript levelsQuantitative real-time RT-PCR(Q-PCR)was used to quan-tify transcript levels of RIC genes,ABA-responsive genes and auxin-responsive genes.Total RNA was extracted from each sample and then reverse transcribed into cDNA. Q-PCR was carried out using a Takara TP800thermal cycler and Takara SYBR RT-PCR Kit(Takara Bio,Kyoto,Japan), following the manufacturer’s instructions.Transcript levels of RIC s,ABA-responsive genes and auxin-responsive genes were normalized against that of tubulin8. Measurement of lateral root formation and primary root growthTo examine the effects of ABA or auxin on lateral root formation and primary root growth,Arabidopsis seedlings were grown for4d under a16h photoperiod and then trans-ferred to fresh half-strength MS agar plates supplemented with the indicated concentrations of ABA or auxin.After an additional5–7d,the net elongation of primary roots was measured and the number of lateral roots was counted using a stereo microscope(Olympus SZX12,Tokyo,Japan). Seed germination assayFor germination assays,the seeds were placed in the dark at 4°C for2d and then sown on MS medium agar plates con-taining1%sucrose in the presence or absence of0–1.5m m concentrations of ABA.The seeds were incubated under a 16h photoperiod at22°C.Germinated seeds(as determined by cotyledon greening or radicle emergence)were scored every12h for6d.Germination ratio refers to the number of germinated seeds as a proportion of the total number of seeds tested.RESULTSRIC1expression is induced by both auxinand ABAMembers of the ROP small GTPase family are reported to mediate ABA and auxin responses(Li et al.2001;Zheng et al.2002;Tao et al.2005).We hypothesized that RIC proteins might serve an important intermediary in the ROP-mediated ABA and auxin signalling pathways.If this hypothesis was true,then the level of the RIC proteins may be substantially regulated by ABA and auxin.Thus,we tested the effect of auxin and ABA on the expression of10out of the11Arabi-dopsis RIC genes(Fig.1).Arabidopsis seedlings were grown on half-strength MS medium for7d and then treated with 1m m NAA or10m m ABA for1h.Total RNA was isolated from these seedlings and RIC gene transcript levels were examined using quantitative real-time PCR(Q-PCR).Upon NAA and ABA treatment,the transcript level of many RIC s (RIC2,3,4,5,7,9and11)increased,but the increase in RIC1 was the highest.Therefore,we chose to focus our analysis on RIC1.Loss of RIC1expression alters gene induction by auxin and ABAAuxin and ABA induce the expression of sets of genes, which are known as auxin-responsive and ABA-responsive genes,respectively(Brady et al.2003;Zhang et al.2005;Li et al.2009).To examine the involvement of RIC1in auxin and ABA signal transduction,we evaluated the effect of RIC1knockout(ric1)on the expression levels of typical auxin-and ABA-responsive genes.ric1,a T-DNA insertion mutant(Stock No.FLAG_075E05),was obtained from INRA-Versailles Genomic Resource Center(http://www-ijpb.versailles.inra.fr/en/cra/cra_accueil.htm).RT-PCR analy-sis conﬁrmed that the T-DNA insertion into the fourth exon completely blocked the expression of RIC1in ric1(Fig.2a). The small auxin-up RNAs(SAURs)encode short tran-scripts that accumulate rapidly upon auxin treatment(Li et al.2009).IAA6and IAA19are members of INDOLE-3-ACETIC ACID/AUXIN(IAA/AUX)genes andtheir Figure1.Expression of RIC genes were induced by auxin and abscisic acid(ABA).Seven-day-old Arabidopsis seedlings were treated without or with1m m auxin[naphthalene-1-acetic acid (NAA)]or10m m ABA for1h,and total RNA was isolated from seedlings for Q-PCR analysis.The transcript levels of RIC genes were normalized against the transcript level of Tubulin8,which served as the internal control,and are presented as values relative to the untreated control.Data are meansϮSEM of three to eight biological replicates.Asterisks indicate values that are statistically signiﬁcantly different from the untreated control(***P<0.005;**P<0.001;*P<0.05).RIC1regulates root development947©2012Blackwell Publishing Ltd,Plant,Cell and Environment,36,945–955948Y.Choi et al.©2012Blackwell Publishing Ltd,Plant,Cell and Environment,36,945–955expressions are induced by auxin(Abel,Nguyen&Theologis 1995;Tatematsu et al.2004).Using Q-PCR analysis,we com-pared the transcript levels of SAUR and IAA genes in ric1 seedlings with those in wild-type seedlings(Fig.2b).Under control conditions without NAA treatment,the transcript levels of these genes in ric1seedlings were similar to or slightly higher than those in wild-type seedlings(Fig.2b).In 7-day-old wild-type seedlings,treatment with1m m NAA for 1h induced a two-to fourfold increase in SAUR gene expres-sion(Fig.2b).Interestingly,however,the induction of SAUR genes by1m m NAA was suppressed in ric1seedlings (Fig.2b);SAUR9transcript level increased3.3Ϯ0.1-fold in the wild type,but1.7Ϯ0.1-fold in ric1(t-test,P<0.005); SAUR15transcript level increased3.9Ϯ0.6-fold in the wild type,but1.8Ϯ0.2-fold in ric1(t-test,P<0.01);SAUR23 transcript level increased3.0Ϯ0.3-fold in the wild type,but 1.4Ϯ0.2-fold in ric1(t-test,P<0.005);SAUR62transcript level increased2.5Ϯ0.2-fold in the wild type but1.4Ϯ0.2-fold in ric1(t-test,P<0.001);and SAUR66transcript level increased3.9Ϯ0.4-fold in the wild type,but1.7Ϯ0.2-fold in ric1(t-test,P<0.001).Similarly,induction of IAA6and IAA19upon NAA treatment was suppressed by ric1(Fig.2b bottom panel),whereas the transcript level of IAA6 increased21.9Ϯ1.3-fold in the wild type,but only11.3Ϯ0.2-fold in ric1(P<0.05),and the transcript level of IAA19 increased27.1Ϯ3.3-fold in the wild type,but only13.1Ϯ1.9-fold in ric1(P<0.05).These results indicate that RIC1is involved in the control of the auxin signalling pathway.ABI3,ABI5,responsive to ABA18(RAB18),and respon-sive to dehydration29A(RD29A)and29B(RD29B)are well-characterized ABA-responsive genes that play critical roles in ABA signalling(Parcy et al.1994;Finkelstein& Lynch2000;Lopez-Molina&Chua2000;Hoth et al.2002; Kang et al.2010).To analyse the involvement of RIC1in ABA signalling,we gauged the effects of ABA on expres-sions of these genes in the roots of wild-type and ric1seed-lings(Fig.2c).Seven-day-old seedlings were incubated in half-strength liquid MS medium in the presence or absence of0.5m m ABA for1h.Under control conditions(i.e.in the absence of ABA),transcript levels of ABI3,ABI5,RD29A, RD29B and RAB18were slightly higher in ric1seedlings than in wild-type seedlings,and this difference was further increased after ABA treatment(Fig.2c).Upon ABA treat-ment,ric1seedlings exhibited much higher transcript levels of thoseﬁve ABA-responsive genes,compared with wild-type seedlings(Fig.2c);ABI3transcript level increased 2.4Ϯ0.5-fold in the wild type,but 5.5Ϯ0.9-fold in ric1 (P<0.01);ABI5transcript level increased5.9Ϯ0.4-fold in the wild type,but12.9Ϯ1.9in ric1(P<0.005);RD29A tran-script level increased12.3Ϯ3.9-fold in the wild type,but 17.4Ϯ6.6-fold in ric1(P<0.06);RD29B transcript level increased5.5Ϯ1.2-fold in the wild type,but10.9Ϯ0.8-fold in ric1(P<0.05);and RAB18transcript level increased 4.6Ϯ1.1-fold in the wild type,but8.0Ϯ1.3-fold in ric1 (P<0.01).In summary,RIC1knockout suppressed the induction of auxin-responsive genes by auxin,but promoted the induction of ABA-responsive genes by ABA.These results suggest that RIC1exerts opposite regulatory functions in the auxin and ABA signalling pathways.RIC1is expressed in the roots ofyoung seedlingsTo identify which auxin-and ABA-mediated processes are regulated by RIC1,weﬁrst determined the tissue-speciﬁc and developmental stage-speciﬁc expression of RIC1.The genomic DNA region containing the RIC1promoter(~2kb) and theﬁrst exon was fused to the GUS-coding region (RIC1p:GUS),and introduced into wild-type plants. RIC1p:GUS expression was observed in T3seeds and seed-lings from six independently transformed Arabidopsis lines (Fig.3a,b).In germinating seeds and young seedlings,the RIC1p:GUS signal was evident in roots.In germinating seeds,RIC1p:GUS signal was limited to the embryonic root tip(Fig.3a,left),and in seedlings at1–3d after sowing,RIC1p:GUS extended the expression to other parts of root including differentiation zone,root hairs and root–shoot junction(Fig.3a,right).In the roots of2-week-old plants,RIC1p:GUS signal was detected in root tips and also in maturation zone,where lateral roots grow out(Fig.3b).RIC1p:GUS signal was strongly detected in columella cells from the root tip, (Fig.3b-d).In maturation zone of root,cells surrounding emerged lateral root(Fig.3b-b)and epidermal cells at the base of lateral roots(Fig.3b-c)showed clear RIC1p:GUS signals.These RIC1expression patterns indicate that RIC1is likely to be involved in the regulation of seed germination, early seedling development and root development.In addition to being expressed in the roots,RIC1p:GUS was also expressed in the hypocotyls,petioles,and weakly in the leaves of young seedlings(Fig.3a,b).In Arabidopsis plants of later development stages,expression of RIC1p:GUS was weak except inﬂowers,where RIC1expression was pre-viously reported(Wu et al.2001;Fu et al.2005,2009;Xu et al.Figure2.ric1knockout mutation altered induction of auxin-and abscisic acid(ABA)-responsive genes.(a)Schematic structure of theRIC1gene(left).The triangle indicates the T-DNA insertion site in ric1.Exons are represented as boxes and introns as lines.RT-PCR analysis using a RIC1-speciﬁc primer set(RT-F and RT-R)shows that ric1is a true null mutant(right).Tubulin8was used as an internal control.(b)Expression of SAUR9,SAUR15,SAUR23,SAUR62,SAUR66,IAA6and IAA19in plants treated or not with1m m auxin [naphthalene-1-acetic acid(NAA)].(c)Expression of ABI3,ABI5,RD29A,RD29B and RAB18in plants treated or not with0.5m m ABA.Q-PCR analyses of transcripts of auxin-and ABA-responsive genes were performed using total RNA isolated from the roots of8-day-old seedlings after1h of treatment without or with auxin or ABA.Data were normalized using Tubulin8as an internal control,and are presented as values relative to the untreated wild type(WT).Data are meansϮSEM of four independent experiments.Asterisks indicate values that are signiﬁcantly different from those of the WT(***P<0.005;**P<0.01;*P<0.05;#P<0.06).RIC1regulates root development949©2012Blackwell Publishing Ltd,Plant,Cell and Environment,36,945–9552010);RIC1p:GUS signal was strongly observed in the anthers and mature pollen grains (Supporting Information Fig.S1).RIC1knockout suppresses the effect of auxin on lateral root formation and primary root elongationAs RIC1is expressed in root (Fig.3)and RIC1expression is up-regulated by auxin (Fig.1),we examined whether auxin-dependent root growth and lateral root formation were affected in the ric1mutant (Fig.4).Arabidopsis seedlings were grown on half-strength MS medium for 4d and then transferred to fresh half-strength MS medium supplemented with various concentrations (0–100n m )of NAA.After 5d,the number of lateral roots (including newly emerged ones)was counted.Auxin promoted the formation of lateral roots in different genotypes,including the wild type,ric1,and ric1/RIC1p:GFP:RIC1(complementation lines,C1and C2;Fig.4a);however,this effect was signiﬁcantly less in ric1than in the wild type and complementation lines (Fig.4b).InFigure 3.RIC1expression in Arabidopsis plants.(a)One dayafter sowing (DAS),an embryo exhibited RIC1p::GUS signal at the root tip (left,indicated by arrow).Seed coat was removed after GUS staining for observation.A young seedling that had justgerminated but had not yet undergone cotyledon expansion (right;1–3DAS)exhibited relatively stronger RIC1p::GUS signal in the root tip and differentiation zone of the root including the root hairs.Bar =300m m.(b)A 2-week-old seedling displayed RIC1p::GUS signal in the root tip and maturation zone withemerged lateral roots,and,weakly,in the shoot.(b-a )Bar =1cm.(b-b ),(b-c )and (b-d )are enlarged images of maturation zone with an emerged lateral root,a lateral root with GUS staining at the base,and a root tip with GUS staining in columellacells.Figure 4.Auxin responses were reduced in ric1plants.Arabidopsis seedlings were grown vertically on half-strength Murashige and Skoog (MS)medium for 4d,transferred to fresh half-strength MS medium supplemented or not with 0–100n m auxin [naphthalene-1-acetic acid (NAA)],and grown for anadditional 5–7d.(a)Levels of RIC1transcript in wild-type (WT),ric1,and ric1/RIC1p:GFP:RIC1(lines C1and C2)plants.RIC1expression in seedlings was quantiﬁed using Q-PCR and presented values relative to that of WT.Data are means ϮSEM of four independent experiments.(b)Number of lateral roots formed in the absence or presence of NAA (means ϮSEM of 28seedlings from four independent experiments),measured 5d after transfer to medium supplemented with or without NAA.Asterisks indicate values that are signiﬁcantly different from those of the WT atP <0.05.(c)Relative values of lateral root number (mean ϮSEM,n =28).Values in (b)were normalized to the values of non-treated controls.Asterisks indicate values that are signiﬁcantly different from those of the WT (***P <0.005).(d)Primary root elongation in the absence or presence of NAA (means ϮSEM of 28seedlings from four independent experiments).The net primary root growth was measured 7d after transfer to medium supplemented with or without NAA.Asterisks indicate values that are signiﬁcantly different from those of the WT (**P <0.05;*P <0.1).(e)Relative values of net primary root elongation (mean ϮSEM,n =28).Values in (d)were normalized to the values ofnon-treated controls.Asterisks indicate values that are signiﬁcantly different from those of the WT (***P <0.005;*P <0.05).950Y.Choi et al .©2012Blackwell Publishing Ltd,Plant,Cell and Environment,36,945–955the absence of exogenous auxin(0n m NAA),ric1plants produced more lateral roots than did the wild type and ric1 complementation lines(n=28,N=4,P<0.05;Fig.4b). However,in the presence of auxin,lateral root number was less in ric1plants than in the wild type(Fig.4b,P<0.05). Whereas80and100n m NAA increased the number of lateral roots per unit length(cm)of primary root in wild-type seedlings to734and920%of non-treated control values, respectively,the same concentration of NAA increased this number in ric1to466and613%of control values,respec-tively(Fig.4c).This alteration in lateral root formation in ric1 plants was completely reversed by the expression of RIC1 driven by its native promoter(ric1/RIC1p:GFP:RIC1).In two independent ric1/RIC1p:GFP:RIC1lines(C1and C2), lateral root formation was recovered to wild-type levels both in the absence and presence of NAA(Fig.4b,c).The effect of RIC1knockout on primary root elongation was also examined(Fig.4d,e).Seven days after transfer to half-strength MS medium supplemented or not with NAA, the net elongation of primary roots was measured.In medium lacking NAA,ric1mutants had reduced primary root elongation compared with wild-type plants(n=28, N=4,P<0.05);the net primary root elongation of wild-type seedlings was4.9Ϯ0.16cm,while that of ric1seedlings was 4.3Ϯ0.22cm(Fig.4d).NAA(50–100n m)inhibited primary root elongation in both ric1and wild-type seedlings (Fig.4d,e).However,ric1seedlings were less sensitive than the wild type to NAA(n=28,N=4,P<0.1);whereas100n m NAA reduced primary root elongation in wild-type seedlings by34.4%(to3.2Ϯ0.16cm),it inhibited that in ric1seedlings by only19.4%(to3.5Ϯ0.13cm).Primary root elongation in the complementation lines(C1and C2)was similar to that in the wild type,in both the absence and presence of NAA (Fig.4d,e).These results suggest that RIC1participates in auxin-regulated lateral root development and primary root elongation.RIC1knockout enhances the effect of ABA on seed germination,lateral root formation and primary root elongationWe then tested whether RIC1knockout also altered the plant’s response to ABA by comparing seed germination and root development in ric1and wild-type plants treated with ABA(Figs5&6).Seeds were sown on half-strength MS medium after2d of stratiﬁcation.In the presence of0.5m m ABA,seed germination,as gauged by cotyledon greening, was delayed to a greater extent in ric1than in wild-type seeds (Fig.5a,b);whereas only25%of ric1seedlings exhibited cotyledon greening84h after sowing,66%of wild-type seed-lings exhibited greening(Fig.5b).Similar enhanced sensitiv-ity to ABA in inhibition of seed germination was observed when germination rate was analysed based on radicle emer-gence(Supporting Information Fig.S2).Seed germination rates in the ric1/RIC1p:GFP:RIC1lines were restored to wild-type levels in the presence of0.5m m ABA,conﬁrming that loss of RIC1expression was responsible for the enhanced suppression of seed germination by ABA(Fig.5b).The delayed germination of ric1seeds in the presence of ABA was not due to a defect in seed development,because the germination rate of ric1seeds in the absence of ABA was not signiﬁcantly different from that of wild type(Fig.5c and Supporting Information Fig.S2b).ABA was reported to inhibit root elongation and lateral root development(Pilet&Chanson1981).We compared primary root elongation and lateral root number in ric1and wild-type plants in the presence and absence ofexogenous Figure5.Inhibition of seed germination by abscisic acid(ABA) was enhanced in the ric1mutant.(a)Representative photographs showing young seedlings of wild type(WT),ric1,and the tworic1/RIC1p:GFP:RIC1lines(C1and C2)in the presence of0.5m m ABA(taken96h after sowing).(b)Seed germination rate in the presence of0.5m m ABA,measured as a percentage of seedlings with green cotyledons at the indicated time points after sowing on half-strength Murashige and Skoog(MS)medium supplemented with ABA.Data are meansϮSEM of three independent experiments.(c)Seed germination rate in the absence of ABA. There was no signiﬁcant difference between genotypes.RIC1regulates root development951©2012Blackwell Publishing Ltd,Plant,Cell and Environment,36,945–955。

