Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments

After partitioning of cytoplasmic contents by cleavage furrow ingression, animal cells remain connected by an intercellular bridge, which subsequently splits by abscission. Here, we examined intermediate stages of abscission in human cells by using live imaging, three-dimensional structured illumination microscopy, and electron tomography. We identified helices of 17-nanometer-diameter filaments, which narrowed the cortex of the intercellular bridge to a single stalk. The endosomal sorting complex required for transport (ESCRT)-III co-localized with constriction zones and was required for assembly of 17-nanometer-diameter filaments. Simultaneous spastin-mediated removal of underlying microtubules enabled full constriction at the abscission site. The identification of contractile filament helices at the intercellular bridge has broad implications for the understanding of cell division and of ESCRT-III-mediated fission of large membrane structures.     

Midbody targeting of the ESCRT machinery by a noncanonical coiled coil in CEP55

The ESCRT (endosomal sorting complex required for transport) machinery is required for the scission of membrane necks in processes including the budding of HIV-1 and cytokinesis. An essential step in cytokinesis is recruitment of the ESCRT-I complex and the ESCRT-associated protein ALIX to the midbody (the structure that tethers two daughter cells) by the protein CEP55. Biochemical experiments show that peptides from ALIX and the ESCRT-I subunit TSG101 compete for binding to the ESCRT and ALIX-binding region (EABR) of CEP55. We solved the crystal structure of EABR bound to an ALIX peptide at a resolution of 2.0 angstroms. The structure shows that EABR forms an aberrant dimeric parallel coiled coil. Bulky and charged residues at the interface of the two central heptad repeats create asymmetry and a single binding site for an ALIX or TSG101 peptide. Both ALIX and ESCRT-I are required for cytokinesis, which suggests that multiple CEP55 dimers are required for function.     

A sense of the end

How a cell distinguishes a double-strand break from the end of a chromosome has long fascinated cell biologists. It was thought that the protection of chromosomal ends required either a telomere-specific complex or the looping back of the 3' TG-rich overhang to anneal with a homologous double-stranded repeat. These models must now accommodate the findings that complexes involved in nonhomologous end joining play important roles in normal telomere length maintenance, and that subtelomeric chromatin changes in response to the DNA damage checkpoint. A hypothetical chromatin assembly checkpoint may help to explain why telomeres and the double-strand break repair machinery share essential components.     

Ceramide triggers budding of exosome vesicles into multivesicular endosomes

Intraluminal vesicles of multivesicular endosomes are either sorted for cargo degradation into lysosomes or secreted as exosomes into the extracellular milieu. The mechanisms underlying the sorting of membrane into the different populations of intraluminal vesicles are unknown. Here, we find that cargo is segregated into distinct subdomains on the endosomal membrane and that the transfer of exosome-associated domains into the lumen of the endosome did not depend on the function of the ESCRT (endosomal sorting complex required for transport) machinery, but required the sphingolipid ceramide. Purified exosomes were enriched in ceramide, and the release of exosomes was reduced after the inhibition of neutral sphingomyelinases. These results establish a pathway in intraendosomal membrane transport and exosome formation.     

Helical structures of ESCRT-III are disassembled by VPS4

During intracellular membrane trafficking and remodeling, protein complexes known as the ESCRTs (endosomal sorting complexes required for transport) interact with membranes and are required for budding processes directed away from the cytosol, including the budding of intralumenal vesicles to form multivesicular bodies; for the budding of some enveloped viruses; and for daughter cell scission in cytokinesis. We found that the ESCRT-III proteins CHMP2A and CHMP3 (charged multivesicular body proteins 2A and 3) could assemble in vitro into helical tubular structures that expose their membrane interaction sites on the outside of the tubule, whereas the AAA-type adenosine triphosphatase VPS4 could bind on the inside of the tubule and disassemble the tubes upon adenosine triphosphate hydrolysis. CHMP2A and CHMP3 copolymerized in solution, and their membrane targeting was cooperatively enhanced on planar lipid bilayers. Such helical CHMP structures could thus assemble within the neck of an inwardly budding vesicle, catalyzing late steps in budding under the control of VPS4.     

