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Is the model of signal amplification by GPCRs/GEFs activating multiple GTPases relevant to a broad spectrum of heterotrimeric and RAS superfamily GTPases? gpcr / gef激活多个gtpase的信号放大模型是否与广谱异源三聚体和RAS超家族gtpase相关?
Pub Date : 2014-05-01 eCollection Date: 2014-06-01 DOI: 10.4161/21592780.2014.943602
Richard A Kahn

Concepts or models of biological processes shape how we think about them, discuss them, and design experiments to test aspects of them. Because of the importance of our models of cell signaling by regulatory GTPases and the desire to extend those models to related signaling modules, I have throughout my career been fascinated by the similarities and differences between the modeling of heterotrimeric G protein and monomeric RAS superfamily GTPases. Recent discussions with colleagues led me to conclude that there is a growing divergence in how researchers model the activation and signaling processes of monomeric and trimeric GTPases and also a surprising lack of consensus within each camp. This series of articles arose in response to these discussions and is intended to spark new ones.

生物过程的概念或模型塑造了我们如何思考、讨论它们,以及设计实验来测试它们的各个方面。由于我们通过调节gtpase建立细胞信号传导模型的重要性以及将这些模型扩展到相关信号传导模块的愿望,我在整个职业生涯中一直着迷于异源三聚体G蛋白和单体RAS超家族gtpase建模之间的异同。最近与同事的讨论使我得出结论,研究人员在如何模拟单体和三聚体gtp酶的激活和信号传递过程方面存在越来越大的分歧,而且每个阵营都令人惊讶地缺乏共识。本系列文章是对这些讨论的回应,旨在引发新的讨论。
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引用次数: 4
Spatial and temporal control of Rho GTPase functions. Rho GTPase功能的时空控制。
Pub Date : 2014-05-01 eCollection Date: 2014-04-01 DOI: 10.4161/21592780.2014.943618
Konstadinos Moissoglu, Martin Alexander Schwartz

Rho family GTPases control almost every aspect of cell physiology and, since their discovery, a wealth of knowledge has accumulated about their biochemical regulation and function. However, each Rho GTPase distributes between multiple cellular compartments, even within the same cell, where they are controlled by multiple regulators and signal to multiple effectors. Thus, major questions about spatial and temporal aspects of regulation remain unanswered. In particular, what are the nano-scale dynamics for their activation, membrane targeting, diffusion, effector activation and GTPase inactivation? How do these mechanisms differ in the different cellular compartments where Rho GTPases function? Addressing these complex aspects of Rho GTPase biology will significantly advance our understanding of the spatial and temporal control of cellular functions.

Rho家族GTPases几乎控制着细胞生理学的各个方面,自发现以来,人们对其生化调节和功能积累了丰富的知识。然而,每个Rho GTPase分布在多个细胞室之间,甚至在同一个细胞内,它们受到多个调节器的控制,并向多个效应器发出信号。因此,关于调控的空间和时间方面的主要问题仍然没有答案。特别是,它们的活化、膜靶向、扩散、效应物活化和GTPase失活的纳米尺度动力学是什么?这些机制在GTPases发挥作用的不同细胞区室中有何不同?解决Rho GTPase生物学的这些复杂方面将大大促进我们对细胞功能的时空控制的理解。
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引用次数: 18
GEF-effector interactions. GEF-effector交互。
Pub Date : 2014-05-01 eCollection Date: 2014-04-01 DOI: 10.4161/21592780.2014.943616
Catherine L Jackson

