中心体蛋白Centlein的敲除促进细胞周期进程（英文）

中心体是大部分动物细胞的微管组织中心,它确保了有序的细胞周期进程以及染色体的精确分离,我们之前报道了中心体蛋白Centlein作为一个分子连接,与C-Nap1和Cep68一起形成复合物维持中心体的连接.然而,关于Centlein的其他功能我们还知之甚少.在本研究中,建立了Centlein的敲除细胞系,并且运用RNA-seq技术分析了敲除细胞系和正常野生型细胞系之间转录水平的差异.发现Centlein敲除细胞系中细胞周期相关基因PLK1、CCNB1、CCNA2和CDC20的表达量上调,流式结果又表明Centlein的敲除促进了细胞周期进程.同时发现Centlein与PLK1之间存在细胞内相互作用,于是我们提出了Centlein通过与PLK1的作用参与细胞周期进程.     

敲除LTA4H基因的PK-15细胞系构建及其对口蹄疫病毒复制的影响

【目的】利用CRISPR/Cas9基因编辑技术构建白三烯A4水解酶(leukotriene A4 hydrolase, LTA4H)基因缺失的猪肾细胞系(porcine kidney-15, PK-15),探究LTA4H对口蹄疫病毒(foot-and-mouth disease virus, FMDV)复制的影响,为开展LTA4H功能研究及调控病毒复制机制研究提供理论依据。【方法】设计2条针对猪LTA4H基因的引导RNA (small guide RNA, sgRNA),分别构建至载体pX459-puro-MCS中;将CRISPR重组质粒转染PK-15细胞,用嘌呤霉素(puromycin)抗生素筛选,并通过有限稀释法筛选单克隆细胞,之后通过Western blotting和测序检测LTA4H基因的敲除,获得LTA4H基因功能缺失细胞系。使用Western blotting、RT-qPCR及病毒滴度测定等方法检测敲除LTA4H基因后对FMDV复制及相关蛋白的表达情况。【结果】获得的单克隆敲除细胞系与野生型细胞相比,能够显著抑制FMDV复制。【结论】本研究成功构建了LTA4H基因敲除的PK-15细胞系,证明LTA4H对FMDV的复制具有促进作用,研究结果为后续LTA4H功能研究提供理论依据。     

杆状病毒凋亡抑制基因IAP1促进家蚕CyclinB的核内积累

[目的]家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)是生产上危害最严重的病原之一。BmNPV感染BmN-SWU1细胞将细胞周期阻滞于G2/M期。CyclinB是调控细胞周期G2期向M期转换的重要细胞周期蛋白。因此,研究BmNPV感染后CyclinB变化对解析病毒调控细胞周期的机制具有重要意义,同时探究这个过程中与CyclinB互作的病毒蛋白,可为构建家蚕转基因品系提供分子靶标。[方法]qRT-PCR检测BmNPV感染后BmCyclinB的表达变化;免疫荧光观察病毒感染前后BmCyclinB的定位变化,通过细胞质细胞核蛋白分离实验验证。免疫共沉淀钓取与BmCyclinB互作的病毒蛋白。BmNPV感染期间敲除BmNPV IAP1观察BmCyclinB的入核比例。[结果]BmNPV感染后BmCyclinB转录水平下调。BmNPV感染前BmCyclinB主要定位于细胞质,而感染后主要定位于细胞核。BmNPV感染BmN-SWU1细胞后促进BmCyclinB在核内积累。共钓取了7个与BmCyclinB互作的病毒蛋白,免疫共沉淀和细胞共定位证明BmNPV IAP1与BmCyclinB之间存在相互作用。敲除BmNPV IAP1后BmCyclinB进入细胞核的数量显著减少。[结论]BmNPV IAP1可通过与BmCyclinB互作,促进BmCyclinB在核内积累。     