Abscisin II, an Abscission-Accelerating Substance from Young Cotton Fruit

Crystalline abscisin II, with a tentative molecular formula of C(15)H(20)O(4), has been isolated from young cotton fruit. It accelerates abscission when applied in amounts as low as 0.01 microg per abscission zone. It inhibits indoleacetic acid-induced straight growth of Avena coleoptiles but has no gibberellin activity on dwarf maize.     

中药复方抗鸡新城疫作用及其对雏鸡免疫内分泌调节的影响

为筛选抗鸡新城疫中药复方并观察中药复方对雏鸡免疫内分泌调节的影响,将152羽30日龄健康免疫雏鸡随机分为5组:3个中药复方组(方1、方2、方3)、病毒对照组和空白对照组.人工感染鸡新城疫病毒(NDV)后,观察鸡群的发病情况,并于试验各期随机心脏采血,测定外周血淋巴细胞转化和血清抗体、白介素-2(IL-2)、白介素-6(IL-6)、干扰素-γ(IFN-γ)、甲状腺素(T4)及胰高血糖素(Glu)的水平.结果表明:中药复方能延缓鸡新城疫的发病时间,减轻病理损伤,促进病鸡康复,有效降低鸡的死亡率,尤以方3效果最好.方1和方3能快速提高抗体水平;方1能提升血清IFN-γ和T4的水平,方2能提高血清IL-2的含量,方3则可提高血清IL-2、IL-6和T4的水平,3个中药复方都可降低鸡死亡高峰期血清IL-6和Glu的水平.研究表明,中药复方能快速刺激免疫鸡体内体液免疫应答,并通过免疫内分泌整体调节途径以抵抗NDV感染,不同组方原则的复方调节途径可能也不一致,但方3的效果整体上优于方1和方2.     

DNA double-strand breaks trigger genome-wide sister-chromatid cohesion through Eco1 (Ctf7)

Faithful chromosome segregation and repair of DNA double-strand breaks (DSBs) require cohesin, the protein complex that mediates sister-chromatid cohesion. Cohesion between sister chromatids is thought to be generated only during ongoing DNA replication by an obligate coupling between cohesion establishment factors such as Eco1 (Ctf7) and the replisome. Using budding yeast, we challenge this model by showing that cohesion is generated by an Eco1-dependent but replication-independent mechanism in response to DSBs in G(2)/M. Furthermore, our studies reveal that Eco1 has two functions: a cohesive activity and a conserved acetyltransferase activity, which triggers the generation of cohesion in response to the DSB and the DNA damage checkpoint. Finally, the DSB-induced cohesion is not limited to broken chromosomes but occurs also on unbroken chromosomes, suggesting that the DNA damage checkpoint through Eco1 provides genome-wide protection of chromosome integrity.     

A conserved checkpoint monitors meiotic chromosome synapsis in Caenorhabditis elegans

We report the discovery of a checkpoint that monitors synapsis between homologous chromosomes to ensure accurate meiotic segregation. Oocytes containing unsynapsed chromosomes selectively undergo apoptosis even if a germline DNA damage checkpoint is inactivated. This culling mechanism is specifically activated by unsynapsed pairing centers, cis-acting chromosome sites that are also required to promote synapsis in Caenorhabditis elegans. Apoptosis due to synaptic failure also requires the C. elegans homolog of PCH2, a budding yeast pachytene checkpoint gene, which suggests that this surveillance mechanism is widely conserved.     