Members of the Arf family of small GTP-binding proteins, or GTPases, are activated by guanine nucleotide exchange factors (GEFs) that catalyze GDP release from their substrate Arf, allowing GTP to bind. In the secretory pathway, Arf1 is first activated by GBF1 at the cis-Golgi, then by BIG1 and BIG2 at the trans-Golgi and trans-Golgi network (TGN). Upon activation, Arf1-GTP interacts with effectors such as coat complexes, and is able to recruit different coat complexes to different membrane sites in cells. The COPI coat is primarily recruited to cis-Golgi membranes, whereas other coats, such as AP-1/clathrin, and GGA/clathrin, are recruited to the trans-Golgi and the TGN. Although Arf1-GTP is required for stable association of these various coats to membranes, and is sufficient in vitro, other molecules, such as vesicle cargo and coat receptors on the membrane, contribute to specificity of coat recruitment in cells. Another mechanism to achieve specificity is interaction of effectors such as coats with the GEF itself, which would increase the concentration of a given coat in proximity to the site where Arf is activated, thus favoring its recruitment. This interaction between a GEF and an effector could also provide a mechanism for spatial organization of vesicle budding sites, similar to that described for Cdc42-mediated establishment of polarity sites such as the emerging bud in yeast. Another factor affecting the amount of freely diffusible Arf1-GTP in membranes is the GEF(s) themselves acting as effectors. Sec7p, the yeast homolog of mammalian BIG1 and BIG2, and Arno/cytohesin 2, a PM-localized Arf1 GEF, both bind to Arf1-GTP. This binding to the products of the exchange reaction establishes a positive feedback loop for activation.

小GTP结合蛋白Arf家族的成员,或GTP酶,被鸟嘌呤核苷酸交换因子(gef)激活,该因子催化GDP从其底物Arf释放,使GTP结合。在分泌途径中,Arf1首先被顺式高尔基体上的GBF1激活,然后被反式高尔基和反式高尔基网络(TGN)上的BIG1和BIG2激活。激活后,Arf1-GTP与衣壳复合物等效应物相互作用,并能够将不同的衣壳复合物招募到细胞的不同膜位点。COPI外壳主要被募集到顺式高尔基膜上,而其他外壳,如AP-1/网格蛋白和GGA/网格蛋白,则被募集到反式高尔基膜和TGN上。虽然Arf1-GTP是这些不同的被膜与膜稳定结合所必需的,并且在体外是足够的,但其他分子,如囊泡货物和膜上的被膜受体,有助于细胞内被膜募集的特异性。另一种实现特异性的机制是效应物(如衣壳)与GEF本身的相互作用,这将增加Arf被激活位点附近特定衣壳的浓度,从而有利于其招募。GEF和效应物之间的相互作用也为囊泡出芽位点的空间组织提供了一种机制,类似于cdc42介导的极性位点的建立,如酵母的出芽。影响膜中自由扩散的Arf1-GTP数量的另一个因素是GEF(s)本身作为效应器。哺乳动物BIG1和BIG2的酵母同源物Sec7p和pm定位的Arf1 GEF Arno/cytohesin 2都与Arf1- gtp结合。这种与交换反应产物的结合为激活建立了一个正反馈回路。
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引用次数: 8
Distinct patterns of phosphatidylserine localization within the Rab11a-containing recycling system. 含 Rab11a 循环系统内磷脂酰丝氨酸定位的不同模式。
Pub Date : 2014-04-03 eCollection Date: 2014-01-01 DOI: 10.4161/cl.28680
Nicholas W Baetz, James R Goldenring

The Rab11 GTPases and Rab11 family-interacting proteins (Rab11-FIPs) define integrated yet distinct compartments within the slow recycling pathway. The lipid content of these compartments is less well understood, although past studies have indicated phosphatidylserine (PS) is an integral component of recycling membranes. We sought to identify key differences in the presence of PS within Rab and Rab11-FIP containing membranes. We used live cell fluorescence microscopy and structured illumination microscopy to determine whether the previously published LactC2 probe for PS displays differential patterns of overlap with various Rab GTPases and Rab11-FIPs. Selective overlap was observed between the LactC2 probe and Rab GTPases when co-expressed in HeLa cells. Rab11-FIP1 proteins consistently overlapped with LactC2 along peripheral and pericentriolar compartments. The specificity of Rab11-FIP1 association with LactC2 was further confirmed by demonstrating that additional Rab11-FIPs (FIP2, FIP3, and FIP5) exhibited selective association with LactC2 containing compartments. Live cell dual expression studies of Rab11-FIPs with LactC2 indicated that PS is enriched along tubular compartments of the Rab11a-dependent recycling system. Additionally, we found that the removal of C2 domains from the Rab11-FIPs induced an accumulation of LactC2 probe in the pericentriolar region, suggesting that inhibition of trafficking through the recycling system can influence the distribution of PS within cells. Finally, we confirmed these findings using structured illumination microscopy suggesting that the overlapping fluorescent signals were on the same membranes. These results suggest distinct associations of Rab GTPases and Rab11-FIPs with PS-containing recycling system membrane domains.