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基于联合三角函数滤波器的水下图像去雾

This article appeared in a journal published by Elsevier.The attached copy is furnished to the author for internal non-commercial research and education use,including for instruction at the authors institutionand sharing with colleagues.Other uses,including reproduction and distribution,or selling or licensing copies,or posting to personal,institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle(e.g.in Word or Tex form)to their personal website orinstitutional repository.Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:/authorsrightsUnderwater image dehazing using joint trilateral ﬁlterqSeiichi Serikawa a ,Huimin Lu a ,b ,⇑aDepartment of Electrical Engineering and Electronics,Kyushu Institute of Technology,Japan b Japan Society for the Promotion of Science,Japana r t i c l e i n f o Article history:Available online 20November 2013a b s t r a c tThis paper describes a novel method to enhance underwater images by image dehazing.Scattering and color change are two major problems of distortion for underwater imaging.Scattering is caused by large suspended particles,such as turbid water which containsabundant particles.Color change or color distortion corresponds to the varying degreesof attenuation encountered by light traveling in the water with different wavelengths,ren-dering ambient underwater environments dominated by a bluish tone.Our key contribu-tions are proposed a new underwater model to compensate the attenuation discrepancyalong the propagation path,and proposed a fast joint trigonometric ﬁltering dehazing algo-rithm.The enhanced images are characterized by reduced noised level,better exposednessof the dark regions,improved global contrast while the ﬁnest details and edges areenhanced signiﬁcantly.In addition,our method is comparable to higher quality than thestate-of-the-art methods by assuming in the latest image evaluation systems.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionUnderwater vision is an important issue in ocean engineering.Different from the natural images,underwater images suf-fer from poor visibility due to the medium scattering and light distortion.First of all,capturing images underwater is difﬁ-cult,mostly due to attenuation caused by light that is reﬂected from a surface and is deﬂected and scattered by particles,and absorption substantially reduces the light energy.The random attenuation of the light is mainly cause of the haze appear-ance while the fraction of the light scattered back from the water along the sight considerable degrades the scene contrast.In particular,the objects at a distance of more than 10meters are almost indistinguishable while the colors are faded due to the characteristic wavelengths are cut according to the water depth [1].There have been many techniques to restore and enhance the underwater images.Schechner and Averbuch [2]exploited the polarization dehazing method to compensate for visibility degradation.They also utilized image fusion method in turbid medium for reconstructing a clear image [3].Hou et al.[4]combined the point spread function (PSF)and modulation trans-fers function to reduce the blurring effect.Although the aforementioned approaches can enhance the image contrast,these methods have demonstrated several drawbacks that reduce their practical applicability.First,the equipment of imaging is difﬁcult in practice (e.g.range-gated laser imaging system).Second,multiple input images are required (e.g.two illumina-tion images [3],white balanced image and color corrected image [1]).In order to solve these above two issues,single image dehazing method is mentioned.Fattal [5]ﬁrstly estimated the scene radiance and derived the transmission image by single image.However,this method cannot well process heavy haze images.He et al.[6]proposed the scene depth information-based dark channel prior dehazing algorithm by using matting Laplacian,which could be computational intensive.To overcome this disadvantage,He et al.also proposed a new guided image ﬁlter [7]0045-7906/$-see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/peleceng.2013.10.016qThis paper is for CAEE special issue SI-40th.Reviews processed and approved for publication by Editor-in-Chief Dr.Manu Malek.⇑Corresponding author at:Department of Electrical Engineering and Electronics,Kyushu Institute of Technology,Japan.Tel.:+818042785999.E-mail addresses:serikawa@elcs.kyutech.ac.jp (S.Serikawa),luhuimin@boss.ecs.kyutech.ac.jp (H.Lu).42S.Serikawa,H.Lu/Computers and Electrical Engineering40(2014)41–50with the foggy image as a reference image,which lead to incomplete haze n et al.[8]proposed a non-local reg-ularized method for haze removal.It fully considered the inﬂuences of sensor noise and multiplicative noise.However,this method is only used for natural images instead of underwater images.In this paper,we introduce a novel approach that is able to enhance underwater images based on single image to over-come the drawbacks of the above methods.We propose a new joint trigonometricﬁlter instead of the matting Laplacian[6] to solve the alpha mattes more efﬁciently.In short summary,our technical contributions are in threefold:ﬁrst,the proposed joint trigonometric guidedﬁlter can perform as an edge-preserving smoothing operator like the popular bilateralﬁlter,but has better behavior near the edges.Second,the novel guidedﬁlter has a fast and non-approximate constant-time algorithm, whose computational complexity is independent of theﬁltering kernel size.Third,the proposed underwater transmission model is suitable in underwater environment.2.Underwater image dehazing2.1.Underwater imaging modelIn the optical model,the acquired image can be modeled as being composed of two components.One is the direct transmission of light from the object,and the other is the transmission due to scattering by the particles of the medium (e.g.airlight).Mathematically,it can be written asIðxÞ¼JðxÞtðxÞþð1ÀtðxÞÞAð1Þwhere I is the achieved image.J is the scene radiance or haze-free image,t is the transmission along the cone of vision,and t(x)=eÀb d(x),b is the attenuation coefﬁcient of the medium,d(x)is the distance between the camera and the object,A is the veiling color constant and x=(x,y)is a pixel.The optical model assumes linear correlation between the reﬂected light and the distance between the object and observer.The light propagation model of Eq.(1)is slightly different from the underwater environment.In the underwater optical imaging model,absorption plays an important role in image degradation.Furthermore,unlike scattering,the absorption coefﬁcient is different for each color channel,being the highest for red and lowest for blue in seawater.These are expressed by the following simpliﬁed hazy image formation model based on Eq.(1):IðxÞ¼JðxÞeÀðb sþb aÞdðxÞþð1ÀeÀb s dðxÞÞA¼JðxÞeÀb s dðxÞÁJðxÞeÀb a dðxÞþð1ÀeÀb s dðxÞÞA;ð2Þ¼KÁJðxÞeÀb s dðxÞþð1ÀeÀb s dðxÞÞA¼KÁJðxÞtðxÞþð1ÀtðxÞÞAwhere b s is the scattering coefﬁcient,b a is the absorption coefﬁcient of light,and K is a constant.In this paper,we simplify the model at a certain water depth,where the transmission t is deﬁned only by the distance between the camera and scene(see Fig.1),which is slightly modiﬁed by Schechner’underwater imaging model[9].In addition,we assume the term for the absorption coefﬁcient is constant.With these simpliﬁcations,Eq.(2)can be easily computed as well as Eq.(1).Fig.1.Underwater optical imaging model(Y.Schechner et al.).imum intensity of the red color channel to compare with the maximum intensity of the green and blue color channels.We deﬁne the dark channel J dark(x)for the underwater image J(x)as,J dark ðxÞ¼minrðminy2XðxÞJcðxÞÞð3Þwhere J c(x)refers to a pixel x in red color channel in the observed image,and X refers to a patch in the image.The dark chan-nel is mainly caused by three factors,shadows,colorful objects or surfaces and dark objects or surfaces.Here,take the min operation in the local patch on the haze imaging function Eq.(1),we assume the transmission as, minðI cðxÞÞ¼~t cðxÞminy2XðxÞðJ cðxÞÞþð1À~tðxÞÞA cð4ÞSince A c is the homogeneous background light and the above equation and the above equation perform one more min oper-ation among all three color channels as follows:minc miny2XðxÞI cðxÞA c¼~t cðxÞmincminy2XðxÞJcðxÞA cþð1À~tðxÞÞð5ÞAs Ref.[11],let us set V(x)=A c(1Àt(x))as the transmission veil,W=min c(I c(x))is the min color components of I(x).We have 05V(x)5W(x).For grayscale image,W=I.Utilize the joint trilateralﬁlter(JTF),we compute the T(x)=median(x)ÀJTF X (|WÀmedian(x)|).And then,we can acquire it by V(x)=max{min[wT(x),W(x)],0},here w is the parameter in(0,1).Finally, the transmission of each patch can be written as,~tðxÞ¼1ÀVðxÞA cð6ÞThe background A c is usually assumed to be the pixel intensity with the highest brightness value in an image.However,in practice,this simple assumption often renders erroneous results due to the presence of self-luminous organisms.Conse-quently,in this paper,we compute the brightest pixel value among all local min corresponds to the background light A c as follows:A c¼maxx2I miny2XðxÞI cðyÞð7Þwhere I c(y)is the local color components of I(x)in each patch.2.3.BilateralﬁlterIn this subsection,we brieﬂy review the bilateralﬁlters.Tomasi and Manduchi[12]proposed the bilateralﬁltering in 1998.The bilateralﬁltering smooths images while preserving edges,by means of a nonlinear combination of nearby image values.This method is non-iterative,local,and simple.The traditional bilateralﬁlter simultaneously weights pixels based on spatial distance from the center pixel as well as distance in tone.The domainﬁlter weights pixels based on their distance from the center,tðxÀyÞ¼12eÀðxÀyÞðxÀyÞ22Dð8Þwhere x and y denote pixel spatial positions.The spatial scale is set by r D,The rangeﬁlter weights pixels based on the pho-tometric difference,wðfðxÞÀfðyÞÞ¼12eÀðfðxÞÀfðyÞÞðfðxÞÀfðyÞÞ2rRð9Þwhere f(Á)is image tonal values.The degree of tonalﬁlter is set by r R.The bilateralﬁlter can be written asR R d fðyÞtðxÀyÞwðfðxÞÀfðyÞÞdyRR dtðxÀyÞwðfðxÞÀfðyÞÞdyð10ÞNote that kernels other than Gaussian kernels are not excluded.2.4.TrilateralﬁlterBilateralﬁltering[12]has been applied in smooth images while preserving the edges.However,to avoid over smoothing structures of sizes comparable to the image resolutions,a narrow spatial window has been used.This leads to the necessity of performing more iterations in theﬁltering process.The standard Gaussian bilateralﬁlter is given~fðxÞ¼1gðxÞZXG rsðyÞG rrðfðxÀyÞÀfðxÞÞfðxÀyÞdyð11ÞS.Serikawa,H.Lu/Computers and Electrical Engineering40(2014)41–5043where gðxÞ¼RXG rsðyÞG rrðfðxÀyÞÀfðxÞÞdy.G rsis the Gaussian spatial kernel and G rris the one-dimensional Gaussian rangekernel.g is the normalization coefﬁcient.Assuming the intensity values f(x)to be restricted to the interval[ÀT,T].G rris approximate by raised cosine kernels.This is motivated by observation that,for alÀT5s5T,lim N!1cosc sﬃﬃﬃﬃNpN¼expÀc2s2 2q2ð12Þwhere c=p/2T and q=c r r are used to control the variance of the target Gaussian on the right,and to ensure that the raised cosines on the left are non-negative and unimodal on[ÀT,T]for every N.The trigonometric function[13]based bilateralﬁlter allows to express the otherwise non-linear transform in Eq.(11)as the superposition of Gaussian convolutions,applied on simple point-wise transforms of the image with a series of spatial ﬁltering,ðF0ÃG rr ÞðxÞ;ðF1ÃG rrÞðxÞ;...;ðF NÃG rrÞðxÞð13Þwhere the image stack F0(x),F1(x),...,F N(x)are obtained from point-wise transform of f(x).Each of these Gaussianﬁltering are computed using O(1)algorithm.And the overall algorithm has O(1)complexity.2.5.Joint trilateralﬁlterIn this subsection,we proposed joint trilateralﬁlter(JTF)to overcome the gradient reversal artifacts occurring.Theﬁlter-ing process of JTF isﬁrstly done under the guidance of the image G which can be another reference image or the input image I itself.Let I p and G p be the intensity value at pixel p of the minimum channel image and guided input image,w k be the kernel window centered at pixel k,to be consistent with bilateralﬁlter.JTF is then formulated byJTFðIÞp ¼1Pq2w kW JTFpqðGÞXq2w kW JTFpqðGÞI qð14Þwhere the kernel weights function W GBTFpqðGÞcan be written byW JTFpq ðGÞ¼1j w j2Xk:ðp;qÞ2w k1þðG pÀl kÞðG qÀl kÞr2kþeð15Þwhere l k and r2k are the mean and variance of guided image G in local window w k,|w|is the number of pixels in this window. When both G p and G q are concurrently on the same side of an edge,the weight assigned to pixel q is large.When G p and G q are on different sides,a small weight will be assigned to pixel q.In this paper,we take a fast bilateralﬁlter to accelerate the computational complex.In the next subsection,we will discuss this algorithm in details.2.6.Recovering the scene radianceWith the transmission depth map,we can recover the scene radiance according to Eq.(1).We restrict the transmission t(x)to a lower bound t0,which means that a small certain amount of haze are preserved in very dense haze regions.Theﬁnal scene radiance J(x)is written as,JðxÞ¼I cðxÞÀA cmaxðtðxÞ;t0ÞþA cð16ÞTypically,we choose t0=0.1.The recovered image may be too dark.So,we take color histogram processing for contrast enhancement.3.Experimental resultsThe performance of the proposed algorithm is evaluated both objectively and subjectively by utilizing ground-truth color patches.We also compare the proposed method with the state-of-the-art methods.Both results demonstrate superior haze removing and color balancing capabilities of the proposed method over the others.In the experiment,we compare our method with Fattal,He and Xiao’s work.Here,we select patch radius r=8, e=0.2Â0.2in Windows XP,Intel Core2(2.0GHz)with1GB RAM in MATLAB7.1.Fig.2shows the results of different meth-ods.The size of the images is345Â292pixels.The drawback of Fattal’s method is elaborated on the Ref.[6].To compare with He’s method,our approach performs better.In He’s approach,because of using soft matting,the visible mosaic artifacts are observed.Some of the regions are too dark(e.g.the right corner of the coral reefs)and hazes are not removed(e.g.the center of the image).There are also some halos around the coral reefs in Xiao’s model[14].Our approach not only works well in haze removal,but also cost little computational complex.We can see the reﬁned transmission depth map to compare these methods clearly in Fig.3.44S.Serikawa,H.Lu/Computers and Electrical Engineering40(2014)41–50The visual assessment demonstrates that our proposed method performs well.In addition to the visual analysis of these ﬁgures,we conducted quantitative analysis,mainly from the perspective of mathematical statistics and the statistical parameters of the images (see Table 1).This analysis includes High-Dynamic Range Visual Difference Predictor2(HDR-VDP2)[15],JPEG-QI [16],and CPU time.In HDR-VDP2,‘‘Ampl.’’means the ampliﬁcation of the image (values are between 0(worst)and 100(best)),and ‘‘Loss’’means the loss of visible contrast (values are between 0(best)and 100(worst)).Sim-ilarly,the Q-MOS value is between 0(worst)and 100(best).Table 1displays the Q-MOS of the pixels that have been ﬁltered by applying HDR-VDP2-IQA.HDR-VDP2-IQA uses the input image as the reference image in these experiments.The results show that the effectiveness of the proposed method is the best.Fig.4presents the probability of detection map by HDR-VDP2-IQA measurement.The metric takes a model of the human visual system being sensitive to three types of structural changes:loss of visible contrast (green)1,ampliﬁcation of invisible contrast (blue),and reversal of visible contras (red).Generally speaking,our approach mainly ampliﬁes the contrast (blue)and a little locations exhibit reverse (red)and only a few losses (green)of contrast.This merit also can be conﬁrmed by experiments in Fig.5.The size of the images is 397Â264pixels.Fig.5shows the depth map of underwater ﬁshes,and then estimated by HDR-VDP2-IQA index.We see that our method only contains the component of amplify of contrast (blue),nearly without the exhibit reverse (red)and losses of contrast (green).underwater image dehazing.(a)Input image.(b)Fattal’s model.(c)He’s model.(d)Xiao’s enhancement.1For interpretation of color in Figs.4and 5,the reader is referred to the web version of this article.and zoom in images.(a)Fattal’s model.(b)He’s model.(c)Xiao’s model.Table1Quantitative analysis of dehazed images.Methods CPU time(s)JPEG-QI HDR-VDP2-IQA(%)Fattal(2008)20.058.79 1.8(Ampl.),16.7(Loss) He et al.(2010)30.858.82 5.9(Ampl.),24.3(Loss) Xiao et al.(2012)14.647.2310.6(Ampl.),30.8(Loss)Our proposed 4.429.3135.4(Ampl.),1.8(Loss)detection map of coral reefs.(a)Fattal’s model.(b)He’s model.(c)Xiao’s model.(d)detection map of underwaterﬁsh.(a)Fattal’s model.(b)He’s model.(c)Xiao’s model.Fig.6shows the underwater tank image with the size of380Â287pixels.We can see that,the Fattal’s model cannot achieve well in underwater image processing.The image is almost blurring.He’s model performs well,but the iron chain on the lower right corner of the image is hardly to see.To Xiao’s model,we canﬁnd that the image also contains a lot of suspended solids,the quality of image is bad.In our processed image,we can clearly watch the cable,iron chain et al.,small objects very well.The processing speed of our proposed method is the fastest in the Table2.That is because the proposed joint trilateralﬁlter can be efﬁcient computed with O(N).These conclude that our model for underwater image or video pro-cessing better than the-state-of-the-art models.In the fourth experiment,we simulate the underwater objects recognition by a tank in darkroom.We take OLYMPUS STYLUS TG-215m/50ft underwater camera for capture images.The distance between light-camera and the objects is 3m.The size of the images is640Â480pixels.The captured image contains a lot of suspended solids caused by plankton,underwater tank images.(a)Fattal’s model.(b)He’s model.(c)Xiao’s model.(d)OurTable2CPU time of dehazed images in processing Figs.5and6.CPU time(s)Fish TankFattal(2008)17.1414.40He et al.(2010)32.2034.54Xiao et al.(2012)12.5910.93Our proposed 6.42 6.75and electrical noise caused by optoelectronic imaging device.The artiﬁcial light also causes some haze in the right corner of the image.The processed result is presented in Fig.7.Before processing,the captured image is distorted seriously.After the proposed dehazing method,the image is much clear than the other methods.The process of our algorithm is fast,nearly cost about30ms by OpenCV2.4and Intel TBB parallel algorithms.The CPU running time,PSNR,SSIM[17]and Q-MOS is the high-est among all of the other methods in Table3.The results indicate that our approach not only works well for haze removal, but also results in lower computation time.4.ConclusionsIn this paper we explored and successfully implemented a simple and effectiveness image dehazing techniques for under-water imaging system.We proposed a simple prior based on the difference in attenuation among the different color chan-nels,which inspire us to estimate the transmission depth map through red color channel in underwater images.Another contribution is to compensate the attenuation discrepancy along the propagation path,and to take the joint trilateralﬁlter forﬁltering the transmission depth map.The trilateralﬁlter utilizes the reﬂectivity in addition to the spatial and intensity information so that geometrical features such as jump and edges are preserved while smoothing.Beside of this,the trilateral ﬁlter can achieve edge-preserving smoothing with a narrow spatial window in only a few iterations.Moreover,the proposed joint trilateralﬁlter can remove the overly darkﬁelds of the underwater images by reﬁning the transmission depth map through trilateralﬁltered source image and estimated transmission depth map.The experiments also demonstrate that our algorithm is faster than the-state-of-the-art algorithms.That is suitable for real-time underwater imaging system in practice from the image processing effects and computation complex.The proposed algorithm also contains some problems,such as the inﬂuence of the possible presence of an artiﬁcial light-ing source is not considering,and the quality assessment may be unsuitable for underwater images.In future,we attempt to solve the artiﬁcial lighting by utilizing the data fusion[18]based method(e.g.Ancuti et al.[19]by using two input images, color correction method[20],and Treibitz and Schechner[3]to fuse different illumination images)and to design a new index for underwater image quality assessment.dehazing.(a)Captured image in clean water.(b)Captured image in turbid water.(c)model.Table3Quantitative analysis of simulation images.Methods CPU time(s)PSNR SSIM Q-MOSFattal(2008)20.6111.2070.422546.1112He et al.(2010)10.6616.9350.699288.4452Xiao et al.(2012) 5.2124.6260.803092.3767Our proposed 5.4628.3370.885993.8854AcknowledgmentsThis work was partially supported by Grants-in-Aid for Scientiﬁc Research of Japan(No.(B)19500478)and the support of Research Fellowship for Young Scientists by Japan Society for the Promotion of Science(No.25-10713).References[1]Ancuti C,Ancuti CO,Haber T,Bekaert P.Enhancing underwater images and videos by fusion.In:Proceeding of IEEE conference on computer vision andpattern recognition;2012.p.81–8.[2]Schechner YY,Averbuch Y.Regularized image recovery in scattering media.IEEE Trans Pattern Anal Mach Intell2007;29(9):1655–60.[3]Treibitz T,Schechner YY.Turbid scene enhancement using multi-directional illumination fusion.IEEE Trans Image Process2012;21(11):4662–7.[4]Hou W,Gray DJ,Weidemann AD,Fournier GR,Forand JL.Automated underwater image restoration and retrieval of related optical properties.In:Proceeding of IEEE international symposium of geoscience and remote sensing;2007.p.1889–92.[5]Fattal R.Signal image dehazing.In:SIGGRAPH;2008.p.1–9.[6]He K,Sun J,Tang X.Single image haze removal using dark channel prior.IEEE Trans Pattern Anal Mac Intell2011;33(12):2341–53.[7]He K,Sun J,Tang X.Guided imageﬁltering.In:Proceeding of the11th European conference on computer vision,vol.1;2010.p.1–14.[8]Lan X,Zhang L,Shen H,Yuan Q,Li H.Single image haze removal considering sensor blur and noise.EURASIP J Adv Signal Process2013;2013:86.Author's personal copy50S.Serikawa,H.Lu/Computers and Electrical Engineering40(2014)41–50[9]Schechner YY,Karpel N.Clear underwater vision.In:Proc of IEEE computer society conference on computer vision and,pattern recognition,vol.1;2004.p.536–43.[10]Chambah M,Semani D,Renouf A,Courtellemont P,Rizzi A.Underwater color constancy:enhancement of automatic liveﬁsh recognition.In:Proc ofSPIE,vol.5293;2004.p.157–68.[11]Tarel J,Hautiere N.Fast visibility restoration from a single color or gray level image.In:Proceeding of IEEE conference of computer vision and patternrecognition;2008.p.2201–08.[12]Tomasi C,Manduchi R.Bilateralﬁltering for gray and color images.In:Proc of IEEE international conference on computer vision,vol.839–846;1998.[13]Chaudhury KN,Sage D,Unser M.Fast O(1)bilateralﬁltering using trigonometric range kernels.IEEE Trans Image Process2011;20(12):3376–82.[14]Xiao C,Gan J.Fast image dehazing using guided joint bilateralﬁlter.Visual Comput2012;28(6–8):713–21.[15]Mantiuk R,Kim KJ,Rempel AG,Heidrich W.HDR-VDP2:a calibrated visual metric for visibility and quality predictions in all luminance conditions.ACM Trans Graph2011;30(4):40–52.[16]Wang Z,Sheikh HR,Bovik AC.No-reference perceptual quality assessment of JPEG compressed images.In:Proc of international conference on imageprocessing,vol.1;2002.p.477–80.[17]Wang Z,Bovik AC,Sheikh HR,Simoncelli EP.Image quality assessment:From error visibility to structural similarity.IEEE Trans Image Process2004;13(4):600–12.[18]Lu H,Zhang L,Serikawa S.Maximum local energy:an effective approach for image fusion in beyond wavelet transform put Math Appl2012;64(5):996–1003.[19]Ancuti CO,Ancuti C,Bekaert P.Effective single image dehazing by fusion.In:Proc of17th IEEE international conference on image processing;2010.p.3541–5.[20]Lu H,Li Y,Serikawa S.Underwater image enhancement using guided trigonometric bilateralﬁlter and fast automatic color correction.In:Proc of20thIEEE international conference on image processing;2013.p.3412–6.Seiichi Serikawa,received the B.S.and M.S.degrees in Electronic Engineering from Kumamoto University in1984and1986.He received the Ph.D.degree in Electronic Engineering from Kyushu Institute of Technology,in1994.Recently,he is the Dean of Department of Electrical Engineering and Electronics in Kyushu Institute of Technology.His current research interests include computer vision,sensors,and robotics.Huimin Lu,received the B.S.degree in Electronics Information Science and Technology from Yangzhou University in2008.He received M.S.degrees in Electrical Engineering from Kyushu Institute of Technology and Yangzhou University in2011,respectively.Recently,he is a Ph.D.candidate in Kyushu Institute of Technology.His current research interests include computer vision,communication and deep-sea information processing.。