DTA-6和S3307对大豆存留荚和脱落荚生理调控的效应

【目的】在半干旱地区,研究植物生长促进型和延缓型调节剂作用下,大豆花荚发育过程中存留和脱落荚生理效应差别,探讨调节剂减少大豆脱落作用的生理效应,为提高大豆产量寻找途径。【方法】试验于2012-2013年在黑龙江省大庆市林甸县,始花期（R1）对3个大豆品种绥农28（SN28）、垦丰16(KF16)、合丰50（HF50）分别叶面喷施调节剂2-N、N-二乙氨基乙基己酸酯（diethyl aminoethyl hexanoate,DTA-6）和烯效唑（uniconazole,S3307）,喷施清水作为对照（CK）。从喷药后第35天（R5）开始第1次取样,每隔7 d取样1次。采集存留和脱落的花荚,并将荚皮与籽粒分开,液氮速冻30 min,取出置于低温冰柜中（-40℃）,待全部样品收集完毕,统一测定。比较研究各处理中存留和脱落荚中氧自由基代谢、相关脱落酶、可溶性物质等生理指标的调控效应。【结果】（1）随着荚发育进程,脱落荚中丙二醛（methane dicarboxylic aldehyde,MDA）含量、超氧化物歧化酶（superoxide dismutase,SOD）活性、过氧化物酶（peroxidase,POD）活性、可溶性糖含量、可溶性蛋白质含量显著高于存留荚,脱落纤维素酶（abscission cellulose,AC）、多聚半乳糖醛酸酶（polygalacturonase,PG）活性显著低于存留荚。（2）DTA-6和S3307都能够调控大豆存留荚和脱落荚生理,二者过程不同,结果相似。二者调控效应表现为：总体上降低了存留荚MDA含量,提高了SOD、POD活性,效果S3307优于DTA-6,在绥农28和垦丰16鼓粒中早期,合丰50鼓粒后期阶段性的降低了存留荚AC活性、PG活性。存留荚和脱落荚生理差别随着荚的生育进程,随之变化,不同荚生育时期,差异幅度不同。存留荚和脱落荚生理指标的差异也受品种内在遗传因素影响。【结论】脱落后的荚生理指标状态与存留荚相比表现为质膜过氧化作用增强,保护酶系统的平衡被破坏,可溶性物质增加,脱落酶活性降低可能受脱落过程环境影响较大。脱落荚和存留荚生理相关指标存在差异,并受荚发育时期和品种影响。DTA-6和S3307调控后存留荚具有较强的生理调控和自我修复能力,其膜损伤、渗透调节、保护酶系统、脱落酶活性降低等均对降低脱落有积极的响应。     

Checkpoint proteins control survival of the postmitotic cells in Caenorhabditis elegans

Checkpoints are evolutionarily conserved signaling mechanisms that arrest cell division and alter cellular stress resistance in response to DNA damage or stalled replication forks. To study the consequences of loss of checkpoint functions in whole animals, checkpoint genes were inactivated in the nematode C. elegans. We show that checkpoint proteins are not only essential for normal development but also determine adult somatic maintenance. Checkpoint proteins play a role in the survival of postmitotic adult cells.     

53BP1, a mediator of the DNA damage checkpoint

53BP1 binds to the tumor suppressor protein p53 and has a potential role in DNA damage responses. We used small interfering RNA (siRNA) directed against 53BP1 in mammalian cells to demonstrate that 53BP1 is a key transducer of the DNA damage checkpoint signal. 53BP1 was required for p53 accumulation, G2-M checkpoint arrest, and the intra-S-phase checkpoint in response to ionizing radiation. 53BP1 played a partially redundant role in phosphorylation of the downstream checkpoint effector proteins Brca1 and Chk2 but was required for the formation of Brca1 foci in a hierarchical branched pathway for the recruitment of repair and signaling proteins to sites of DNA damage.     

Analysis of Calcium Content, Hormones, and Degrading Enzymes in Tomato Pedicel Explants During Calcium-Inhibited Abscission

This study was designed to analyze the changes of phytohormone concentrations, calcium fraction, and the activities of degrading enzymes during calcium-inhibited and ethyleneglycol-bis-（β-aminoethyl ether）N, N＇-tetraacetic acid （EGTA）-induced abscission of tomato pedicel explants. Added calcium caused an increase in the total calcium content within the abscission zone and produced a corresponding reduction （20%） in pedicel explant abscission. As expected, EGTA treatment produced the opposite effect and resulted in a decrease in the total calcium content, while accelerating abscission of pedicel explants. Hormone analysis revealed that indole-3-acetic acid （IAA） concentrations in the abscission zone first decreased and then increased before the occurrence of abscission in all treatments, with the greatest effect produced by addition of EGTA. Similarly, abscisic acid （ABA）, and gibberellin （GA1＋3） concentrations, and ethylene production were elevated in the abscission zone during the first 16 h before abscission when explants imbibed EGTA. With calcium treatment, the concentrations of ABA, ethylene, and GA1＋3 also increased in pedicels throughout the first 16 h following exposure, but the increase was slower and less dramatic than with EGTA. Both cellulase and polygalacturonase were induced in the explants during abscission and the activities were also strengthened by treatment with EGTA. Calciumtreated explants produced lower hydrolysing enzyme activities than controls throughout abscission. Calcium played a role of mediating hormone balance and degrading enzymes activities and affected on abscission.     

Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes

The function of the ATR (ataxia-telangiectasia mutated- and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA. Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner. A checkpoint-deficient mutant of RPA, rfa1-t11, is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro. Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.     

棉花蕾铃离层脱落相关基因的克隆及其表达分析

[目的]研究激素与棉花蕾铃脱落的关系,为解析离层脱落相关基因的表达、调控及功能鉴定提供基础资料.[方法]以50 mg/L赤霉素(GA3)和250 mg/L乙烯利(CEPA)分别处理的棉花蕾铃离层cDNA为Tester,H2O处理的棉花蕾铃离层cDNA为Driver,利用抑制消减杂交(SSH)技术构建经GA3和乙烯处理后的消减文库,反向Northern blo-tting筛选出离层差异表达的EST序列,然后进行基因功能注释与功能分类,并分析蕾铃离层差异表达基因的组织表达模式和GA3诱导表达规律.[结果]以GA3处理的离层cDNA为Tester、H2O处理的离层cDNA为Driver,所构建的cDNA文库命名为GA文库;以CEPA处理的离层cDNA为Tester、H2O处理的离层cDNA为Driver,所构建的cDNA文库命名为CE文库.从构建的两个消减文库(GA和CE)中共筛选出29个在离层差异表达的EST序列,通过基因功能注释与功能分类,可将这些EST序列分为翻译相关、代谢相关、信号传导、转录相关、蛋白合成、能量代谢、抗逆相关、基因组序列和未知功能等九大类,其中与翻译相关的EST序列占24.14%,说明经激素处理后棉花蕾铃离层有大量的蛋白合成.从消减文库中挑选10个差异表达基因(EST序列)进行RT-PCR分析,结果发现以GA3处理棉花蕾铃离层后部分相关基因的表达模式会发生变化.[结论]棉花蕾铃离层经激素处理后能激活脱落相关基因的表达,进而影响器官脱落,是其对逆境胁迫反应的结果.     

乙烯代谢途径中基因表达与器官脱落研究进展

器官脱落是一种由各种因素共同调节的复杂生理过程,多种激素对其都有影响,其中乙烯对脱落的影响最大.乙烯含量的上升和器官脱落正相关,但乙烯含量的上升和器官脱落之间并非相邻的两个步骤.器官脱落首先须使植物体本身乙烯合成量增加,之后通过乙烯受体基因和乙烯进行结合,再通过乙烯信号传递体把乙烯上升信号传递下去,从而引起离区细胞壁水解酶活性增强和基因表达量上升,进而导致离区细胞壁发生破碎,最终导致器官脱落.综述了乙烯代谢途径中的乙烯合成、膜上乙烯受体、乙烯膜内信号传递体与器官脱落的研究进展.     