单增李斯特菌vip基因缺失

单增李斯特菌(Listeria monocytogenes)是广泛存在于自然界及食物中的食源性致病菌,作为胞内寄生菌,它可以引起强烈的细胞免疫,是潜在的优良疫苗载体。vip是单增李斯特菌的毒力基因,与其侵袭能力密切相关。因此构建vip基因敲除株可为单增李斯特菌疫苗载体的研发打下重要基础。从单增李斯特菌EGDe基因组中扩增出vip基因上、下游序列,连接到穿梭载体pKSV7中得到敲除载体pKSV7-Δvip,将其以电穿孔的方式转入单增李斯特菌后,通过同源重组利用氯霉素和温度双重压力筛选得到vip基因的敲除突变株,并对敲除菌株的生长曲线进行分析发现vip敲除对细菌的生长没有显著影响,为进一步研究vip基因功能、单增李斯特的致病机制和疫苗载体的研发提供参考。     

CRISPR/Cas9介导的USP30基因敲除细胞系的建立及其对塞内卡病毒增殖的影响

【目的】利用规律成簇的间隔短回文重复序列/Cas9核酸酶（clustered regularly interspaced short palindromic repeats/Cas9 nuclease,CRISPR/Cas9）技术建立USP30基因敲除的人胚胎肾细胞（human embryonic kidney 293T cells,HEK-293T）细胞系,为开展宿主泛素特异性蛋白酶30（ubiquitin-specific protease 30,USP30）蛋白的功能研究建立了细胞模型;同时,初步探究USP30蛋白在病毒感染过程中的作用。【方法】根据Ensemble数据库查询USP30基因序列,定位USP30在基因组中不同转录本重叠区的第一个外显子段,设计并合成2对引导RNA （single guide RNAs,sgRNA）,分别构建在pX459载体中;将pX459-USP30-sgRNA质粒转染HEK-293T细胞,并用嘌呤霉素处理,筛选出转染阳性的细胞,然后通过有限稀释法筛选单克隆细胞,通过Western blotting及测序检测USP30基因的敲除。通过Western blotting及实时荧光定量PCR分析比较塞内卡病毒（Senecavirus A,SVA）在野生型和USP30基因敲除HEK-293T细胞中的复制差异。【结果】Western blotting及测序证实USP30基因敲除单克隆细胞系构建成功。进一步实验发现,SVA在USP30基因敲除细胞中的复制水平显著低于野生型细胞。【结论】成功构建USP30基因敲除的HEK-293T细胞系,首次证明USP30对SVA的复制具有促进作用,为进一步揭示USP30相关免疫反应和SVA感染过程的作用机制提供了良好的细胞模型,也为开展宿主USP30蛋白调控病毒复制的机制研究提供了关键工具和一定的理论依据。     

利用CRISPR-Cas9技术构建敲除VDH的产香兰素拟无枝酸菌

[目的] 建立适用于拟无枝酸菌（Amycolatopsis sp.）的CRISPR-Cas9基因编辑系统,敲除其编码香兰素脱氢酶基因（VDH）,减少发酵副产物香草酸。[方法] 以VDH为靶标基因,将pKCcas9dO质粒上的tipA、j23119启动子分别替换为pRLE6质粒Km^r启动子、链霉菌中常用的强启动子permE*,同时将sgRNA替换为能识别靶基因香兰素脱氢酶的特异性sgRNA,获得质粒pKCKmCas9VDH。然后将其与靶基因的上下游同源臂连接,获得敲除质粒pLYZYP01。将pLYZYP01质粒电转进Amycolatopsis sp.感受态细胞,筛选获得VDH的敲除突变体菌株。[结果] 利用上述方法,成功获得VDH敲除菌株Amycolatopsis sp.ΔVDH。[结论] 建立了适用于拟无枝酸菌CCTCC M 2011265的基因敲除系统,成功敲除VDH基因,在添加12 g/L底物阿魏酸的情况下,香兰素产量达到9.19 g/L,摩尔转化率由88.6%提高到97.7%。     

斜纹夜蛾核多角体病毒抑制苜蓿丫纹夜蛾核多角体病毒诱导的斜纹夜蛾细胞凋亡

野生型苜蓿丫纹夜蛾核多角体病毒(Autographa californica multicapsid nucleopolyhedrovirus, AcMNPV)感染斜纹夜蛾(Spodoptera litura)细胞系Sl-zsu-1,可引起典型的细胞凋亡;但可以在草地夜蛾(Spodoptera frugiperda)细胞Sf-9中复制并形成多角体.比较了AcMNPV p35基因在病毒感染两种细胞的复制和转录情况,认为p35在非受纳细胞中及时有效的表达能阻止细胞发生凋亡;共感染实验结果表明,斜纹夜蛾核多角体病毒(Spodoptera litura multicapsid nucleopolyhedrovirus, SpltMNPV)可以抑制AcMNPV诱导的细胞凋亡并可帮助病毒进行复制,推测SpltMNPV基因组中与p35同源的p49基因挽救了细胞的自杀行为.     

金黄色葡萄球菌tst-1基因敲除菌株的构建

抽提金黄色葡萄球菌834菌株的基因组DNA,PCR克隆扩增tst-1及tst-1的上、下游基因,通过将tst-1上、下游基因分别重组到载体质粒pAULA中,形成同源重组质粒pAULA Δtst-1,将pAULA-Δtst-1电转入细菌内,进行同源重组,以PCR、Western blot鉴定tst-1基因敲除菌株无tst-1基因片段,且无TSST-1蛋白表达,表明已成功构建金黄色葡萄球菌tst-1基因的敲除菌株。     

p14ARF对人黑色素瘤细胞增殖的影响及其作用机理的初探

ARF(alternative reading frame)作为INK4a/ARF的β转录产物,能够稳定p53, 诱导细胞周期阻断或凋亡.利用高表达p14^ARF的人黑色素瘤细胞模型,探讨了ARF抑制细胞增殖的分子作用机理.研究发现p14^ARF高表达能将细胞周期阻断在G1和G2期, p53, p21^cip1和p27^kip1蛋白水平明显增强, 而p-ERK1/2,CyclinD1和CyclinE蛋白水平下降, 明显抑制细胞生长. 提示p14^ARF能通过ERK(extracellular signal-regulated kinase)信号通路相互协调作用抑制A375细胞增殖.     

RNAi介导珠子参PjCAS基因沉默对皂苷合成的影响

该研究利用Gateway技术构建珠子参环阿屯醇合成酶基因(Panax japonicus cycloartenol synthase,PjCAS)的RNAi表达载体,利用农杆菌介导转化在珠子参细胞中成功实现了PjCAS 的RNA干扰;采用实时荧光定量PCR分析珠子参皂苷生物合成途径中关键酶基因的表达情况,同时检测转基因细胞中皂苷和植物甾醇含量的变化,探讨PjCAS基因对珠子参皂苷合成的调控作用。结果表明：(1)成功获得PjCAS基因的RNAi片段,并成功构建了PjCAS基因RNAi载体pHellsgate PjCAS。(2)经农杆菌遗传转化,获得6株实现PjCAS基因RNA干扰的转基因阳性细胞系。(3)与普通细胞系相比,转基因细胞系中PjCAS基因的表达量大约下降了85%,同时与珠子参皂苷合成直接相关的关键酶基因PjDS、PjAS表达量最高分别上调了90%和150%。(4)转基因细胞系中6种单体皂苷的含量均显著高于对照组,其中达玛烷型单体皂苷Re、Rb1、Rd和齐墩果烷型单体皂苷R0、IV、IVa的平均含量比普通珠子参细胞系分别提高了28%、49%、40%、36%、59%、50%。说明珠子参皂苷含量的变化受PjCAS基因的间接调控。(5)6株转基因细胞系中植物甾醇含量较对照显著降低了53%~73%。研究发现,沉默PjCAS基因可促进珠子参皂苷合成的关键酶基因PjDS、PjAS显著上调表达,并提高转PjCAS基因细胞系中单体皂苷的含量,从而促进了珠子参皂苷合成量的显著增加,证明通过抑制植物甾醇合成通路关键基因PjCAS的表达可以有效降低植物甾醇合成支路的代谢通量,使更多的代谢流朝着珠子参皂苷合成方向流动,最终促进了珠子参皂苷的生物合成。     

PLK1 phosphorylation of pericentrin initiates centrosome maturation at the onset of mitosis