《2024年高通量测序分析角膜移植排斥反应中CD4~+T细胞受体CDR3免疫组库的研究》范文

《高通量测序分析角膜移植排斥反应中CD4~+T细胞受体CDR3免疫组库的研究》篇一高通量测序分析角膜移植排斥反应中CD4+T细胞受体CDR3免疫组库的研究摘要：本文通过高通量测序技术，对角膜移植排斥反应中CD4+T细胞受体CDR3免疫组库进行了深入研究。

通过对CD4+T细胞受体CDR3的序列分析，揭示了排斥反应的免疫应答机制，为临床诊断和治疗角膜移植排斥反应提供了新的思路和方法。

一、引言角膜移植是治疗角膜疾病的有效手段之一，但移植排斥反应是常见的并发症之一。

CD4+T细胞在角膜移植排斥反应中发挥着重要作用。

近年来，高通量测序技术的发展为研究CD4+T细胞受体CDR3免疫组库提供了新的途径。

本文旨在通过高通量测序技术，分析角膜移植排斥反应中CD4+T细胞受体CDR3的序列特征，以揭示其免疫应答机制。

二、材料与方法1. 实验材料实验所用样本为角膜移植患者的临床样本，包括正常角膜、移植排斥反应及非排斥反应的角膜样本。

2. 方法（1）样本处理与DNA提取：采用适当的处理方法提取样本中的DNA。

（2）高通量测序：对提取的DNA进行高通量测序，获取CD4+T细胞受体CDR3的序列信息。

（3）数据分析：利用生物信息学方法对高通量测序数据进行处理和分析，包括序列比对、注释、多样性分析和差异表达分析等。

三、实验结果1. CD4+T细胞受体CDR3的序列特征通过高通量测序，我们获得了大量的CD4+T细胞受体CDR3序列信息。

与正常角膜相比，在移植排斥反应的角膜样本中，CD4+T细胞受体CDR3的序列多样性明显增加，且出现了一系列新的序列类型。

2. 免疫应答机制分析通过差异表达分析，我们发现了一些在移植排斥反应中特异性表达的CD4+T细胞受体CDR3序列。

这些序列可能与排斥反应的发病机制有关。

进一步的功能注释和生物信息学分析表明，这些序列可能与免疫应答、细胞激活和信号传导等过程相关。

四、讨论本研究通过高通量测序技术，揭示了角膜移植排斥反应中CD4+T细胞受体CDR3的序列特征和免疫应答机制。

Adaptive Soft Frequency Reuse for LTE

This work may not be copied or reproduced in whole or in part for any commercial purpose. Permission to copy in whole or in part without payment of fee is granted for nonproﬁt educational and research purposes provided that all such whole or partial copies include the following: a notice that such copying is by permission of Mitsubishi Electric Research Laboratories, Inc.; an acknowledgment of the authors and individual contributions to the work; and all applicable portions of the copyright notice. Copying, reproduction, or republishing for any other purpose shall require a license with payment of fee to Mitsubishi Electric Research Laboratories, Inc. All rights reserved. Copyright c Mitsubishi Electric Research Laboratories, Inc., 2008 201 Broadway, Cambridge, Massachusetts 02139

换热器设计

Abstract—This paper is intended to assist anyone with some general technical experience, but perhaps limited specific knowledge of heat transfer equipment. A characteristic of heat exchanger design is the procedure of specifying a design, heat transfer area and pressure drops and checking whether the assumed design satisfies all requirements or not. The purpose of this paper is how to design the oil cooler (heat exchanger) especially for shell-and-tube heat exchanger which is the majority type of liquid-to-liquid heat exchanger. General design considerations and design procedure are also illustrated in this paper and a flow diagram is provided as an aid of design procedure. In design calculation, the MatLAB and AutoCAD software are used. Fundamental heat transfer concepts and complex relationships involved in such exchanger are also presented in this paper. The primary aim of this design is to obtain a high heat transfer rate without exceeding the allowable pressure drop. This computer program is highly useful to design the shell-and-tube type heat exchanger and to modify existing deign. Keywords—Shell-and-Tube Heat Exchanger, MatLAB and AutoCAD I. INTRODUCTION HE process of heat exchange between two fluids that are at different temperatures and separated by a solid wall occurs in many engineering applications. The device used to implement this exchange is termed a heat exchanger, and specific applications may be found in space heating and airconditioning, power production, waste heat recovery, and chemical processing. The problem of heat transmission is encountered in many industries and because of the diversity in the fields of application there exist countless difference in detail. However, the calculation principle underlying the problem to design a heat exchanger are everywhere the same, and it is the purpose of this paper to present design calculations rather than to deal with the details of individual problems and special cases [1]. A computer program in MatLAB has been written for shell and tube heat exchanger design. Normally, the heat exchanger design calculations are seems to be complex and repeated calculations are required with assumed and/or fixed data. So, many thermodynamic and fluid dynamic parameters are encountered into designs procedure and therefore this condition will satisfy and save the time by using a computer.

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∗ Corresponding author. Tel.: +86 10 62772907; fax: +86 10 62771160. E-mail address: panf@ (F. Pan). 0043-1648/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.wear.2008.04.025
Wear 265 (2008) 1808–1813
Contents lists available at ScienceDirect
Wear
journal homepage: /locate/wear
Investigation of the wear behaviors of Ag/Cu multilayers by nanoscratch
Article history: Received 23 April 2007 Accepted 30 April 2008 Available on0.Qp 62.25.+g Keywords: Multilayer Nanoscratch Friction coefﬁcient Wear
3.2. Coefﬁcient of friction Fig. 2 shows the proﬁles of the coefﬁcient of friction of two typical samples as a function of the normal ramping load and scratch length. The coefﬁcient of friction was calculated by taking the ratio of the lateral force and normal load on the indenter. The coefﬁcient of friction increases with the increase of the scratch length as shown in Fig. 2. At the initial stage the coefﬁcient is not stable, which may be ascribed to the multilayer structure consisting of two materials with different friction coefﬁcients. At a very shallow scratch depth, every individual layers will inﬂuence the apparent friction coefﬁcient with the penetration of the indenter, but at a relative deep scratch depth the apparent friction coefﬁcient will be determined by many layers and an individual layers will have negligible effect on it. From Fig. 2, one also can ﬁnd that the coefﬁcient of multilayer with = 100 nm is larger than that of multilayer with = 20 nm. To investigate the periodicity dependence of the friction coefﬁcient, Fig. 3 presents the coefﬁcient at 250 ␮m scratch length as a function of the periodicity. It can be found that friction coefﬁcient of multilayers with = 4 nm, 10 nm, 20 nm has approx-

Real-time imaging of the intracellular glutathione redox potential

Real-time imaging of the intracellular glutathione redox potentialMarcus Gutscher 1,Anne-Laure Pauleau 1,Laurent Marty 2,Thorsten Brach 2,Guido H Wabnitz 3,Yvonne Samstag 3,Andreas J Meyer 2&Tobias P Dick 1Dynamic analysis of redox-based processes in living cells is now restricted by the lack of appropriate redox biosensors.Conventional redox-sensitive GFPs (roGFPs)are limited by undeﬁned speciﬁcity and slow response to changes in redox potential.In this study we demonstrate that the fusion of human glutaredoxin-1(Grx1)to roGFP2facilitates speciﬁc real-time equilibration between the sensor protein and the glutathione redox couple.The Grx1-roGFP2fusion proteinallowed dynamic live imaging of the glutathione redox potential (E GSH )in different cellular compartments with high sensitivity and temporal resolution.The biosensor detected nanomolar changes in oxidized glutathione (GSSG)against a backdrop of millimolar reduced glutathione (GSH)on a scale of seconds to minutes.It facilitated the observation of redox changes associated with growth factor availability,cell density,mitochondrial depolarization,respiratory burst activity and immune receptor stimulation.Intracellular redox changes accompany major transitions in the life cycle of metazoan cells,such as proliferation,differentiation,senescence and death 1–4.Redox changes can be either short-lived,as exempliﬁed by transient and localized production of reactive oxygen species (ROS)in the course of normal growth factor signaling 5,or long-lived,as observed in the case of cellular senes-cence 6,7.Pro-oxidative redox states can also be inﬂicted by the activation of host defense,notably inﬂammation 8,as well as abiotic factors.Elevated and prolonged oxidative stress has been associated with various age-related pathophysiologies,including neurodegen-erative,malignant and cardiovascular disorders.Mounting evi-dence suggests that oxidative processes can have a causal role in disease initiation or progression 9.Although there appears to exist a broad consensus that pro-oxidative redox changes are important in health and disease,their labile and context-dependent nature has made their undisturbed observation notoriously difﬁcult.Recent evidence suggests that informative measurements of cellular redox states might be more challenging than previously realized.Cells harbor several redox systems,which appear to be kinetically controlled and not in equilibrium with each other 10.They have distinct regulatory functions and are themselves subject to independent regulation.Measurement of one redox pair does not necessarily inform about the state of other redox couples 11.Moreover,the spatiotemporal pattern of intracellular redox changes may be highly nonuniform between and within cellular compart-ments 12,13.Understanding the relevance and speciﬁcity of redox changes will require nondisruptive measurements with speciﬁcity for deﬁned redox pairs and considerable spatiotemporal resolution.The lack of tools for measuring the status of deﬁned intracellular redox couples has been a profound limitation for basic research.Conventional approaches either lack well-deﬁned speciﬁcity or disrupt cellular integrity.For example,redox-sensitive ﬂuorescent dyes,frequently used to detect oxidant generation within cells,interact with multiple oxidants and may even promote artiﬁcial ROS formation 14.Customary redox measurements of glutathione and thioredoxin,though speciﬁc for the respective redox couple,require cell lysis and analytical separation procedures,making measurements prone to artifacts.Genetically encoded biosensors promise to overcome the limita-tions of conventional redox measurements.Most recently,the H 2O 2-sensing properties of the bacterial protein OxyR had been exploited to develop HyPer,a yellow ﬂuorescent protein (YFP)-based biosensor for intracellular hydrogen peroxide 15.Before it,others 16,17developed redox-sensitive ﬂuorescent proteins by insert-ing an artiﬁcial dithiol-disulﬁde pair into the structures of YFP and GFP ,respectively.A major advantage of the latter (called redox-sensitive GFPs or roGFPs)is that they are ratiometric by excitation,thus minimizing measurement errors resulting from variable con-centration or photobleaching.However,endogenously expressed roGFPs do not appear to respond to physiologically relevant redox signals in animal cells 12.It has been concluded that the usefulness of existing roGFPs is limited by their slow response to changes in redox potential.Moreover,it has remained unclear which endo-genous cellular redox systems actually interact with roGFPs.We reasoned that conventional roGFPs might be oblivious to most endogenous redox changes because their equilibration with cellular redox systems is too slow and unselective.Following this consideration,we explored the concept of equipping the biosensorRECEIVED 15JANUARY;ACCEPTED 7APRIL;PUBLISHED ONLINE 11MAY 2008;DOI:10.1038/NMETH.12121RedoxRegulation Research Group,German Cancer Research Center (DKFZ/A160),Im Neuenheimer Feld 280,D-69120Heidelberg,Germany.2Heidelberg Institute ofPlant Sciences,University of Heidelberg,Im Neuenheimer Feld 360,D-69120Heidelberg,Germany.3Institute of Immunology,University of Heidelberg,Im Neuenheimer Feld 305,D-69120Heidelberg,Germany.Correspondence should be addressed to T.P .D.(t.dick@dkfz.de).NATURE METHODS |VOL.5NO.6|JUNE 2008|553ARTICLES©2008 N a t u r e P u b l i s h i n g G r o u p h t t p ://w w w .n a t u r e .c o m /n a t u r e m e t h o d swith an enzyme to speciﬁcally catalyze the equilibration between the redox pair of interest,reduced glutathione (GSH)andoxidized glutathione (GSSG),and the reporting redox couple (roGFP2red and roGFP2ox ).Here we demonstrate that the fusion of roGFP2and human glutaredoxin-1(Grx1)allows dynamic live imaging of intracellular glutathione redox potential,E GSH ,with unprecedented sensitivity and temporal resolution.The sensor detected both short-and long-lived deﬂections of endogenous E GSH and facilitated the observation of physiologically relevant redox-based signals.RESULTSGrx1catalyzes equilibration between glutathione and roGFP2T o enforce rapid and speciﬁc equilibration,we fused either human Grx1or thioredoxin-1(Trx1)to the N terminus of roGFP2,linked by a 30-amino-acid spacer,(Gly-Gly-Ser-Gly-Gly)6,to allow for ﬂexible interactions between the linked entities.For control experi-ments,we generated a catalytically inactive Grx1fusion protein by replacing active site cysteines with serines (Grx1(ss)-roGFP2).We ﬁrst conﬁrmed that the fusion of either Grx1or Trx1to roGFP2did not interfere with its intrinsic redox-sensing and ratiometric properties (Supplementary Fig.1online).We also veriﬁed that the ﬂuorescence excitation spectrum was not inﬂuenced by the fusion (data not shown)and that the emission ratio of the sensor constructs was insensitive to pH changes between 5.5and 8.5(Supplementary Fig.2online).Next we examined the response of unfused and fused roGFP2to consecutive exposure to reduced and oxidized glutathione.Only the catalytically active Grx1-roGFP2fusion protein responded to glutathione injections,thus demonstrating that Grx1,but not Trx1,confers dynamic responsiveness to the GSH/GSSG redox state (Fig.1a ).T o estimate the kinetic advantage mediated by fused Grx1,we measured the rate of GSSG-mediated oxidation of Grx1-coupled and uncoupled roGFP2at various GSSG concentrations,as exempliﬁed by the response to 10m M GSSG (Fig.1b and see Supplementary Fig.3online for corresponding emission intensity measurements).Kinetic analysis revealed that the oxidation of Grx1-roGFP2by GSSG is at least 100,000times faster as compared to uncoupled roGFP2.As the midpoint redox potential of roGFP2(–280mV 18)is more reducing compared to that of glutathione at physiological concen-trations (ranging from –151mV at 1mM to –181mV at 10mM),we anticipated that even a small increase in the amount of glutathione oxidation (OxD GSH ,for a mathematical deﬁnition see Supplementary Methods online)within a highly reduced glutathione pool should give rise to a large and disproportionate degree of sensor oxidation (OxD roGFP2).Calculation of corre-sponding Nernst potentials (see Supplementary Methods )con-ﬁrmed that the redox sensor is expected to be highly sensitive to glutathione oxidation because the physiologically relevant potential range between –320mV and –240mV is covered by only minor changes in OxD GSH (Fig.1c ).T o verify the predicted sensitivity ofR a t i o 390/480 n mGSHGSSGGSSGTime (s)Time (s)Potential (mV)aR a t i o 390/480 n m0.0250.0500.075OxD GSH0.1000.1250.1500.1750.200060120180240300360Time (s)420480540600660720780840900No GSH H 2O2dbcFigure 1|Grx1dynamically catalyzes rapid redox equilibration between glutathione and roGFP2.(a )Oxidized Grx1-roGFP2,Grx1(SS)-roGFP2,Trx1-roGFP2and roGFP2were reduced by injection of 15mM GSH after 45s and oxidized with 100m M GSSG after 7min.Incomplete reduction of Grx1-roGFP2by GSH is due to the presence of trace amounts of GSSG.(b )Oxidation of reduced Grx1-roGFP2and roGFP2with 10m M GSSG,injected after 110s.(c )Nernst relationship between redox potential,OxD roGFP2and OxD GSH (at 10mM total glutathione).The vertical lines are visual guides to indicate that within the physiological range small changes in OxD GSH lead to large changes in OxD roGFP2.(d )Grx1-roGFP2was incubated with glutathione solutions (20mM total)containing increasing fractions of GSSG.The indicated OxD GSH is calculated from the amount of GSSG supplied to the redox buffer and does not take into account traces of GSSG already present in the GSH preparation.(e )After 75s,100m M H 2O 2was injected into a solution of reduced Grx1-roGFP2,in buffer alone,with 5mM fully reduced GSH with or without 0.1units of glutathione peroxidase (GPX).To remove residual GSSG from the GSH solution before use,the least amount of NADPH (30m M)required to achieve near-complete reduction by 0.1m M glutathione reductase was added.Data are representative of two (a ,d ,e )or three (b )experiments.554|VOL.5NO.6|JUNE 2008|NATURE METHODSARTICLES©2008 N a t u r e P u b l i s h i n g G r o u p h t t p ://w w w .n a t u r e .c o m /n a t u r e m e t h o d sthe sensor,we measured the response of the Grx1-roGFP2sensor at various OxD GSH and obtained a curve consistent with theoreticalexpectations (Fig.1d ).Of note,commercially available GSH contained traces of GSSG (typically corresponding to an OxD GSH between 0.0015and 0.002),which led to substantial sensor oxida-tion,for example,50%in a 20mM GSH solution (Fig.1d )or 96%in a 1mM GSH solution (data not shown).T o verify that sensor oxidation in freshly prepared GSH solutions is due to trace amounts of GSSG,we treated GSH solutions with glutathione reductase and NADPH,thus clamping Grx1-roGFP2in the fully reduced state,corresponding to a E GSH below –320mV (Supple-mentary Fig.4online).Coupled Grx1mediates a glutathione-speciﬁc sensor response Previous studies have shown that glutaredoxins selectively interact with glutathione and discriminate against other redox-active com-pounds 19.T o conﬁrm that Grx1confers glutathione speciﬁcity to the biosensor,we evaluated its in vitro response to reduction by ascorbate or cysteine and to oxidation by cystine or hydroxyethyl disulﬁde.As expected,only incubation with glutathione led to a substantial sensor response (Supplementary Fig.5online).Thus,the previously described speciﬁcity of Grx1for glutathione is also given in the context of the Grx1-roGFP2fusion protein.Notably,the presence of micromolar H 2O 2by itself did not lead to substantial sensor oxidation within the measurement period.Instead,H 2O 2-mediated sensor oxidation depended on theconcomitant presence of glutathione (Fig.1e ).Catalytic amounts of glutathione peroxidase further enhanced the sensor response to H 2O 2(Fig.1e ),thus conﬁrming that glutathione oxidation pre-cedes and facilitates sensor oxidation.Grx1-roGFP2does not engage in disulﬁde exchange with Trx1Although fusion of Trx1did not inﬂuence the response of roGFP2to glutathione (Fig.1a ),we considered the possibility that Trx1might be able to directly reduce oxidized roGFP2or Grx1-roGFP2.When we exposed fully oxidized Grx1-roGFP2to the complete thioredoxin system,we observed only slight sensor responses (Supplementary Fig.6online).This ﬁnding demonstrated that the engineered disulﬁde bond of roGFP2is not efﬁciently targeted by Trx1and suggested that the Trx system is unlikely to interfere with Grx1-roGFP2redox behavior.Grx1-roGFP2facilitates imaging of intracellular redox changes T o investigate the redox-sensing properties of Grx1-roGFP2in living cells,we stably expressed Grx1-roGFP2and roGFP2in HeLa cells.We treated cells with a single bolus of 50m M H 2O 2,followed 2min later by exposure to 0.5mM dithiothreitol (DTT).Addition of H 2O 2to the growth medium led to an immediate and strong oxidative response in Grx1-roGFP2-positive cells (Fig.2a ).The sensor remained in the oxidized state until injection of DTT induced its reduction.In contrast,uncoupled roGFP2respondedR a t i o 408/488 n m0.10.20.30.40.50.60.70.80.91R a t i o 408/488 n m 0306090Time (s)120150180270210240Grx1-roGFP2 (cell 2)roGFP2 (cell 1)roGFP2 (cell 2)0.5 mM DTT50 µM H 2O 2a1.01.52.02.53.0M a x i m u m r a t i o c h a n g e3.54.04.55.0151020H 2O 2 (µM)501005001,000cR a t i o 408/488 n m0.20.30.40.50.60.70.80.90306090Time (s)12015018021025 µM H 2O 2100 µM H 2O 2H 2O 2bFigure 2|Grx1-roGFP2fusion protein allows live imaging of rapid intracellular redox changes.(a )HeLa cells expressing Grx1-roGFP2or roGFP2were excited with 408and 488nm lasers,and the ratio of emissions in the green channel (500–530nm)was calculated.After 50s,cells were treated with 50m M H 2O 2followed by addition of 500m M DTT 2min later.False-color ratio pictures of the cells at indicated time points exemplify the kinetic difference between Grx1-roGFP2and roGFP2(left).Scale bar,10m m.In each experiment,the ratio was quantiﬁed for two individual cells (arrowheads)and plotted against time (right).(b )Grx1-roGFP2–expressing HeLa cells were treated with the indicated amounts of H 2O 230s after starting the measurement and the ratiometric sensor response was measured.(c )Based on experiments similar to those in b ,the maximal ratio change of cells expressing Grx1-roGFP2was plotted against H 2O 2concentration.For each point in the graph at least 6cells from 3different experiments were analyzed.A ratio value of 1.0represents the fully reduced (DTT-treated)state.Error bars represent s.d.from the mean.Data are representative of three experiments.NATURE METHODS |VOL.5NO.6|JUNE 2008|555ARTICLES©2008 N a t u r e P u b l i s h i n g G r o u p h t t p ://w w w .n a t u r e .c o m /n a t u r e m e t h o d sslowly and did not reach the same oxidation level within the given timeframe.We obtained similar results with various transiently transfected cell lines (data not shown).T o estimate the sensitivity of the Grx1-roGFP2sensor protein,we tested its response to exogenously added H 2O 2.Addition of H 2O 2in the low-micromolar range led to short-lived deﬂections of E GSH lasting 90–120s,suggestive of an efﬁcient anti-oxidative response (Fig.2b ).Accordingly,addition of larger amounts of H 2O 2led to increased and prolonged shifts of E GSH (Fig.2b ).Addition of extracellular H 2O 2in the low micromolar range,likely leading to nanomolar concentrations of intracellular GSSG 20,21,was sufﬁcient to elicit a sensor response (Fig.2c ).A response to limiting amounts of H 2O 2was barely detectable in cells with unfused roGFP2(Supplementary Fig.7online),thus conﬁrming that slow equilibration impedes the detection of short-lived oxidative events by roGFP2.Redox changes caused by growth conditionsWe then asked whether the fusion protein would allow detection of endogenous redox changes.Previously,it has been shown that growth factor deprivation causes an increase in intracellular ROS 22.We cultured Grx1-roGFP2–expressing HeLa cells in thepresence0.20.4Ratio 407/488 nm0.60.81aP e r c e n t a g e o f m a x i m u m0.20.4Ratio 407/488 nm0.60.81Medium LowL o wMe di u m Cell densityH i g hbFigure 3|Redox response of HeLa cells to growth-factor starvation and changes in cell density.(a )HeLa cells expressing Grx1-roGFP2were incubated in medium containing 0or 10%FCS for 20h and analyzed by microscopy (left;scale bar,10m m)and ﬂow cytometry (middle).For quantiﬁcation of microscopy data,emission ratios from 12cells in 3different experiments were averaged.Both techniques revealed similar results (right).(b )Grx1-roGFP2-expressing HeLa cells seeded at different densities (average cell density:low,1.6Â104;medium,4Â104;and high,8Â104cells/cm 2)were analyzed by ﬂow cytometry after 24h (left).We analyzed 30,000cells from three different experiments,and the average of the mean ratio was calculated (right).Error bars represent s.d.from the mean.Data are representative of three experiments.0.10.20.30.40.50.60.70.80.90.20.30.40.50.60.70.80.91515516165171751818519195200TMRM intensity TMRM intensityR a t i o 405/488 n m5018212427303336394245485154570Time (min)Time (min)Ratio (cell 1)Ratio (cell 2)Ratio (cell 3)TMRM (cell 1)TMRM (cell 2)TMRM (cell 3)R a t i oT M R MR a t i oT M R MabTMRM (cell 1)TMRM (cell 2)Ratio (cell 1)Ratio (cell 2)Figure 4|Grx1-roGFP2visualizes redox changes associated with TRAIL-induced apoptosis.(a )Apoptosis was induced in Grx1-roGFP2–expressing HeLa cells by the addition of 400ng/ml TRAIL.D C was visualized with TMRM.False-color ratio and TMRM intensity images at different time points are depicted (left).Cell 1maintained D C during the measurement period whereas cells 2and 3responded to the TRAIL treatment (arrowheads).The emission ratio of Grx1-roGFP2(excitation lines 405and 488nm)and the TMRM intensity for the selected cells were plotted against time (right).(b )A mitochondrial-targeted Grx1-roGFP2was used in experiments similar to a .False-color ratio and TMRM intensity pictures (left)as well as quantiﬁcation diagrams (right)are shown for a responding (arrowhead 1)and a non-responding cell (arrowhead 2).Scale bars,20m m.Data are representative of ﬁve (a )or three (b )experiments.556|VOL.5NO.6|JUNE 2008|NATURE METHODSARTICLES©2008 N a t u r e P u b l i s h i n g G r o u p h t t p ://w w w .n a t u r e .c o m /n a t u r e m e t h o d sor absence of serum and determined E GSH by microscopy and ﬂow cytometry (Fig.3a ).T o analyze the distribution of redox potential in cell populations by ﬂow cytometry,we made use of the fact that the thiol-disulﬁde status of the biosensor canbe ‘clamped’by a brief treatment of intact cells with the mem-brane-permeable alkylating agent N -ethyl maleimide (NEM),thus allowing for ﬁxation and additional handling without impairing sensor redox status (Supplementary Fig.8online).Both techniques revealed very similar ratiometric changes (Fig.3a ),corresponding to an oxidative shift in E GSH approximately from –300to –280mV .We independently conﬁrmed an increase in endogenous ROS in the same cells using the redox-sensitive dye 2¢,7¢-dichlorodihydroﬂuor-escein diacetate (H 2DCFDA;Supplementary Fig.9online).It has also been suggested that endogenous oxidant levels depend on cell density 23.T o determine whether intracellular E GSH is inﬂuenced by cell density,we seeded Grx1-roGFP2–expressing HeLa cells at different densities,allowed them to adhere in fresh medium and analyzed them by ﬂow cytometry.We observed a reproducible correlation between redox state and cell density;greater cell density led to more reduced E GSH (Fig.3b ).A ﬁvefold increase in cell density decreased E GSH by approximately 40mV ,from –280to –320mV .The change in E GSH correlated with corresponding changes in the amount of endogenous ROS,as conﬁrmed by H 2DCFDA staining (Supplementary Fig.9).Endogenous redox changes associated with apoptosisApoptosis is known to be associated with oxidative redox changes,caused by mitochondrial ROS production and,possibly,activeextrusion of reduced glutathione 4,24,25.Redox changes inside mitochondria are con-nected to the permeability transition,a cri-tical control point in apoptosis 26.We followed E GSH during apoptosis in both the cytosolic and mitochondrial compart-ments.T o this end,we targeted the Grx1-roGFP2fusion protein to the mitochon-drial matrix using a signal sequence fromNeurospora crassa ATP synthase protein 9(‘mito-Grx1-roGFP2’).We treated sensor-expressing cells with TNF-related apoptosis-inducing ligand (TRAIL)to induce apoptosis,using a concentration that led to 60%cell death within 16h.Additionally,we loaded the cells with tetramethylrhodamine methyl ester (TMRM)to monitor the mitochondrial membrane potential (D C ).As this dye may produce ROS upon strong illumination 27,we chose very mild exposition conditions to minimize this effect.Between 2and 5h after addition of TRAIL,individual cells lost D C .Mitochondrial depolarization typically occurred over 10–15min.The cytosolic E GSH remained stable before the loss of D C .Following loss of D C and an additional delay of 15–20min,cytosolic E GSH increased continuously over the next 3–4h,from a ground state potential of approximately –290mV to 4–240mV ,thus reaching sensor satu-ration (Fig.4a ).Control experiments testing Sytox orange uptake conﬁrmed that the plasma membrane remained intact during the measurement period and that loss of cellular integrity led to an overall loss of sensor signal without measurable sensor oxidation (data not shown).The observed changes in D C and E GSH did not occur in the absence of TRAIL treatment (data not shown).In contrast to the observations in the cytoplasm,the increase in glutathione oxidation inside mitochondria occurred concurrently with the decrease of D C ,and maximal oxidation was reached within 15min (Fig.4b ).Thus it appeared that mitochondrial glutathione oxidation is directly coupled to the drop in D C ,whereas oxidation of the cytosolic glutathione pool followed with a temporal delay.Oxidant formation mediated by NADPH oxidaseThe generation of oxidants by phagocyte NADPH oxidase (Phox)is one important cellular defense against pathogens upon ﬁrstR a t i o 405/488 n mR a t i o 405/488 n mTime (s)abFigure 5|Grx1-roGFP2visualizes physiological redox changes associated with immune-cell acti-vation.(a )Grx1-roGFP2–positive mouse RAW264.7cells were stimulated with 1m M PMA after 6min.False-color ratio pictures of PMA-treated cells are depicted at representative time points (top).Scale bar,10m m.The sensor ratio of two stimulated cells (arrowheads)and one control cell was calculated and plotted over time (bottom).(b )Isolatedprimary human T cells were transiently transfected with Grx1-roGFP2and analyzed by time-resolved ﬂow cytometry.After 150s (left arrow),cells were subjected to either anti-CD3or isotype controlantibody for another 350s.Cross-linking of primary antibody was then induced by injection of goat-anti-mouse antibody (right arrow).The emission ratio (excitation wavelengths 405and 488nm)was plotted against time.Data from approximately 40–60cells were averaged per second.Data are representative of three experiments.NATURE METHODS |VOL.5NO.6|JUNE 2008|557ARTICLES©2008 N a t u r e P u b l i s h i n g G r o u p h t t p ://w w w .n a t u r e .c o m /n a t u r e m e t h o d scontact.In vitro ,Phox-mediated oxidant formation can be induced by phorbol ester 28.Treatment of biosensor-expressing RA W264.7macrophages with phorbol 12-myristate 13-acetate (PMA)during live-cell imaging demonstrated a continuous increase in cytosolic oxidation,which leveled after approximately 2h (Fig.5a ).We conﬁrmed the oxidative response in the same experiment by H 2DCFDA staining (Supplementary Fig.10online).Oxidant formation induced by T-cell receptor stimulationT-cell receptor stimulation is known to induce rapid cytosolic oxidant generation 29,and H 2O 2has been found to function as an essential second messenger in T-cell receptor signaling 30.T o clarify whether the Grx1-roGFP2biosensor allows the detection of a rapid receptor-triggered ROS signal,we transiently transfected primary peripheral human T cells with the biosensor and used time-resolved ﬂow cytometry to follow the cytosolic redox state.T-cell stimula-tion through cross-linking of T-cell receptors led to a substantial increase in cytosolic oxidation,reaching approximately 75%sensor oxidation within 10min (Fig.5b ).Addition of soluble antibodies to CD3alone or cross-linking of isotype-matched control anti-bodies did not induce an oxidative response.DISCUSSIONIdeally,redox sensors should be non-disruptive,dynamic,sensitive and speciﬁc for a deﬁned redox pair of interest.The genetically encoded redox biosensor reported here is based on the ability of roGFP2to mimic a natural Grx1target protein in that it becomes dynamically oxidized and reduced in response to E GSH ,comparable to physiological Grx1target proteins 31.The relevance of this concept is also emphasized by our recent observation that roGFP2expressed in Arabidopsis thaliana speciﬁcally responds to the cytosolic glutathione redox buffer,a response most likely mediated by plant glutaredoxins,which are far more abundant than their counterparts in animal cells 32.Moreover,quantitative analysis of roGFP2ﬂuorescence in Arabidopsis mutants partially deﬁcient in glutathione biosynthesis conﬁrmed that measured changes in E GSH are consistent with theoretical predictions derived from the Nernst equation 32.The operating mode of Grx1-roGFP2appears to rest upon the established monothiol mechanism of Grx1,as removal of the second thiol in the active site (Cys26)did not notably change the redox-sensing behavior of Grx1-roGFP2in live-cell experiments (data not shown).Nucleophilic Cys23is known to interact with GSSG to form a mixed protein-glutathione disulﬁde intermediate.According to the reaction mechanism of glutaredoxins 33,34,subsequent oxidation of roGFP2is likely mediated via glutathionylation of one of the two roGFP2cysteines.This notion is also supported by the absence of detectable mixed disulﬁde intermediates between Grx1and roGFP2(data not shown).Our observations are in line with a recent in vitro study using the fusion of yeast glutaredoxin-1(Grx1p)to redox-sensitive yellow ﬂuorescent protein (rxYFP)to obtain mechanistic insight into the enzymatic mechanism of glutaredoxin 35.However,the rxYFP-Grx1p fusion protein differs from Grx1-roGFP2in ways that appear to limit its use as a biosensor:rxYFP does not show ratiometric behavior,and its dithiol-disulﬁde pair is less negative in mid-point potential (–265mV),thus making it less responsive to small changes in glutathione oxidation.Responding to low-micromolar concentrations of exogenously added H 2O 2,Grx1-roGFP2in HeLa cells was no less sensitive than HyPer under comparable conditions 15,although the latter is a dedicated H 2O 2sensor,directly oxidized by H 2O 2.In contrast to HyPer,the intracellular response of Grx1-roGFP2to low amounts of H 2O 2depends on the formation of GSSG,likely mediated by endogenous glutathione peroxidases.Of note,the maximal ratio-metric change for HyPer in living cells is about 2.4,as compared to a more favorable factor of 4.4for Grx1-roGFP2.Another difference between Grx1-roGFP2and HyPer is that the redox state of the former is driven in both directions by the same redox pair (GSH and GSSG),whereas the redox state of the latter represents the interplay between H 2O 2-mediated oxidation and an intracellular reducing system.The expression of Grx1-roGFP2allowed observation of the real-time response of the intracellular glutathione system to exogenously applied agents.In principle,such measurements can be applied to any compound—for example,antioxidants or xenobiotics—thus suggesting applications in drug screening and toxicology.Expres-sion of Grx1-roGFP2also permitted the measurement of endogen-ous redox changes,including those associated with growth-factor deprivation,cell density and apoptosis.Similar to HyPer 15,Grx1-roGFP2revealed a close relationship between D C and the mito-chondrial redox state.By targeting one version of Grx1-roGFP2to the mitochondrial matrix,we resolved the temporal delay between mitochondrial and cytoplasmic glutathione oxidation.Finally,the detection of a receptor-triggered ROS signal in primary human lymphocytes suggests that the Grx1-roGFP2biosensor affords the observation of physiological redox signals similar to those that would be experienced by cells in vivo under normal circumstances.METHODSIn vitro measurements of the roGFP2redox state.We expressed recombinant proteins (Grx1-roGFP2,Grx1(SS)-roGFP2,Trx1-roGFP2,roGFP2and Trx1)in Escherichia coli strains BL21(Stra-tagene)or M15(Qiagen)and puriﬁed via hexahistidine afﬁnity chromatography.The puriﬁed recombinant proteins were desalted (Slide-A-Lyzer dialysis cassettes;Pierce)and diluted into a standard reaction buffer (100mM potassium phosphate,1mM EDTA;pH 7.0)to a ﬁnal concentration of 1m M.We measured the emis-sion of roGFP2(505–515nm)after excitation at 390and 480nm in a plate reader (FLUOstar Optima;BMG Labtech)equipped with built-in injectors.We calculated the ratio of the emission (390/480nm)and plotted it against time.For better comparability,we set the ratio of fully reduced protein to 0.1.For oxidation experiments,we ﬁrst reduced the proteins with 20mM DTT for 20min on ice and desalted them with Zeba Desalt spin columns (Pierce).Cell culture and imaging of living cells.We grew HeLa and RAW264.7cells in DMEM (Gibco)supplemented with 10%heat-inactivated FBS,2mM L -glutamine,100U/ml penicillin and 100m g/ml streptomycin (Gibco).We generated HeLa cells stably expressing Grx1-roGFP2or roGFP2by retroviral transduction and subsequent selection with 0.5m g/ml puromycin.We created Grx1-roGFP2–expressing RAW264.7cells by lentiviral infection.T o monitor D C ,we loaded HeLa cells expressing Grx1-roGFP2with 30nM TMRM (Molecular Probes).Apoptosis was induced by addition of 400ng/ml of TRAIL.We monitored membrane integrity by staining with 50nM Sytox orange (Molecular Probes).558|VOL.5NO.6|JUNE 2008|NATURE METHODSARTICLES©2008 N a t u r e P u b l i s h i n g G r o u p h t t p ://w w w .n a t u r e .c o m /n a t u r e m e t h o d s。

实验性青光眼视网膜神经节细胞p53基因表达的检测

实验性青光眼视网膜神经节细胞p53基因表达的检测骆荣江;葛坚;卓业鸿【期刊名称】《中华实验眼科杂志》【年(卷),期】2001(019)003【摘要】目的检测不同眼压状态下实验性青光眼视网膜神经节细胞(RGCs)p53基因的表达。

方法家兔16只,任选1眼作实验,另眼对照2%甲基纤维素0.10～0.15ml前房注射制做高眼压模型,根据眼压值低中高不同对动物进行分组分期取材石蜡切片免疫组化染色检测p53蛋白的表达情况采用计算机图像分析技术对RGCs密度进行定量分析。

结果凋亡率高低依次为:眼压较低组(1.86%)>眼压中等升高组(0.93%)>眼压较高组(0.53%)>对照组(0.05%)(P<0.01)。

结论与前人对POAG患者的研究。

结果相反,在眼压急速升高的模型眼中,凋亡主要发生在实验的早期%ObjectiveTo detect the expression on p53 gene in retinal ganglion cells(RGCs) of rabbit eyes with different degrees of intraocular hypertension.MethodsIntraocular pressure(IOP)was elevated by intracameral injection of 0.10～0.15 ml of 2% methylcellulose.Animals were grouped up by different values of IOP.The rabbit eyes were enucleated,fixed,paraffin-embedded and cut into serial 6- μm thin sections.For histological evaluation,the slides were stained with DAB chromogen(conducting an immuohistochemical experiment),and were examined by light microscopic and automatic image analysis.ResultsThe number of RGCs with a positive reaction for p53 monoclonal antibody was statistically higher in the experimental eyes than in their control felloweyes(P<0.01),and apoptosis was mainly found in the early stage of research.ConclusionOur research presents a new alternative view of apoptosis in experimental glaucoma(According to formerreports,apoptosis was mainly found in the advanced stage of the patients with POAG)【总页数】3页(P208-210)【作者】骆荣江;葛坚;卓业鸿【作者单位】中山医科大学中山眼科中心广州,510060;中山医科大学中山眼科中心广州,510060;中山医科大学中山眼科中心广州,510060【正文语种】中文【中图分类】R775【相关文献】1.频域OCT检测青光眼视网膜神经节细胞复合体厚度的研究 [J], 樊宁;黄丽娜;何靖;申晓丽;曾琨;成洪波2.频域OCT检测青光眼视网膜神经节细胞复合体厚度的研究 [J], 樊宁;黄丽娜;何靖;申晓丽;曾琨;成洪波3.益气养阴方对实验性糖尿病大鼠视网膜P53、Bcl-2基因表达的影响 [J], 颉瑞萍;刘莹;车红霞;杨维杰;张玲玲4.神经生长因子β对兔实验性青光眼视网膜神经节细胞的保护作用 [J], 陆志荣;管怀进;程新梁;张天一;鄂群;龚启荣5.不同眼压状态下实验性青光眼视网膜神经节细胞p53基因表达 [J], 李素美;王文奇因版权原因，仅展示原文概要，查看原文内容请购买。

第一个宽带太赫兹隔离器

一团队开发出第一个宽带太赫兹隔离器
来源：electronics news.；电子科技大学太赫兹研究中心四川太赫兹应用研究联合课题组孙翰编译
一个在加拿大的INRS Énergie Matériaux Télécommunications研究中心的团队已经开发出一种装置，该装置对于太赫兹信号源的各种应用是至关重要的。

他们的非互易性电磁隔离器是最近发表在自然通讯上文章的主题，旨在说明这一新发展的重要性。

直到现在，在没有对应太赫兹波段光谱的隔离器，这一现状阻碍了某些技术的发展。

新的设备为包括太赫兹激光器和放大器在内的太赫兹技术发展铺平了道路，因此科学界现在对其予以了重视。

太赫兹波源和探测器领域的最新进展，促使了成像、通信和光谱技术的发展——这些最终可应用于探测爆炸物。

所有这些技术都使用了电磁频谱宽带的电流隔离器，现有的隔离器是不适合的。

隔离器在抑制扭曲测量的反射波或防止损坏其他原件中是必要的。

因此，缺乏一个可行的隔离器代表了太赫兹波源使用的主要限制。

该小组在INRS的工作提供了这个问题的第一种解决方案，使用锶铁氧化物（SrFe12O19）的磁铁，这一材料具有无需外部附加磁场的优点。

Publication:
The article, entitled “A magnetic non-reciprocal isolator for broadband terahertz operation,” appeared March 5, 2013, in Nature Communications (4:1558, DOI: 10.1038/natcomms2572).。

BT将与清华大学联合建立研究实验室

BT将与清华大学联合建立研究实验室
佚名
【期刊名称】《数字通信世界》
【年(卷),期】2012(000)010
【摘要】英国电信（BT）日前宣布进一步加强与清华大学经济管理学院（TsinghuaSEM）的合作关系，并在清华大学经济管理学院建立一个新的研究实验室，该研究实验室旨在开发和演示新知识产权（IPR）与先进技术，为中国创新与BT技术搭建桥梁。

BT与清华大学经济管理学院正在考虑包括“云计算”和“电子医疗”的商业应用等众多的研究合作项目。

【总页数】1页(P29-29)
【正文语种】中文
【中图分类】TP393.09
【相关文献】
1.BT与清华大学建立研究实验室 [J], 叶惠
2.BT与清华大学联合建立研究实验室 [J],
3.阿尔卡特朗讯贝尔实验室与INRIA建立联合研究实验室携手开发下一代互联网关键技术 [J],
4.阿尔卡特朗讯贝尔实验室与INRIA建立联合研究实验室 [J],
5.阿尔卡特朗讯贝尔实验室与INRIA建立联合研究实验室携手开发下一代互联网关键技术 [J],
因版权原因，仅展示原文概要，查看原文内容请购买。