柑橘CitPG34的克隆、定位与表达分析

【目的】 多聚半乳糖醛酸酶是一类参与细胞壁降解的水解酶,在植物生长发育和器官脱落过程中发挥着重要作用。本研究克隆柑橘CitPG34及其启动子（CitPG34-P）并进行表达分析,为深入研究柑橘PG在幼果脱落过程的生物功能奠定基础。【方法】 以‘塔罗科’血橙（Citrus sinensis L. Osbeck）为材料,克隆CitPG34及其启动子,利用ProtParam、Cello、CLUSTALX、MEGA5.2、PlantCARE等软件对其蛋白特性及启动子顺式作用元件进行分析预测;利用实时荧光定量PCR（qRT-PCR）分析CitPG34在不同组织以及柑橘幼果脱落过程中的表达水平。采用同源重组的方法构建pCAMBIA1302-CitPG34-GFP融合蛋白表达载体和CitPG34启动子表达载体（CitPG34-P::gus）,分别用于亚细胞定位和启动子活性分析。【结果】 从‘塔罗科’血橙幼果离层中克隆获得CitPG34,其ORF为1 194 bp,编码397个氨基酸,预测蛋白分子量为41.47 kD,理论等电点为5.19,其不稳定系数为30.23,表明CitPG34属于稳定蛋白;通过在线软件TMHMM分析发现：CitPG34为跨膜蛋白,具有一个跨膜结构,位于第7—29位氨基酸之间。在CitPG34二级结构中,α-螺旋结构约占15.37%,扩展链约占29.72%,无规则卷曲约占54.91%,与其三级结构预测基本一致。NJ树分析显示CitPG34与西洋梨PcPG3（BAF42034）亲缘关系最近,表明CitPG34可能与果实脱落和软化相关。qRT-PCR分析表明,CitPG34在花中表达量最高,在根、叶、离层A、离层C中表达量较低,在幼果中几乎不表达。1-氨基环丙烷羧酸（ACC）处理果梗后能显著提高离层A中CitPG34的表达水平,相反IAA抑制其转录。此外,在柑橘幼果正常脱落过程中,CitPG34表达明显升高。亚细胞定位发现,CitPG34主要位于细胞壁。克隆获取CitPG34起始密码子（ATG）前2 075 bp启动子序列（CitPG34-P）,PlantCare预测发现,在CitPG34-P序列上存在多种顺式调控元件,如核心启动元件TATA-box、增强子元件CAAT-box以及脱落酸响应元件ABRE等。将CitPG34-P::gus转入烟草,通过GUS组织化学染色发现,该启动子受乙烯诱导,主要在叶脉和毛状体中表达。【结论】 CitPG34的ORF长度为1 194 bp,可编码397个氨基酸,其蛋白主要位于细胞壁;该基因具有明显的组织特异性,在花中表达最高;CitPG34表达量与柑橘幼果脱落显著相关。上述结果表明,CitPG34在柑橘幼果脱落和花发育过程中可能发挥着重要的生物功能。     

Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response

The BRCT repeats of the breast and ovarian cancer predisposition protein BRCA1 are essential for tumor suppression. Phosphopeptide affinity proteomic analysis identified a protein, Abraxas, that directly binds the BRCA1 BRCT repeats through a phospho-Ser-X-X-Phe motif. Abraxas binds BRCA1 to the mutual exclusion of BACH1 (BRCA1-associated C-terminal helicase) and CtIP (CtBP-interacting protein), forming a third type of BRCA1 complex. Abraxas recruits the ubiquitin-interacting motif (UIM)-containing protein RAP80 to BRCA1. Both Abraxas and RAP80 were required for DNA damage resistance, G(2)-M checkpoint control, and DNA repair. RAP80 was required for optimal accumulation of BRCA1 on damaged DNA (foci) in response to ionizing radiation, and the UIM domains alone were capable of foci formation. The RAP80-Abraxas complex may help recruit BRCA1 to DNA damage sites in part through recognition of ubiquitinated proteins.     

Separase regulates INCENP-Aurora B anaphase spindle function through Cdc14

The inner centromere-like protein (INCENP) forms a complex with the evolutionarily conserved family of Aurora Bkinases. The INCENP-Aurora complex helps coordinate chromosome segregation, spindle behavior, and cytokinesis during mitosis. INCENP-Aurora associates with kinetochores in metaphase and with spindle microtubules in anaphase, yet the trigger for this abrupt transfer is unknown. Here we show that the conserved phosphatase Cdc14 regulated the yeast INCENP-Aurora complex, Sli15-Ipl1. Cdc14 dephosphorylated Sli15 and thereby directed the complex to spindles. Activation of Cdc14 by separase was sufficient for Sli15 dephosphorylation and relocalization. Cdc14 not only regulates mitotic exit but also modulates spindle midzone assembly through Sli15-Ipl1.