Rab11 GTPases 和 Rab11 家族互作蛋白(Rab11-FIPs)定义了慢循环途径中完整而又不同的区室。尽管过去的研究表明磷脂酰丝氨酸(PS)是再循环膜不可或缺的组成部分,但人们对这些区室的脂质含量了解较少。我们试图找出在含有 Rab 和 Rab11-FIP 的膜中 PS 存在的关键差异。我们使用活细胞荧光显微镜和结构照明显微镜来确定之前发表的 LactC2 PS 探针是否与各种 Rab GTPases 和 Rab11-FIPs 显示出不同的重叠模式。在 HeLa 细胞中共同表达时,观察到 LactC2 探针与 Rab GTP 酶之间存在选择性重叠。Rab11-FIP1 蛋白与 LactC2 始终沿外周和皮层周围重叠。通过证明其他 Rab11-FIP(FIP2、FIP3 和 FIP5)表现出与含有 LactC2 的区室的选择性结合,进一步证实了 Rab11-FIP1 与 LactC2 结合的特异性。Rab11-FIPs 与 LactC2 的活细胞双重表达研究表明,PS 富集在依赖 Rab11a 的循环系统的管状区室中。此外,我们还发现,从 Rab11-FIPs 中移除 C2 结构域会诱导 LactC2 探针在细胞周区聚集,这表明抑制通过循环系统的运输会影响 PS 在细胞内的分布。最后,我们使用结构照明显微镜证实了这些发现,表明重叠的荧光信号位于相同的膜上。这些结果表明,Rab GTP酶和Rab11-FIPs与含PS的循环系统膜域有不同的关联。
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引用次数: 0
Altered trafficking of mutated growth factor receptors and their associated molecules: implication for human cancers. 突变生长因子受体及其相关分子的运输改变:对人类癌症的影响。
Pub Date : 2014-03-18 eCollection Date: 2014-01-01 DOI: 10.4161/cl.28461
Shunsuke Kon, Nobuhide Kobayashi, Masanobu Satake

Ligand-stimulated receptor tyrosine kinases (RTKs) are phosphorylated/ubiquitinated, endocytosed and transported to the lysosomes via endosomes/multivesicular bodies, resulting in the attenuation of signal transmission. If this physiological mechanism of RTK signal downregulation is perturbed, signal transduction persists and may contribute to cellular transformation. This article presents several such examples. In some cases, endocytosis is impaired, and the activated RTK remains on the plasma membrane. In other cases, the activated RTK is endocytosed into endosomes/multivesicular bodies, but not subsequently sorted to the lysosomes for degradation. The latter cases indicate that even endocytosed RTKs can transmit signals. Transport of RTKs is accomplished via the formation and movement of membrane vesicles. Blockage or delay of endocytosis/trafficking can be caused by genetic alterations in the RTK itself or by mutations in CBL, Arf GAPs, or other components involved in internalization and vesicle transport. A survey of the literature indicates that, in some cases, even RTKs synthesized de novo can initiate signaling at the endoplasmic reticulum/Golgi before reaching the plasma membrane. The spectrum of molecules targeted by the signal is likely to be different between cell surface- and endoplasmic reticulum/Golgi-localized RTKs.