色心晶体微弱信号测试抗干扰技术的研究

色心晶体微弱信号测试抗干扰技术的研究
李荣华
【期刊名称】《华侨大学学报（自然科学版）》
【年(卷),期】1990(011)003
【摘要】色心晶体红外荧光信号在检测过程中,尤其检测其变化值会遇到一种十分微弱的光信号.为了将这种微弱光信号从各种干扰噪声中准确地检测记录下来,本文分析了各种干扰的起因,研究了相应的抗干扰技术,并在色心晶体荧光光谱仪组装过程中得到实际应用,提高了整机的信噪比.结果较为满意.
【总页数】5页(P262-266)
【作者】李荣华
【作者单位】华侨大学材料物理化学研究所
【正文语种】中文
【中图分类】O4
【相关文献】
1.Ce:YAG晶体色心和缺陷的伽马射线辐照研究 [J], 李抒智;杨卫桥;董永军;华伟;向卫东;梁晓娟;钟家松;赵寅生;吕春燕
2.CVT小信号测试法中抗干扰技术的应用研究 [J], 李鹏程;张秋雁;肖监;丁鸣
3.氟化锂晶体的色心效应在环境热点剂量监测中的研究 [J], 徐伟;乌晓燕
4.微弱电信号测试计量的植物生长信息传递研究展望 [J], 王兰州;胡安雨
5.含色心BaMgF_4晶体电子结构的研究 [J], 康玲玲;刘廷禹;张启仁;徐灵芝
因版权原因，仅展示原文概要，查看原文内容请购买。

