网格蛋白介导的内吞作用机制

受体介导的内吞作用是目前公认的生物体摄取生物大分子的途径,而网格蛋白介导的内吞又是最主要的受体介导方式.结合国内外最新报道,介绍了网格蛋白和衔接蛋白的结构、分子特性和功能;从衔接蛋白、网格蛋白的招募;包被小凹的内陷、缢缩和包被液泡的芽殖和包被液泡的脱壳等过程,阐释了网格蛋白介导的内吞作用机制.     

卵黄脂磷蛋白通过网格蛋白介导的内吞作用进入培养的人成纤维细胞

为了探究从斑马鱼(Danio rerio)胚胎裂解液中纯化的卵黄脂磷蛋白(lipovitellin, Lv)进入培养的人成纤维细胞的分子途径,该研究用Sephadex G-200色谱柱结合QAE-Sephadex A50阴离子交换色谱柱从斑马鱼0 h胚胎裂解液中分离纯化出Lv。FITC标记的Lv(FITC-Lv)预先分别与Hepes、牛组蛋白、鱼精蛋白、卵磷脂、0 h胚胎胰蛋白酶水解物或者斑马鱼胚胎裂解液孵育,再加入至opti-MEM培养基中培养人成纤维细胞,培养一定时间后,用荧光倒置显微镜观察,发现Hepes和组蛋白可以促进Lv进入细胞。制霉菌素是陷窝介导的内吞作用(caveolae-mediated endocytosis)的抑制剂,氯丙嗪和蔗糖是网格蛋白介导的内吞作用(clathrin-mediated endocytosis)的抑制剂,阿米洛利是巨胞饮作用(macropinocytosis)的抑制剂。为了证实Lv进入细胞的分子途径,在FITC标记Lv与人成纤维细胞的培养体系中,分别加入上述抑制剂。结果发现氯丙嗪和蔗糖可以抑制Lv进入细胞。该研究获得如下结论:纯化的斑马鱼Lv单独...     

SARS-CoV-2入胞活细胞示踪平台的建立

新型冠状病毒（SARS-CoV-2）由于其高传染性已造成全球大流行。目前关于SARS-CoV-2依赖内吞途径进入细胞的研究偏向于药物抑制实验或固定样品的蛋白共定位实验,而对于其入胞时与细胞内吞结构相互作用的动力学机制研究较少。本研究首先基于慢病毒系统包装出可在生物安全二级实验室（BSL-2）进行操作的SARS-CoV-2假病毒,之后利用膜染料对假病毒的囊膜进行荧光标记,并进行鉴定。通过免疫荧光方法观察到假病毒与网格蛋白包被的内吞结构共定位,进一步在网格蛋白敲低的Caco-2细胞系上进行假病毒感染,检测到荧光素酶活性显著下降,这些结果确定了网格蛋白介导的内吞作用参与假病毒感染。最后基于单病毒示踪技术,通过共聚焦显微镜对假病毒入胞过程进行活细胞实时拍摄,选取两个具有代表性的假病毒粒子依赖网格途径入胞的典型视野,并对假病毒动力学进行分析。本研究成功建立了SARS-CoV-2假病毒入胞活细胞示踪平台,可应用于研究单个假病毒粒子入胞过程动力学机制。该平台对SARS-CoV-2入胞机制的研究具有重大意义。     

Dynamin-I的功能结构域及其在突触囊泡内吞过程中的作用

在神经系统中,突触是神经元间信息传递的桥梁.突触囊泡通过胞吐和内吞作用完成一次突触囊泡循环.Dynamin-I(又称发动蛋白-1),为三磷酸鸟苷酶(GTPase)家族的一员,由四个功能结构域组成,分别为N末端GTPase域、普列克底物蛋白同源域、GTPase效应结构域和C末端脯氨酸和精氨酸富集域.目前大量研究证明,dyanmin-I的四个功能结构域各自具有特定的功能、接受不同的调控机制,在突触囊泡胞吞过程中发挥重要作用.因此深入研究dyanmin-I的四个功能结构域以及其在突触囊泡内吞过程中的生理调控的方式和位点,具有重要的理论和实践意义.     

蛋白磷酸酶2A在真菌细胞中的研究进展

蛋白质可逆的磷酸化修饰在真菌细胞生命活动中发挥着重要作用.磷酸化与去磷酸化过程相互协调.蛋白质中磷酸丝氨酸/苏氨酸位点的去磷酸化反应主要由蛋白磷酸酶2A(Protein Phosphatase 2A,PP2A)负责催化.PP2A由催化亚基、调节亚基与结构亚基组合成有活性的三聚体全酶形式发挥功能.本文以模式真菌酿酒酵母、...     