The microtubule-organizing activity of the centrosome oscillates during the cell cycle, reaching its highest level at mitosis. At the onset of mitosis, the centrosome undergoes maturation, which is characterized by a drastic expansion of the pericentriolar matrix (PCM) and a robust increase in microtubule-organizing activity. It is known that PLK1 is critical for the initiation of centrosome maturation. In this paper, we report that pericentrin (PCNT), a PCM protein, was specifically phosphorylated by PLK1 during mitosis. Phosphoresistant point mutants of PCNT did not recruit centrosomal proteins, such as CEP192, GCP-WD (γ-complex protein with WD repeats), γ-tubulin, Aurora A, and PLK1, into the centrosome during mitosis. However, centrosomal recruitment of CEP215 depended on PCNT irrespective of its phosphorylation status. Furthermore, ectopic expression of PLK1-PCNT fusion proteins induced the centrosomal accumulation of CEP192, GCP-WD, and γ-tubulin even in interphase cells, mimicking centrosome maturation. Based on these results, we propose that PLK1-mediated phosphorylation of PCNT initiates centrosome maturation by organizing the spindle pole-specific PCM lattice.     

Pharmacological inhibition of Polo-like kinase 1 (PLK1) by BI-2536 decreases the viability and survival of hamartin and tuberin deficient cells via induction of apoptosis and attenuation of autophagy

The mechanistic target of rapamycin complex 1 (mTORC1) increases translation, cell size and angiogenesis, and inhibits autophagy. mTORC1 is negatively regulated by hamartin and tuberin, the protein products of the tumor suppressors TSC1 and TSC2 that are mutated in Tuberous Sclerosis Complex (TSC) and sporadic Lymphangioleiomyomatosis (LAM). Hamartin interacts with the centrosomal and mitotic kinase polo-like kinase 1 (PLK1). Hamartin and tuberin deficient cells have abnormalities in centrosome duplication, mitotic progression, and cytokinesis, suggesting that the hamartin/tuberin heterodimer and mTORC1 signaling are involved in centrosome biology and mitosis. Here we report that PLK1 protein levels are increased in hamartin and tuberin deficient cells and LAM patient-derived specimens, and that this increase is rapamycin-sensitive. Pharmacological inhibition of PLK1 by the small-molecule inhibitor BI-2536 significantly decreased the viability and clonogenic survival of hamartin and tuberin deficient cells, which was associated with increased apoptosis. BI-2536 increased p62, LC3B-I and GFP-LC3 punctae, and inhibited HBSS-induced degradation of p62, suggesting that PLK1 inhibition attenuates autophagy. Finally, PLK1 inhibition repressed the expression and protein levels of key autophagy genes and proteins and the protein levels of Bcl^-2 family members, suggesting that PLK1 regulates both autophagic and apoptotic responses. Taken together, our data point toward a previously unrecognized role of PLK1 on the survival of cells with mTORC1 hyperactivation, and the potential use of PLK1 inhibitors as novel therapeutics for tumors with dysregulated mTORC1 signaling, including TSC and LAM.     

Proteomic and functional analysis of the mitotic Drosophila centrosome

Regulation of centrosome structure, duplication and segregation is integrated into cellular pathways that control cell cycle progression and growth. As part of these pathways, numerous proteins with well‐established non‐centrosomal localization and function associate with the centrosome to fulfill regulatory functions. In turn, classical centrosomal components take up functional and structural roles as part of other cellular organelles and compartments. Thus, although a comprehensive inventory of centrosome components is missing, emerging evidence indicates that its molecular composition reflects the complexity of its functions. We analysed the Drosophila embryonic centrosomal proteome using immunoisolation in combination with mass spectrometry. The 251 identified components were functionally characterized by RNA interference. Among those, a core group of 11 proteins was critical for centrosome structure maintenance. Depletion of any of these proteins in Drosophila SL2 cells resulted in centrosome disintegration, revealing a molecular dependency of centrosome structure on components of the protein translation machinery, actin‐ and RNA‐binding proteins. In total, we assigned novel centrosome‐related functions to 24 proteins and confirmed 13 of these in human cells.     