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Polar T argeting and Endocytic Recycling in Auxin-Dependent Plant DevelopmentJ ¨urgen Kleine-Vehn and Ji ˇr ´ıFrimlDepartment of Plant Systems Biology,VIB,and Department of Molecular Genetics,Ghent University,9052Ghent,Belgium;email:jiri.friml@psb.ugent.beAnnu.Rev.Cell Dev.Biol.2008.24:447–73The Annual Review of Cell and Developmental Biology is online at This article’s doi:10.1146/annurev.cellbio.24.110707.175254Copyright c2008by Annual Reviews.All rights reserved1081-0706/08/1110-0447$20.00Key Wordstrafﬁcking,endocytosis,polar auxin transport,PIN proteinsAbstractPlant development is characterized by a profound phenotypic plastic-ity that often involves redeﬁning of the developmental fate and polar-ity of cells within differentiated tissues.The plant hormone auxin and its directional intercellular transport play a major role in these pro-cesses because they provide positional information and link cell polar-ity with tissue patterning.This plant-speciﬁc mechanism of transport-dependent auxin gradients depends on subcellular dynamics of auxin transport components,in particular on endocytic recycling and polar targeting.Recent insights into these cellular processes in plants have revealed important parallels to yeast and animal systems,including clathrin-dependent endocytosis,retromer function,and transcytosis,but have also emphasized unique features of plant cells such as diversity of polar targeting pathways;integration of environmental signals into subcellular trafﬁcking;and the link between endocytosis,cell polarity,and cell fate speciﬁcation.We review these advances and focus on the translation of the subcellular dynamics to the regulation of whole-plant development.447Click here for quick links to Annual Reviews content online, including:• Other articles in this volume • Top cited articles• Top downloaded articles • Our comprehensive searchFurtherANNUAL REVIEWSA n n u . R e v . C e l l D e v .B i o l . 2008.24:447-473. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b yC h i n e s e A c a d e m y o f S c i e n c e s - L i b r a r y o n 10/15/08. F o r p e r s o n a l u s e o n l y .Polar auxin transport:thedirectional transport of the plant hormone auxin from cell to cellContentsDEVELOPMENTALINTRODUCTION...............448POLAR TARGETING...............449Passengers and Destinations:Polar Cargos and Polar Domains......450Tickets to Go or to Stay:Polar T argeting Signals..........452Staying at the Station:Retention at the Polar Domains............453How to Get There:PolarT argeting Pathways.............453ENDOCYTIC RECYCLINGIN PLANT CELLS (454)The Back and Forth:Constitutive Endocytic Recycling of Plasma Membrane Proteins.............454Getting Away:Endocytosisin Plant Cells...................455Getting Back:Recycling inPlant Cells (457)Going to the Other Side:T ranscytosis Linking Endocytic Recycling and Polar T argeting...459Separating the Daughters:Endocytic Recycling in Cytokinetic Cells (461)EXEMPLIFIED CASES:POLAR TARGETING ANDENDOCYTIC RECYCLINGIN PLANT DEVELOPMENT....462Induced Endocytosis in Plants (462)Integrating Developmental and Environmental Signalsthrough Polarity Modulations (463)Canalization Hypothesis and the Effect of Auxin on ItsOwn Efﬂux.....................465DEVELOPMENTAL INTRODUCTIONAnimals and plants evolved basic biologi-cal differences that characterize their survival strategies.Animals developed elaborate sensory and locomotory capacities that enable complexbehavioral responses,such as the ﬁght-or-ﬂight response,to overcome environmental stress.In contrast,during their evolution plants emphasized increased physiological tolerance and phenotypic plasticity.These different life strategies are also adequately reﬂected in the various ways in which animals and plants establish their body architecture.Whereas during embryogenesis animals are already deﬁning their adult shape to a large extent,in plants this early developmental phase just sketches a basic body plan,and the ﬁnal shape of a plant will be largely deﬁned by an elaborate postembryonic development (Weigel&J ¨urgens 2002).T o achieve this develop-mental plasticity,plants maintain permanent populations of stem cells (meristems)at the growing root and shoot apices and are able to redeﬁne the developmental programs as well as the polarity of already speciﬁed tissues.Thus,plants can sustain and regulate their growth rate,can postembryonically form new organs,and possess a high capacity for tissue regeneration (Steeves &Sussex 1989,Weigel&J ¨urgens 2002).Different animal species also retain these capabilities to some extent;how-ever,plants are far superior in utilizing these mechanisms for individually shaping their body according to the demands of the environment.The plant signaling molecule auxin determines many aspects of this ﬂexible plant development.Auxin acts as a prominent signal,providing,by its local accumulation in selected cells,a spatial and temporal reference for changes in the developmental program (Reinhardt et al.2000,Friml 2003,Leyser 2006,Esmon et al.2006,T anaka et al.2006,Dubrovsky et al.2008).Auxin is distributed through tissues by a di-rectional cell-to-cell transport system,termed polar auxin transport,that depends on speciﬁc auxin carrier proteins (Figure 1)(Benjamins et al.2005,Blakeslee et al.2005,Kramer &Bennett 2006,Vieten et al.2007).Auxin efﬂux carriers of the PIN-FORMED (PIN)family (G¨a lweiler et al.1998,Luschnig et al.1998,Chen et al.1998,Utsono et al.1998,Petr´a ˇsek et al.2006)show a polar subcellular localiza-tion that correlates with and determines the448Kleine-V ehn·FrimlA n n u . R e v . C e l l D e v .B i o l . 2008.24:447-473. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b yC h i n e s e A c a d e m y o f S c i e n c e s - L i b r a r y o n 10/15/08. F o r p e r s o n a l u s e o n l y .Rubery &Sheldrake postulated in the mid-1970s the so-called chemiosmotic hypothesis for directional intercellular auxin movement (Rubery &Sheldrake 1974and,independently,Raven 1975).Accordingly,the auxin indole acetic acid (IAAH)is largely pro-tonated at the lower pH of the cell wall and can pass through the plasma membrane into the cell.In the higher-pH cytosol,part of the IAAH is deprotonated,and the resulting charged IAA −is largely membrane impermeable and requires transporter activity to exit the cell.The localization of the PIN-FORMED (PIN)auxin efﬂux carrier at the plasma membrane determines the auxin exit site from an individual cell.Coordinated polar localization of PINs in a given tissue hence determines the direction of cell-to-cell auxin transport.AUX1/LAX1denotes auxin inﬂux carriers AUXIN RESISTANT1/LIKE AUX1.direction of auxin ﬂow through tissues (Friml et al.2004,Wi´s niewska et al.2006).In plants,polarities of tissue and of individual cells are closely connected by the ﬂow of auxin (Sauer et al.2006),and the cell biological processes depending on vesicle trafﬁcking and polar targeting have an immediate developmentalRecycling:membranes and other molecules recycle from intracellular endocytic compartments back to the plasma membrane Tight junctions:anchored protein complexes forming a physical barrier between polardomains;limit lateral diffusion and are involved in polarity establishment and maintenance in animal epithelial cellsoutput related to auxin-mediated signaling.At the level of polar auxin transport,many developmental and environmental signals are integrated.By rearranging the subcellular localization of PIN auxin efﬂux carriers,such signals inﬂuence auxin-dependent patterning and contribute substantially to the adaptive and ﬂexible nature of plant development.Our aim is to review recent advances on subcellular trafﬁcking and polar targeting in plants and to highlight links with physiol-ogy and development.A special focus is given to auxin-dependent regulation of development because this area is intimately linked to en-docytic recycling and polar targeting.Most of these concepts were formulated on the basis of studies in the model plant Arabidopsis thaliana ;nonetheless,they seem to apply to a large extent to higher plants in general.POLAR TARGETINGThe establishment and maintenance of cell polarity are central themes of developmental and cell biology because individual cell polar-ities,transmitted by cell divisions,are trans-lated into tissue and organ polarity and,ulti-mately,shape.In addition,cell polarity plays a key role in directional signaling and intercellu-lar communication.At the level of individual cells,polarity is typically reﬂected by the asymmetric distribu-tion of intracellular components that can form functionally and/or morphologically distinct domains (Bonifacino &Lippincott-Schwartz 2003).Mechanisms for generating or maintain-ing cell polarity have been extensively studied in different model organisms,such as worms,ﬂies,mammals,and yeasts (e.g.,Knoblich 2000,Irazoqui &Lew 2004,Margolis &Borg 2005,Nance 2005).Animal epithelial cells are a fa-vorite model system because their plasma mem-brane harbors two distinct domains that are separated by tight junctions:an apical domain facing the lumen and a basolateral domain (Mostov et al.2003,Janssens &Chavrier 2004).These protein-based barriers in the membrane prevent lateral diffusion of proteins and lipids •Directional V esicle Transport Pinpoints Plant Growth449A n n u . R e v . C e l l D e v .B i o l . 2008.24:447-473. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b yC h i n e s e A c a d e m y o f S c i e n c e s - L i b r a r y o n 10/15/08. F o r p e r s o n a l u s e o n l y .trans-Golgi network (TGN):the main sorting compartment of the secretorypathway in eukaryotic cells;may act as an early-endosomalcompartment in plantsbetween the two distinct polar domains,main-taining the distribution of various polar-competent proteins.Researchers have identi-ﬁed numerous polar cargos that reside in a cell-line-speciﬁc manner preferentially at the apical and/or basolateral plasma membranes in polarized epithelial cells.Apical and baso-lateral components are recruited differentially by the targeted delivery of membrane and se-EpidermisCortexEndodermis SteleQuiescent center Columella Lateral capPolar distribution of PINsPIN2PIN3PIN2PIN2PIN2PIN7PIN7PIN3PIN4PIN1Figure 2Patterns of PIN protein localization in the Arabidopsis root tip.Schematic and immunolocalizations of PIN proteins in the Arabidopsis root tip.Arrows indicate polar PIN localization at the plasma membrane,illustrating celltype–dependent decisions in the PIN polar localization.Note the differential PIN2targeting in the epidermis (apical)and young cortex (basal)cells.cretory proteins to these domains as a result of three processes.(a )Newly synthesized pro-teins are sorted in the trans-Golgi network (TGN)into carrier vesicles that speciﬁcally de-liver them to the apical surface or the basolat-eral surface.(b )Some proteins are selectively retained at the plasma membrane.(c )Proteins that are not retained are rapidly endocytosed and either recycled back through recycling en-dosomes or,alternatively,delivered to a differ-ent,polar plasma membrane domain by a pro-cess called transcytosis (Rodriguez-Boulan et al.2005).Passengers and Destinations:Polar Cargos and Polar DomainsEven though in no other kingdom is the relation between individual cell polarity and macro-scopic patterning as prominent as in plants,knowledge on cell polarity and mechanisms of targeted cargo delivery is still lacking in plants.Most of our understanding on polar targeting has been gained by study of the polar delivery of auxin efﬂux carriers from the PIN family (Figure 2).PIN proteins have emerged in recent years from genetic studies in A.thaliana as key regulators of a plethora of auxin-mediated developmental processes,including axis formation in embryogenesis (Friml et al.2003b),postembryonic organogenesis (Okada et al.1991,Benkov´a et al.2003,Reinhardt et al.2003,Heisler et al.2005),root meristem maintenance (Friml et al.2002a,Blilou et al.2005),vascular tissue differentiation and regeneration (G¨a lweiler et al.1998,Sauer et al.2006,Scarpella et al.2006),and tropic growth (Luschnig et al.1998,Friml et al.2002b).PIN proteins act as mediators of the auxinefﬂux from cells (Petr´a ˇsek et al.2006)and have different subcellular distributions—including apolar,basal,apical,and lateral plasma mem-brane localizations—depending on the PIN protein as well as the cell type (Wi´s niewska et al.2006).The most typical are basal (root tip–facing)localization of the PIN1protein in the inner cells of both shoots and roots,apical (shoot apex–facing)localization of PIN2in the450Kleine-V ehn·FrimlA n n u . R e v . C e l l D e v .B i o l . 2008.24:447-473. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b yC h i n e s e A c a d e m y o f S c i e n c e s - L i b r a r y o n 10/15/08. F o r p e r s o n a l u s e o n l y .root epidermis and lateral root cap cells,and lateral localization of PIN3at the inner side of shoot endodermis cells (G¨a lweiler et al.1998;M ¨uller et al.1998;Friml et al.2002b,2003a).Other components of auxin transport,such as the auxin inﬂux carrier AUXIN RESISTANT1/LIKE AUX1(AUX1/LAX)(Bennett et al.1996,Yang et al.2006,Swarup et al.2008)and multiple drug resistance/P-glycoprotein (MDR/PGP)trans-porters (Geisler et al.2005,T erasaka et al.2005),are also localized in a polar manner in some cells while being symmetrically local-ized in most cells (Mravec et al.2008).For example,AUX1localizes to the apical side of protophloem cells opposite to PIN1or to the same side as PIN1in the shoot apical meristem (Swarup et al.2001,Reinhardt et al.2003).In contrast,PGP4has a basal or an apical local-ization in root epidermal cells (T erasaka et al.2005).In addition to components of the auxin transport,other polar cargos in plants,includ-ing transporters for boron (BOR1and BOR4)and for silicon in rice (LSI1and LSI2),have been identiﬁed.Such cargos are localized at ei-ther the inner or the outer lateral sides of cells,as well as the regulator of anisotropic expansion,COBRA,which is similarly polarly targeted to both longitudinal cell sides (Roudier et al.2005;T akano et al.2005;Ma et al.2006,2007;Miwa et al.2007).The PLEIOTROPIC DRUG RESISTENCE (PDR)-type transporter for the auxin-like compounds PIS1/PDR9re-sides at the outer lateral side of root epider-mis cells.The lateral cargo POLAR AUXIN TRANSPORT INHIBITOR-SENSITIVE1(PIS1),the basal cargo PIN1,and the apical cargo PIN2have been simultaneously visual-ized in the same cells,highlighting that plant cells are able to maintain at least three polar do-mains within a single cell (R ˚uˇz iˇc ka et al.2008).Future studies will address whether epidermal root cells are potent to maintain,besides the apical,basal,and outer lateral domains,an ad-ditional inner lateral polar domain.Nonethe-less,although apical-basal targeting in plants and apical-basolateral delivery in animals canEndocytosis:the uptake of material into a cell by the formation of a membrane-bound vesiclereﬂect a comparable polar competence among the divergent kingdoms,the simultaneous de-livery of lateral cargos hints at a more complex situation for cell polarity in plant cells that may once again stress the ﬂexibility and enormous importance of cell polarity regulation in plants (Figure 3).Figure 3The black box of plant polarity.A schematic representation of various polar cargos in plant cells.Plants are competent to deliver cargos to apical,basal,inner,and outer polar domains.Apical cargos include PGP4(root epidermis cells),AUX1(root protophloem cells),PIN1(epidermal cells of shoot apex and embryonal protoderm cells),and PIN2(root epidermis and older cortex cells).Basal cargos encompass PGP4(root epidermis cells),PIN1(e.g.,root stele),PIN2(young cortex cells),and PIN4(in the proximal part of the rootmeristem).Outer lateral cargos are represented by,e.g.,PIS1(root epidermis),BOR4(root epidermis),LSI1(root exodermis and endodermis cells),andCOBRA (root epidermis).Inner lateral polarity can be deﬁned by PIN3(shoot endodermis and root pericycle),BOR1(root pericycle cells),LSI2(root exodermis and endodermis cells),and COBRA (root epidermis).Moreover,several PIN cargos undergo rapid polarity alterations (depicted in the middle ),including the establishment of basal localization of PIN1during embryogenesis or lateral root development,an apical-to-basal polarity shift of PIN7during embryogenesis in suspensor cells,a basal-to-apical shift in upper cortex cells of PIN2,and dynamic relocation of PIN3to the bottom sides of root cap cells after gravity stimulation. •Directional V esicle Transport Pinpoints Plant Growth451A n n u . R e v . C e l l D e v .B i o l . 2008.24:447-473. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b yC h i n e s e A c a d e m y o f S c i e n c e s - L i b r a r y o n 10/15/08. F o r p e r s o n a l u s e o n l y .Transcytosis:the dynamic translocation of the same molecules from one distinct plasma membrane domain to another via recycling endosomes Basal polarity:polarity of the lower cell side,the polar plasma membrane domain that faces the root apexApical polarity:polarity of the upper cell side,the polar plasma membrane domain that faces the shoot apexInner lateral polarity:polarity of the inner periclinal cell side,which points away from the body surface Outer lateralpolarity:polarity of the outer periclinal cell side,which points to the body surfaceTickets to Go or to Stay:Polar Targeting SignalsIn animal systems,polar cargo proteins carry signals that determine their residence at different polar domains.These signals may be a combination of plasma membrane retention,internalization,and polar sorting signals (Dugani &Klip 2005,Rodriguez-Boulan et al.2005).In plants,different polar cargos such as PIN1,PIN2,and PIS1localize to different polar destinations in the same cell type,suggesting polarity determinants in the protein sequence itself.Moreover,an insertion of green ﬂuorescent protein (GFP)at a speciﬁc position within the middle hydrophilic loop causes PIN1localization to shift to the oppo-site side of the cell compared with wild-type PIN1(Wi´s niewska et al.2006).These results demonstrate the presence of polarity signals in the sequence of polar cargos,but detailed insight is still lacking.Polarity signals probably decide to recruit PINs to the distinct apical and basal targeting machineries that are related to phosphorylation sides,because the Ser/Thr protein kinase PINOID (PID)(Friml et al.2004)as well as the protein phosphatase 2A (PP2A)(Michniewicz et al.2007)act on PIN phosphorylation and play a decisive role in the apical-versus-basal targeting of PIN proteins.Loss of the PID function causes an apical-to-basal shift in the PIN polarity corresponding with defects in embryo and shoot organogenesis (Christensen et al.2000,Benjamins et al.2001,Friml et al.2004).Accordingly,PID gain of function results in an opposite basal-to-apical PIN polarity shift,leading to auxin depletion from the root meristem and collapse of the root growth (Friml et al.2004).Similar phenotypes,including the basal-to-apical shift of PIN po-larity,can be observed in the loss-of-function mutants of the A regulatory subunits of PP2A (Michniewicz et al.2007).Importantly,PID directly phosphorylates the hydrophilic loop of PIN proteins,and PP2A antagonizes this action (Michniewicz et al.2007).A possible scenario may be that phosphory-lated PIN proteins are preferentially recruitedinto the apical pathway,whereas dephospho-rylated PINs become a substrate of the basal targeting pathway (Figure 4).This model in-corporates important features of mammalian epithelial cells,in which cargos are phosphory-lated to inﬂuence their polar delivery (Casanova et al.1990).Importantly,phosphorylation-dependent PIN targeting provides a means for any signaling pathway upstream of PID and PP2A activities to modulate PIN polar target-ing and thus directional auxin ﬂuxes.Different relative expression levels of PID and PP2A in various cell types in combination with divergentFigure 4Contribution of PIN-FORMED (PIN)phosphorylation to the decision on the PIN polar distribution.PINOID (PID)-dependentphosphorylation of PIN proteins may affect afﬁnity to distinct apical and basal targeting pathways.An increase in PID kinase or a decrease in proteinphosphatase 2A (PP2A)activities leads to a basal-to-apical PIN polarity shift.On the contrary,increased PP2A activity counteracts the PID effect and leads to preferential GNOM-dependent basal PINtargeting.The place of PID and PP2A action is not entirely clear,but PID and PP2A are also partially associated with the plasma membrane.ARF denotes adenosyl ribosylation factor.452Kleine-V ehn·FrimlA n n u . R e v . C e l l D e v .B i o l . 2008.24:447-473. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b yC h i n e s e A c a d e m y o f S c i e n c e s - L i b r a r y o n 10/15/08. F o r p e r s o n a l u s e o n l y .phosphorylation sites (some of which would be phosphorylated more or less efﬁciently)would explain how both PIN-speciﬁc and cell type–speciﬁc signals are integrated to determine the polar localization of the given PIN protein into a given cell type.The regulation of PID kinase may also be connected with phospho-lipid signaling.The plant 3-phosphoinositide-dependent kinase 1(PDK1)binds PID in vitro and increases PID kinase activity (Zegzouti et al.2006).The involvement of phosphory-lation events for the polar delivery of other car-gos besides PINs has not been thoroughly ad-dressed.Outer lateral delivery of PIS1seems to occur independently of the PID activity,but comparable information for other polar cargos is missing.Extensive work in the coming years is ex-pected to focus on the identiﬁcation and thor-ough characterization of polar targeting signals for different polar cargos in plants.The other crucial issues that are completely unknown in plants concern where and how the polar target-ing signals are recognized as well as where and how the polar cargos are sorted.Staying at the Station:Retention at the Polar DomainsDespite the pronounced importance of polar lo-calization of proteins in plant cells for plant de-velopment,mechanisms for this phenomenon are still ill deﬁned.So far no indications exist for anything analogical to tight junctions,and we lack even fundamental knowledge of how polar-competent cargos are kept in their polar domains.Cytoskeleton-and membrane sterol–dependent constitutive endocytosis and targeted recycling may be involved in main-taining the localization of proteins localized in their polar domains.All these cellular components and processes are required for localization of different cargos.Both basal PIN1localization and even more apical AUX1localization are sensitive to the disruption of the actin cytoskeleton,leading to internaliza-tion and loss of polar localization (Kleine-VehnSterol:plant sterols are amphiphilic molecules and vital constituents of all membranes,including the plasma membraneet al.2006).In contrast,disruption of micro-tubules affects only indirectly the localization of AUX1and PIN proteins that is observed only when the overall cell morphology is altered (Kleine-Vehn et al.2006).In contrast,intact microtubules are required to maintain the outer lateral localization of PIS1;following its disruption,PIS1is found predominantly atapical and basal positions (R ˚uˇz iˇc ka et al.2008).Polar localization of PIN and AUX1proteins as well as auxin signaling depend on the sterol composition of plasma mem-branes (Souter et al.2002,Grebe et al.2003,Willemsen et al.2003,Kleine-Vehn et al.2006).Arabidopsis plantlets defective in the STEROL METHYLTRANSFERASE1(SMT1)gene,which is involved in sterol biosynthesis and affects membrane sterol composition,have cell polarity defects,including impaired polar local-ization of PIN proteins and AUX1(Willemsen et al.2003,Kleine-Vehn et al.2006).Fur-thermore,sterols and PIN proteins have overlapping subcellular trafﬁcking pathways (Grebe et al.2003).Detergent-resistant,sterol-enriched plasma membrane microdomains,sometimes called lipid rafts,are important for various types of plasma membrane–based signaling processes and are present in higher plants as well (reviewed in Bhat &Panstruga 2005).There are indications that PIN and PGP proteins are directly associated with these structures (Titapiwatanakun et al.2008),but what the functional relevance could be of such associations remains to be established.In conclusion,despite the obvious require-ments of cytoskeleton and sterol composition for polar localization of various cargos,it is still unclear whether or eventually in which cases they are involved in keeping cargos at their po-lar positions or whether they play a role in the polar delivery of these proteins to the plasma membrane.How to Get There:Polar Targeting PathwaysThe existence of diverse polar cargos with vari-ous polar targeting signals implies a diversiﬁed •Directional V esicle Transport Pinpoints Plant Growth453A n n u . R e v . C e l l D e v .B i o l . 2008.24:447-473. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b yC h i n e s e A c a d e m y o f S c i e n c e s - L i b r a r y o n 10/15/08. F o r p e r s o n a l u s e o n l y .Adenosylribosylation factor (ARF):a class of small GTPases;a regulator of clathrin-and COPI-dependent vesicle budding Guanine nucleotide exchange factor (GEF):induces GDP-to-GTPexchange and hence the activation of small GTPasesBrefeldin A (BFA):a speciﬁc inhibitor of some ARF GEFsnetwork of distinct polar targeting pathways.Indeed,for example,AUX1and PIN1po-lar delivery occurs by two distinct targeting machineries with different molecular require-ments and different sensitivities to inhibitors of cellular processes (Dharmasiri et al.2006,Kleine-Vehn et al.2006).An important factor for the delivery of PIN proteins to the plasma membrane is an endoso-mal regulator of the vesicle budding,GNOM,which encodes a guanine nucleotide exchange factor for adenosyl ribosylation factors (ARF GEF)(Shevell et al.1994;Geldner et al.2001,2003).In gnom (also designated emb30)mutant embryos,the coordinated polar localization of PIN1is impaired (Steinmann et al.1999),seem-ingly the result of a failure to establish the initial basal localization of PIN1at the globular stage (Kleine-Vehn et al.2008a).Also,in the postem-bryonic roots,GNOM function seems to be crucial for basal targeting,whereas apical local-ization of PINs or AUX1is unaffected in gnom mutants (Kleine-Vehn et al.2006,2008a).Col-lectively,these studies demonstrate that apical cargos utilize a targeting pathway that is molec-ularly distinct from that used by basally local-ized PIN proteins (Kleine-Vehn et al.2006,2008a).In addition,outer lateral PIS1targeting appears to differ fundamentally from apical and basal pathways because PIS1polar localization does not involve any known molecular compo-nents of apical/basal targeting,such as GNOMor PINOID (R ˚uˇz iˇc ka et al.2008).Although api-cal and basal PIN targeting appears to be inter-connected and,thus,to be used alternatively byPIN proteins,the relation between apical/basal and outer lateral polar targeting needs to be unraveled.In summary,genetic and pharmacological interference with different cellular processes as well as the simultaneous localization of cargos to the apical,basal,and outer lateral domains in single cells strongly suggest that there are at least three distinct polar target-ing mechanisms in plants.However,molecu-lar insight into these pathways remains very limited.ENDOCYTIC RECYCLING IN PLANT CELLSThe Back and Forth:Constitutive Endocytic Recycling of Plasma Membrane ProteinsThe internalization of proteins from the plasma membrane is a critical event for all eukaryotic cells.Whereas many internalized molecules are degraded in the lysosomal/vacuolar pathway,other cell surface proteins and molecules un-dergo sequential rounds of recycling back to the plasma membrane.Eukaryotic cells possess the remarkable ability to turn over the entire plasma membrane on an hourly basis (T uvim et al.2001).As such,endocytic recycling is a key for the regulation of the cell surface identity and contributes to rapid cellular responses to in-trinsic and extrinsic cues.Regarding the funda-mental importance of endocytic recycling,var-ious integral plasma membrane proteins,such as signaling components and transporters,ap-pear to display recycling events to the plasma membrane in plants.Pharmacological inhibitors have been valu-able tools for unraveling the internalization of plant proteins to endosomal compartments and subsequent recycling back to the plasma mem-brane (Carter et al.2004).The fungal toxin brefeldin A (BFA)interferes with various vesicle trafﬁcking processes in cells and speciﬁcally tar-gets ARF GEFs.Cytosolic GDP-bound ARF proteins are inactive and become recruited to the target membrane by ARF GEF–dependent GDP-to-GTP exchange.ARF proteins play an important role in the formation of vesicle coats required for their budding and cargo se-lection in different subcellular compartments.BFA is a noncompetitive inhibitor that stabi-lizes ARF/ARF GEF intermediates and freezes both proteins inactively at the place of action (reviewed by Donaldson &Jackson 2000).In cultured cells of tobacco,BFA interferes with ARF GEF–dependent endoplasmic reticulum (ER)-to-Golgi trafﬁcking,leading to ER-Golgi hybrids (Ritzenthaler et al.2002).In contrast,in Arabidopsis ,this process is catalyzed by the454Kleine-V ehn·FrimlA n n u . R e v . C e l l D e v .B i o l . 2008.24:447-473. D o w n l o a d e d f r o m a r j o u r n a l s .a n n u a l r e v i e w s .o r g b yC h i n e s e A c a d e m y o f S c i e n c e s - L i b r a r y o n 10/15/08. F o r p e r s o n a l u s e o n l y .。