配体刺激受体酪氨酸激酶(RTKs)被磷酸化/泛素化、内吞并通过内体/多泡体转运到溶酶体,导致信号传递的衰减。如果这种RTK信号下调的生理机制受到干扰,信号转导就会持续存在,并可能导致细胞转化。本文提供了几个这样的例子。在某些情况下,内吞作用受损,激活的RTK留在质膜上。在其他情况下,活化的RTK被内吞到核内体/多泡体中,但随后没有被分类到溶酶体中降解。后一种情况表明,即使是内吞的rtk也可以传递信号。rtk的转运是通过膜泡的形成和运动来完成的。内吞作用/运输的阻滞或延迟可由RTK本身的遗传改变或CBL、Arf GAPs或参与内化和囊泡运输的其他成分的突变引起。文献综述表明,在某些情况下,即使是重新合成的rtk也可以在到达质膜之前在内质网/高尔基体启动信号传导。信号靶向的分子谱在细胞表面和内质网/高尔基定位的rtk之间可能是不同的。
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引用次数: 8
Quantitative Analysis of Guanine Nucleotide Exchange Factors (GEFs) as Enzymes. 鸟嘌呤核苷酸交换因子(GEFs)酶的定量分析。
Pub Date : 2014-01-09 eCollection Date: 2013-01-01 DOI: 10.4161/cl.27609
Paul A Randazzo, Xiaoying Jian, Pei-Wen Chen, Peng Zhai, Olivier Soubias, John K Northup

The proteins that possess guanine nucleotide exchange factor (GEF) activity, which include about ~800 G protein coupled receptors (GPCRs),1 15 Arf GEFs,2 81 Rho GEFs,3 8 Ras GEFs,4 and others for other families of GTPases,5 catalyze the exchange of GTP for GDP on all regulatory guanine nucleotide binding proteins. Despite their importance as catalysts, relatively few exchange factors (we are aware of only eight for ras superfamily members) have been rigorously characterized kinetically.5-13 In some cases, kinetic analysis has been simplistic leading to erroneous conclusions about mechanism (as discussed in a recent review14). In this paper, we compare two approaches for determining the kinetic properties of exchange factors: (i) examining individual equilibria, and; (ii) analyzing the exchange factors as enzymes. Each approach, when thoughtfully used,14,15 provides important mechanistic information about the exchange factors. The analysis as enzymes is described in further detail. With the focus on the production of the biologically relevant guanine nucleotide binding protein complexed with GTP (G•GTP), we believe it is conceptually simpler to connect the kinetic properties to cellular effects. Further, the experiments are often more tractable than those used to analyze the equilibrium system and, therefore, more widely accessible to scientists interested in the function of exchange factors.

具有鸟嘌呤核苷酸交换因子(GEF)活性的蛋白,包括约800 G蛋白偶联受体(gpcr),1 15个Arf GEF,2 81个Rho GEF,3 8个Ras GEF,4和其他gtpase家族的其他蛋白,5在所有调节鸟嘌呤核苷酸结合蛋白上催化GTP与GDP的交换。尽管它们作为催化剂很重要,但相对较少的交换因子(我们知道ras超家族成员只有8个)已经被严格地动力学表征。5-13在某些情况下,动力学分析过于简单,导致对机理的错误结论(如最近的一篇综述所讨论的14)。在本文中,我们比较了确定交换因子动力学性质的两种方法:(i)检查个体平衡;(ii)分析交换因子如酶。每一种方法,如果经过深思熟虑的使用,14,15都提供了关于交换因素的重要机制信息。进一步详细地描述了作为酶的分析。随着重点关注鸟嘌呤核苷酸结合蛋白与GTP复合物(G•GTP)的生产,我们认为将动力学特性与细胞效应联系起来在概念上更简单。此外,这些实验通常比用于分析平衡系统的实验更容易处理,因此,对交换因子功能感兴趣的科学家更容易使用。
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引用次数: 10
Binding of the vesicle docking protein p115 to the GTPase Rab1b regulates membrane recruitment of the COPI vesicle coat. 囊泡对接蛋白p115与GTPase Rab1b的结合调节COPI囊泡外壳的膜募集。
Pub Date : 2014-01-09 eCollection Date: 2013-01-01 DOI: 10.4161/cl.27687
Yusong Guo, Adam D Linstedt