Pharmacological inhibition of Polo-like kinase 1 (PLK1) by BI-2536 decreases the viability and survival of hamartin and tuberin deficient cells via induction of apoptosis and attenuation of autophagy

The mechanistic target of rapamycin complex 1 (mTORC1) increases translation, cell size and angiogenesis, and inhibits autophagy. mTORC1 is negatively regulated by hamartin and tuberin, the protein products of the tumor suppressors TSC1 and TSC2 that are mutated in Tuberous Sclerosis Complex (TSC) and sporadic Lymphangioleiomyomatosis (LAM). Hamartin interacts with the centrosomal and mitotic kinase polo-like kinase 1 (PLK1). Hamartin and tuberin deficient cells have abnormalities in centrosome duplication, mitotic progression, and cytokinesis, suggesting that the hamartin/tuberin heterodimer and mTORC1 signaling are involved in centrosome biology and mitosis. Here we report that PLK1 protein levels are increased in hamartin and tuberin deficient cells and LAM patient-derived specimens, and that this increase is rapamycin-sensitive. Pharmacological inhibition of PLK1 by the small-molecule inhibitor BI-2536 significantly decreased the viability and clonogenic survival of hamartin and tuberin deficient cells, which was associated with increased apoptosis. BI-2536 increased p62, LC3B-I and GFP-LC3 punctae, and inhibited HBSS-induced degradation of p62, suggesting that PLK1 inhibition attenuates autophagy. Finally, PLK1 inhibition repressed the expression and protein levels of key autophagy genes and proteins and the protein levels of Bcl^-2 family members, suggesting that PLK1 regulates both autophagic and apoptotic responses. Taken together, our data point toward a previously unrecognized role of PLK1 on the survival of cells with mTORC1 hyperactivation, and the potential use of PLK1 inhibitors as novel therapeutics for tumors with dysregulated mTORC1 signaling, including TSC and LAM.     

Centrosomal Chk2 in DNA damage responses and cell cycle progession

Two major control systems regulate early stages of mitosis: activation of Cdk1 and anaphase control through assembly and disassembly of the mitotic spindle. In parallel to cell cycle progression, centrosomal duplication is regulated through proteins including Nek2. Recent studies suggest that centrosome-localized Chk1 forestalls premature activation of centrosomal Cdc25b and Cdk1 for mitotic entry, whereas Chk2 binds centrosomes and arrests mitosis only after activation by ATM and ATR in response to DNA damage. Here, we show that Chk2 centrosomal binding does not require DNA damage, but varies according to cell cycle progression. These and other data suggest a model in which binding of Chk2 to the centrosome at multiple cell cycle junctures controls co-localization of Chk2 with other cell cycle and centrosomal regulators.Key words: Chk2, centrosome, checkpoint, DNA damage, wild type, kinase-defective     

PLK2 phosphorylation is critical for CPAP function in procentriole formation during the centrosome cycle

Control of centrosome duplication is tightly linked with the progression of the cell cycle. Recent studies suggest that the fundamental process of centriole duplication is evolutionally conserved. Here, we identified c entrosomal P 4.1‐a ssociated p rotein (CPAP), a human homologue of SAS‐4, as a substrate of PLK2 whose activity oscillates during the cell cycle. PLK2 phosphorylates the S589 and S595 residues of CPAP in vitro and in vivo. This phosphorylation is critical for procentriole formation during the centrosome cycle. PLK4 also phosphorylates S595 of CPAP, but PLK4 phosphorylation is not a critical step in the PLK4 function in procentriole assembly. CPAP is phosphorylated in a cell cycle stage‐specific manner, so that its phosphorylation increases at the G1/S transition phase and decreases during the exit of mitosis. Phosphorylated CPAP is preferentially located at the procentriole. Furthermore, overexpression of a phospho‐resistant CPAP mutant resulted in the failure to form elongated centrioles. On the basis of these results, we propose that phosphorylated CPAP is involved in procentriole assembly, possibly for centriole elongation. This work demonstrates an example of how procentriole formation is linked to the progression of the cell cycle.     

Geminin is partially localized to the centrosome and plays a role in proper centrosome duplication

Background information. Centrosome duplication normally parallels with DNA replication and is responsible for correct segregation of replicated DNA into the daughter cells. Although geminin interacts with Cdt1 to prevent loading of MCMs (minichromosome maintenance proteins) on to the replication origins, inactivation of geminin nevertheless causes centrosome over‐duplication in addition to the re‐replication of the genome, suggesting that geminin may play a role in centrosome duplication. However, the exact mechanism by which loss of geminin affects centrosomal duplication remains unclear and the possible direct interaction of geminin with centrosomal‐localized proteins is still unidentified. Results. We report in the present study that geminin is physically localized to the centrosome. This unexpected geminin localization is cell‐cycle dependent and mediated by the actin‐related protein, Arp1, one subunit of the dynein—dynactin complex. Disruption of the integrity of the dynein—dynactin complex by overexpression of dynamitin/p50, a well‐characterized inhibitor of dynactin, reduces the centrosomal localization of both geminin and Arp1. Enrichment of geminin on centrosomes was enhanced when cellular ATP production was suppressed in the ATP‐inhibitor assay, whereas the accumulation of geminin on the centrosome was disrupted by depolymerization of the microtubules using nocodazole. We further demonstrate that the coiled‐coil motif of geminin is required for its centrosomal localization and the interaction of geminin with Arp1. Depletion of geminin by siRNA (small interfering RNA) in MDA‐MB‐231 cells led to centrosome over‐duplication. Conversely, overexpression of geminin inhibits centrosome over‐duplication induced by HU in S‐phase‐arrested cells, and the coiled‐coil‐motif‐mediated centrosomal localization of geminin is required for its inhibition of centrosome over‐duplication. Centrosomal localization of geminin is conserved among mammalian cells and geminin might perform as an inhibitor of centrosome duplication. Conclusions. The results of the present study demonstrate that a fraction of geminin is localized on the centrosome, and the centrosomal localization of geminin is Arp1‐mediated and dynein—dynactin‐dependent. The coiled‐coil motif of geminin is required for its targeting to the centrosome and inhibition of centrosome duplication. Thus the centrosomal localization of geminin might perform an important role in regulation of proper centrosome duplication.     

Centrobin/NIP2 Is a Microtubule Stabilizer Whose Activity Is Enhanced by PLK1 Phosphorylation during Mitosis

Centrobin/NIP2 is a centrosomal protein that is required for centrosome duplication. It is also critical for microtubule organization in both interphase and mitotic cells. In the present study, we observed that centrobin is phosphorylated in a cell cycle stage-specific manner, reaching its maximum at M phase. PLK1 is a kinase that is responsible for M phase-specific phosphorylation of centrobin. The microtubule forming activity of centrobin was enhanced by PLK1 phosphorylation. Furthermore, mitotic spindles were not assembled properly with the phospho-resistant mutant of centrobin. Based on these results, we propose that centrobin functions as a microtubule stabilizing factor and PLK1 enhances centrobin activity for proper spindle formation during mitosis.     

CEP215 is involved in the dynein-dependent accumulation of pericentriolar matrix proteins for spindle pole formation

CEP215 is a human orthologue of Drosophila centrosomin which is a core centrosome component for the pericentriolar matrix protein recruitment. Recent investigations revealed that CEP215 is required for centrosome cohesion, centrosomal attachment of the g-TuRC, and microtubule dynamics. However, it remains to be obscure how CEP215 functions for recruitment of the centrosomal proteins during the centrosome cycle. Here, we investigated a role of CEP215 during mitosis. Knockdown of CEP215 resulted in characteristic mitotic phenotypes, including monopolar spindle formation, a decrease in distance between the spindle pole pair, and detachment of the centrosomes from the spindle poles. We noticed that CEP215 is critical for centrosomal localization of dynein throughout the cell cycle. As a consequence, the selective centrosomal proteins were not recruited to the centrosome properly. Finally, the centrosomal localization of CEP215 also depends on the dynein-dynactin complex. Based on the results, we propose that CEP215 regulates a dynein-dependent transport of the pericentriolar matrix proteins during the centrosome maturation.