Dynamin-Ⅰ的功能结构域及其在突触囊泡内吞过程中的作用

在神经系统中,突触是神经元间信息传递的桥梁。突触囊泡通过胞吐和内吞作用完成一次突触囊泡循环。Dynamin-Ⅰ(又称发动蛋白-1),为三磷酸鸟苷酶(GTPase)家族的一员,由四个功能结构域组成,分别为N末端GTPase域、普列克底物蛋白同源域、GTPase效应结构域和C末端脯氨酸和精氨酸富集域。目前大量研究证明,dyanmin-Ⅰ的四个功能结构域各自具有特定的功能、接受不同的调控机制,在突触囊泡胞吞过程中发挥重要作用。因此深入研究dyanmin-Ⅰ的四个功能结构域以及其在突触囊泡内吞过程中的生理调控的方式和位点,具有重要的理论和实践意义。     

受体酪氨酸激酶ErbB2靶向治疗策略和内吞降解调控的研究进展

受体酪氨酸激酶ErbB2/HER2是人类表皮生长因子受体家族成员之一,在乳腺癌、胃癌、卵巢癌等多种肿瘤中过表达并与患者不良预后密切相关.过表达的ErbB2受体通过与家族成员形成同源或异源二聚体发挥激酶活性,激活下游信号转导通路,进而促进肿瘤细胞的生长和增殖.ErbB2已经成为肿瘤治疗的重要靶标,目前临床应用的靶向Erb...     

蛋白磷酸酶2A对微管的调控作用研究进展

微管是细胞骨架的主要成分之一,几乎存在于所有真核生物细胞之中,参与细胞众多生理功能。PP2A是真核生物体内存在最广泛的蛋白磷酸酶之一,可以调控大部分细胞生命活动,其中,包括微管所介导的许多生命活动。该文从以下方面介绍了PP2A在微管功能行使中的重要作用,包括PP2A参与微管蛋白翻译后修饰、调控分子马达和微管相关蛋白的活性、维持细胞周期中微管的动态平衡以及PP2A异常与微管类疾病的相关性。     

肠道病毒71型2A蛋白的研究进展

肠道病毒71(enterovirus 71,EV71)是手足口病(hand,foot and mouth disease,HFMD)主要致病原之一。手足口病临床上常表现为发热,手掌、脚掌及口腔黏膜皮疹或疱疹。然而,EV71感染导致的手足口病易伴随神经系统并发症,甚至死亡。EV71非结构蛋白2A作为蛋白酶和转录激活因子,在EV71生命周期中发挥重要作用。本文对2A的结构与功能研究进展进行综述,揭示2A的双重功能如何促进病毒复制和调控靶细胞,为进一步研究靶向2A的抗病毒疫苗和药物提供理论基础。     

非肌肉肌球蛋白ⅡA的相互作用蛋白研究进展

非肌肉肌球蛋白ⅡA(NMⅡA)是肌球蛋白超家族的成员之一,其生物学功能已引起国内外学者的广泛重视。研究发现,NMⅡA可作为分子马达为细胞内分子运动提供动力;还可通过与其他蛋白质发生相互作用而参与细胞黏附与迁移、细胞吞噬、胞质分裂等多种生理病理活动。本文综述了与NMⅡA存在相互作用的蛋白质及其产生的生物学效应,以期为全面了解NMⅡA的生理病理功能,深入探讨肿瘤等重大疾病的发病机制及新药研发提供一定线索和依据。     

Endophilin A2 regulates B‐cell endocytosis and is required for germinal center and humoral responses

Antigen‐specific B‐cell responses require endosomal trafficking to regulate antigen uptake and presentation to helper T cells, and to control expression and signaling of immune receptors. However, the molecular composition of B‐cell endosomal trafficking pathways and their specific roles in B‐cell responses have not been systematically investigated. Here, we report high‐throughput identification of genes regulating B‐cell receptor (BCR)‐mediated antigen internalization using genome‐wide functional screens. We show that antigen internalization depends both on constitutive, clathrin‐mediated endocytosis and on antigen‐induced, clathrin‐independent endocytosis mediated by endophilin A2. Although endophilin A2‐mediated endocytosis is dispensable for antigen presentation, it is selectively required for metabolic support of B‐cell proliferation, in part through regulation of iron uptake. Consequently, endophilin A2‐deficient mice show defects in GC B‐cell responses and production of high‐affinity IgG. The requirement for endophilin A2 highlights a unique importance of clathrin‐independent intracellular trafficking in GC B‐cell clonal expansion and antibody responses.     

Endophilin B1 is required for the maintenance of mitochondrial morphology

We report that a fatty acyl transferase, endophilin B1, is required for maintenance of mitochondrial morphology. Down-regulation of this protein or overexpression of endophilin B1 lacking the NH(2)-terminal lipid-modifying domain causes striking alterations of the mitochondrial distribution and morphology. Dissociation of the outer mitochondrial membrane compartment from that of the matrix, and formation of vesicles and tubules of outer mitochondrial membrane, was also observed in both endophilin B1 knockdown cells and after overexpression of the truncated protein, indicating that endophilin B1 is required for the regulation of the outer mitochondrial membrane dynamics. We also show that endophilin B1 translocates to the mitochondria during the synchronous remodeling of the mitochondrial network that has been described to occur during apoptosis. Double knockdown of endophilin B1 and Drp1 leads to a mitochondrial phenotype identical to that of the Drp1 single knockdown, a result consistent with Drp1 acting upstream of endophilin B1 in the maintenance of morphological dynamics of mitochondria.     