Membrane recruitment of the COPI vesicle coat is fundamental to its function and contributes to compartment identity in the early secretory pathway. COPI recruitment is triggered by guanine nucleotide exchange activating the Arf1 GTPase, but the key exchange factor, GBF1, is a peripheral membrane component whose membrane association is dependent on another GTPase, Rab1. Inactive Rab GTPases are in a soluble complex with guanine nucleotide dissociation inhibitor (GDI) and activation of Rab GTPases by exchange factors can be enhanced by GDI dissociation factors (GDFs). In the present study, we investigated the vesicle docking protein p115 and it's binding to the Rab1 isoform Rab1b. Inhibition of p115 expression induced dissociation of Rab1b from Golgi membranes. Rab1b bound the cc2 domain of p115 and p115 lacking this domain failed to recruit Rab1b. Further, p115 inhibition blocked association of the COPI coat with Golgi membranes and this was suppressed by constitutive activation of Rab1b. These findings show p115 enhancement of Rab1b activation leading to COPI recruitment suggesting a connection between the vesicle docking machinery and the vesicle coat complex during the establishment of post-ER compartment identity.

COPI囊泡外壳的膜募集是其功能的基础,并有助于早期分泌途径中的室识别。COPI的募集是由鸟嘌呤核苷酸交换激活Arf1 GTPase触发的,但关键的交换因子GBF1是外周膜组分,其膜结合依赖于另一种GTPase Rab1。失活的Rab gtpase可与鸟嘌呤核苷酸解离抑制剂(GDI)形成可溶复合物,GDI解离因子(GDFs)可增强Rab gtpase被交换因子激活。在本研究中,我们研究了囊泡对接蛋白p115及其与Rab1异构体Rab1b的结合。抑制p115表达诱导Rab1b从高尔基膜分离。Rab1b结合p115的cc2结构域,缺少该结构域的p115无法招募Rab1b。此外,p115抑制阻断了COPI外壳与高尔基膜的结合,这被Rab1b的组成性激活所抑制。这些发现表明,p115增强Rab1b激活导致COPI募集,这表明在建立er后腔室身份过程中,囊泡对接机制与囊泡外壳复合物之间存在联系。
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引用次数: 6
Multipronged interaction of the COG complex with intracellular membranes. COG复合物与胞内膜的多管齐下相互作用。
Pub Date : 2014-01-01 Epub Date: 2014-02-13 DOI: 10.4161/cl.27888
Rose Willett, Irina Pokrovskaya, Tetyana Kudlyk, Vladimir Lupashin

The conserved oligomeric Golgi complex is a peripheral membrane protein complex that orchestrates the tethering and fusion of intra-Golgi transport carriers with Golgi membranes. In this study we have investigated the membrane attachment of the COG complex and it's on/off dynamic on Golgi membranes. Several complimentary approaches including knock-sideways depletion, FRAP, and FLIP revealed that assembled COG complex is not diffusing from Golgi periphery in live HeLa cells. Moreover, COG subunits remained membrane-associated even in COG4 and COG7 depleted cells when Golgi architecture was severely affected. Overexpression of myc-tagged COG sub-complexes revealed that different membrane-associated COG partners including β-COP, p115 and SNARE STX5 preferentially bind to different COG assemblies, indicating that COG subunits interact with Golgi membranes in a multipronged fashion.

保守的低聚高尔基复合物是一种外周膜蛋白复合物,它协调了高尔基内运输载体与高尔基膜的捆绑和融合。在这项研究中,我们研究了COG复合物的膜附着及其在高尔基膜上的开/关动态。包括侧敲耗散、FRAP和FLIP在内的几种互补方法显示,在活的HeLa细胞中,组装的COG复合物不会从高尔基外围扩散。此外,当高尔基结构受到严重影响时,即使在COG4和COG7耗尽的细胞中,COG亚基仍然与膜相关。myc标记的COG亚复合物的过表达表明,不同的膜相关COG伙伴包括β-COP, p115和SNARE STX5优先结合不同的COG组件,表明COG亚基以多管齐下的方式与高尔基膜相互作用。
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引用次数: 23
Unique and conserved features of the plant ER-shaping GTPase RHD3. 植物er成形GTPase RHD3的独特和保守特征。
Pub Date : 2014-01-01 Epub Date: 2014-02-28 DOI: 10.4161/cl.28217
Giovanni Stefano, Federica Brandizzi

The architectural integrity of the endoplasmic reticulum (ER) network depends on the function of membrane-associated dynamin-like GTPases that include metazoan atlastins, plant RHD3 and yeast Sey1p. The evidence that these proteins are sufficient to drive membrane fusion of reconstituted proteoliposomes, and that loss-of-function mutations lead to conspicuous ER shape defects indicates that atlastins, RHD3 and Sey1p promote ER membrane fusion. However, complementation experiments in reciprocal loss-of-function backgrounds have also suggested that RHD3 and Sey1p may be not functionally equivalent, supporting that ER fusion mechanisms may be not entirely conserved in eukaryotes. In this Letter, we provide a brief overview of the field as well as evidence that may explain the functional differences of the plant and yeast ER-shaping dynamin-like GTPases.

内质网(ER)网络的结构完整性取决于膜相关动力蛋白样GTPases的功能,包括后生动物的atlastins、植物的RHD3和酵母的Sey1p。这些蛋白足以驱动重组蛋白脂质体的膜融合,以及功能缺失突变导致明显的内质网形状缺陷的证据表明,atlastins、RHD3和Sey1p促进内质网膜融合。然而,在互失功能背景下的互补实验也表明,RHD3和Sey1p可能在功能上并不等同,这支持了内质网融合机制在真核生物中可能并不完全保守。在这封信中,我们提供了该领域的简要概述以及可能解释植物和酵母er形成动力蛋白样GTPases的功能差异的证据。
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引用次数: 16
Novel effects of Brefeldin A (BFA) in signaling through the insulin receptor (IR) pathway and regulating FoxO1-mediated transcription. Brefeldin A (BFA)在胰岛素受体(IR)信号通路和fox01介导的转录调控中的新作用
Pub Date : 2014-01-01 Epub Date: 2014-01-09 DOI: 10.4161/cl.27732
Paulina Wyrozumska, Jason W Ashley, Sasanka Ramanadham, Qinglan Liu, W Timothy Garvey, Elizabeth Sztul

Brefeldin A (BFA) is a fungal metabolite best known for its ability to inhibit activation of ADP-ribosylation factor (Arf) and thereby inhibit secretory traffic. BFA also appears to regulate the trafficking of the GLUT4 glucose transporter by inducing its relocation from intracellular stores to the cell surface. Such redistribution of GLUT4 is normally regulated by insulin-mediated signaling. Hence, we tested whether BFA may intersect with the insulin pathway. We report that BFA causes the activation of the insulin receptor (IR), IRS-1, Akt-2, and AS160 components of the insulin pathway. The response is mediated through phosphoinositol-3-kinase (PI3K) and Akt kinase since the PI3K inhibitor wortmannin and the Akt inhibitors MK2206 and perifosine inhibit the BFA effect. BFA-mediated activation of the insulin pathway results in Akt-mediated phosphorylation of the insulin-responsive transcription factor FoxO1. This leads to nuclear exclusion of FoxO1 and a decrease in transcription of the insulin-responsive gene SIRT-1. Our findings suggest novel effects for BFA in signaling and transcription, and imply that BFA has multiple intracellular targets and can be used to regulate diverse cellular responses that include vesicular trafficking, signaling and transcription.

Brefeldin A (BFA)是一种真菌代谢物,以其抑制adp核糖基化因子(Arf)的激活从而抑制分泌流量的能力而闻名。BFA似乎还通过诱导GLUT4葡萄糖转运体从细胞内储存转移到细胞表面来调节其运输。这种GLUT4的再分配通常由胰岛素介导的信号传导调节。因此,我们测试了BFA是否可能与胰岛素途径相交。我们报道BFA导致胰岛素通路中胰岛素受体(IR)、IRS-1、Akt-2和AS160组分的激活。这种反应是通过磷酸肌醇-3激酶(PI3K)和Akt激酶介导的,因为PI3K抑制剂wortmannin和Akt抑制剂MK2206和perifosine抑制BFA的作用。bfa介导的胰岛素通路激活导致akt介导的胰岛素应答转录因子fox01的磷酸化。这导致FoxO1的核排斥和胰岛素应答基因SIRT-1的转录减少。我们的研究结果表明,BFA在信号传导和转录方面具有新的作用,并暗示BFA具有多种细胞内靶点,可用于调节多种细胞反应,包括囊泡运输、信号传导和转录。
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引用次数: 5
期刊
Cellular logistics
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