Ca2+ triggers a novel clathrin-independent but actin-dependent fast endocytosis in pancreatic beta cells

The existence of clathrin-independent recycling of secretory vesicles has been controversial. By combining patch-clamp capacitance recording, optical methods and specific molecular interventions, we dissect two types of mechanistically different endocytosis in pancreatic β cells, both of which require GTP and dynamin. The fast one is a novel clathrin-independent but actin-dependent endocytosis that is triggered by high cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). Large fluorescent dextran (10 nm in diameter) was able to be internalized by this pathway, indicating that it was not likely to be 'kiss and run'. The slow endocytosis is a clathrin-dependent process in which actin plays a complementary role. For the first time, we show that the rate constants for both types of endocytosis exhibit supralinear dependence on increase in [Ca²⁺]_i. Compared with the slow endocytosis, higher [Ca²⁺]_i level was required to fully accelerate the fast one, indicative of distinct Ca²⁺ sensors for different endocytosis. In the end, we show that physiologically relevant stimulation induces clathrin-independent endocytosis in intact β cells, implying that it may contribute to the normal recycling of secretory vesicles in vivo .     

Clathrin-independent endocytosis: A cargo-centric view

Clathrin-independent endocytosis occurs in all cells and interest in this mode of cellular entry has grown. Although this form of endocytosis was first described for entry of bacterial toxins, here we focus our attention on the endogenous cell surface “cargo” proteins that enter cells by this mechanism. The cargo proteins entering by this mechanism are varied and include nutrient transporters, ion channels, cell adhesion molecules and proteins associated with the immune system. Despite the apparent lack of selection at the cell surface, we provide some examples of specific sorting of these cargo proteins after entry, leading to distinct itineraries and cellular fates.     

Endophilin A1 regulates dendritic spine morphogenesis and stability through interaction with p140Cap

Dendritic spines are actin-rich membrane protrusions that are the major sites of excitatory synaptic input in the mammalian brain, and their morphological plasticity provides structural basis for learning and memory. Here we report that endophilin A1, with a well-established role in clathrin-mediated synaptic vesicle endocytosis at the presynaptic terminal, also localizes to dendritic spines and is required for spine morphogenesis, synapse formation and synaptic function. We identify p140Cap, a regulator of cytoskeleton reorganization, as a downstream effector of endophilin A1 and demonstrate that disruption of their interaction impairs spine formation and maturation. Moreover, we demonstrate that knockdown of endophilin A1 or p140Cap impairs spine stabilization and synaptic function. We further show that endophilin A1 regulates the distribution of p140Cap and its downstream effector, the F-actin-binding protein cortactin as well as F-actin enrichment in dendritic spines. Together, these results reveal a novel function of postsynaptic endophilin A1 in spine morphogenesis, stabilization and synaptic function through the regulation of p140Cap.     

Systems Dynamics in Endocytosis

The endocytic system acts at the crossroads of different cellular activities to play a central role in the regulation of cell signaling and membrane dynamics. An European Molecular Biology Organization (EMBO) conference held in October 2013 in Villars‐sur‐Ollon gathered researchers from all over the world to present their latest findings on the endolysosomal system and identify major challenges for the future. The conference covered the entire spectrum of research in this rapidly evolving field ranging from the cellular mechanics of endocytosis to the role of proteins and lipids in the biogenesis and function of endolysosomal organelles and the analysis of higher order system properties in multicellular contexts. In particular, the meeting highlighted current efforts to complement the insights that can be gained by biochemical and cell biological approaches with the use of quantitative biophysics, systems biology and animal model systems to achieve an integrated view of the properties of the endomembrane system and its role in cellular information processing.     

Clathrin-independent endocytosis contributes to uptake of glucose into BY-2 protoplasts

In eukaryotic cells, several pathways exist for the internalization of plasma membrane proteins and extracellular cargo molecules. These endocytic pathways can be divided into clathrin-dependent and clathrin-independent pathways. While clathrin-dependent pathways are known to be involved in a variety of cellular processes in plants, clathrin-independent pathways have so far only been identified in animal and yeast cells. Here we show that internalization of fluorescent glucose into BY-2 cells leads to accumulation of the sugar in compartments of the endocytic pathway. This endocytic uptake of glucose was not blocked by ikarugamycin, an inhibitor of clathrin-dependent endocytosis, suggesting a role for clathrin-independent endocytosis in glucose uptake. Investigations of fusion and fission of single vesicles by membrane capacitance measurements revealed stimulation of endocytic activity by extracellular glucose. Glucose-stimulated fission of vesicles was not affected by addition of ikarugamycin or blocking of clathrin coat formation by transient over-expression of HUB1 (the C-terminal part of the clathrin heavy chain). These data demonstrate that clathrin-independent endocytosis does occur in plant cells. This pathway may represent a common mechanism for the uptake of external nutrients.     

Actin and Endocytosis in Budding Yeast

Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed.