Organization and Dynamics of Endomembrane Pathways and Organelles

人类免疫缺陷病毒（HIV）完成感染周期所依赖的关键宿主因子之一是运输所需的内体分选复合物（ESCRT）。通过招募这种机制，HIV能够介导病毒颗粒从膜分裂的最后一步。否则，病毒的逸出将受到严重抑制。尽管在证明ESCRT机制在介导HIV脱落中的作用方面取得了很大进展，但ESCRT亚复合物在HIV天然组装位点的纳米级组织及其功能仍然不明确。ESCRT机制参与重要的细胞膜重塑事件，如细胞分裂和多泡体生物发生（MVB），组织成三种一般的亚复合物。其中一组包括ESCRT-0、-I和-II，它们直接与货物蛋白或支架蛋白相互作用，指导下游ESCRT复合物的组装。另一组包括ESCRT- iii亚基，它们被认为在膜上聚合成螺旋结构，以收缩和脱落由早期ESCRT/货物复合体形成的膜芽。最后一组由AAA+ atp酶Vps4A/B组成，它们被认为是通过ESCRT-III纤维重塑和/或再循环实现HIV分离所必需的。总之，三组ESCRT亚复合物协同驱动细胞膜重塑。通过体外组装和细胞过表达研究获得的结构信息表明，在一个芽形成和脱落的模型中，ESCRT-III细丝包围并收缩膜突起的孔，作用于颈部基部，从突起头部转过来。另一方面，在细胞分裂过程中，当被中间支架引导时，ESCRT-III细丝似乎从支架上聚合并收缩膜，顺时针作用于支架结构。由于出芽HIV病毒粒子的尺寸很小（直径120-140 nm）， ESCRT-III细丝在HIV芽位点的聚合方式是顺式还是反式尚不清楚。因此，目前尚不清楚ESCRT机制如何介导病毒膜脱落以传播HIV感染。我们使用干涉光激活定位显微镜（iPALM）破译了ESCRT组件在HIV组装位点的三维（3D）纳米级组织，从而深入了解ESCRT病毒膜脱落的机制。我们观察到ESCRT亚基定位于出芽病毒粒子和释放颗粒的头部，在病毒分离时，头部定位的CHMP2A水平相对于Tsg101和CHMP4B下降。因此，HIV释放的驱动力可能来自病毒芽内部ESCRT亚基的初始支架，随后支架附着在质膜上并在病毒出芽期间重塑。

One of the key host factors human immunodeficiency virus (HIV) relies on to complete its infection cycle is the endosomal sorting complexes required for transport (ESCRT). By recruiting this machinery, HIV is able to mediate the final step of virus particle fission from the membrane. Otherwise, virus egress is severely inhibited. Despite great progress in demonstrating the ESCRT machinerys role in mediating HIV abscission, the nanoscale organization, and thus function, of ESCRT subcomplexes at native HIV assembly sites remains poorly defined. ESCRT machinery participates in important cellular membrane remodeling events, such as cytokinesis and multivesicular body biogenesis (MVB), organizing into three general sets of subcomplexes. One set includes ESCRT-0, -I, and -II, which interact directly with cargo or scaffold proteins to direct assembly of downstream ESCRT complexes. Another set includes ESCRT-III subunits, which are thought to polymerize on membranes into a helical architecture to constrict and abscise membrane buds formed by early ESCRT/cargo complexes. The final set consists of the AAA+ ATPases Vps4A/B, which are thought to be necessary for HIV abscission through ESCRT-III filament remodeling and or recycling. Together, the three sets of ESCRT subcomplexes cooperate to drive cellular membrane remodeling. Structural information obtained by in vitro assembly and cellular overexpression studies suggest a model of bud formation and abscission where ESCRT-III filaments encircle and constrict the aperture of a membrane protrusion, acting on the base of the neck, in trans from the protrusion head. On the other hand, when directed by the midbody scaffold during cytokinesis, ESCRT-III filaments appear to polymerize from the scaffold and constrict the membrane, acting in cis with respect to the scaffolding structure. Whether ESCRT-III filaments polymerize at HIV bud sites in cis or trans to the scaffolding structure is not known because of the small dimensions of a budding HIV virion (120-140 nm diameter). Consequently, it remains unclear how ESCRT machinery acts to mediate viral membrane abscission in order to propagate HIV infection. We used interferometric photoactivation-localization microscopy (iPALM) to decipher the three-dimensional (3D) nanoscale organization of ESCRT components at HIV assembly sites, and thereby gain insight into the mechanism for viral membrane abscission by ESCRTs. We observed ESCRT subunits localize within the head of budding virions and released particles, with head-localized levels of CHMP2A decreasing relative to Tsg101 and CHMP4B upon virus abscission. Thus, the driving force for HIV release may derive from initial scaffolding of ESCRT subunits within the viral bud interior followed by scaffold attachment to the plasma membrane and remodeling during viral budding. MicroRNAs (miRNAs) are small, 1822 nt long, noncoding RNAs that act as potent negative gene regulators in a variety of physiological and pathological processes. To repress gene expression, miRNAs are packaged into RNA-induced silencing complexes (RISCs) that target mRNAs for degradation and/or translational repression in a sequence-specific manner. Recently, miRNAs have been shown to also interact with proteins outside RISCs, impacting cellular processes through mechanisms not involving gene silencing. In this study, we defined a previously unappreciated activity of miRNAs in inhibiting RNAprotein interactions that in the context of HIV-1 biology blocks HIV virus budding and reduces virus infectivity. This occurred by miRNA binding to the nucleocapsid domain of the Gag protein, the main structural component of HIV-1 virions. The resulting miRNAGag complexes interfered with viralRNA-mediated Gag assembly and viral budding at the plasma membrane, with imperfectly assembled Gag complexes endocytosed and delivered to lysosomes. The blockade of virus production by miRNA was reversed by adding the miRNAs target mRNA and stimulated by depleting Argonaute-2,suggesting that when miRNAs are not mediating gene silencing, they can block HIV-1 production through disruption of Gag assembly on membranes. Overall, our findings have significant implications for understanding how cells modulate HIV-1 infection by miRNA expression and raise the possibility that miRNAs can function to disrupt RNA-mediated protein assembly processes in other cellular contexts. To address how cells control their shape, we combined 3D superresolution analyses of crawling cells with the development of a biophysical modeling scheme to show that the seemingly complex process of lamella flattening in the crawling cell can be explained based on mechanical principles and cytoskeletal reorganization. Structured illumination microscopy (SIM) helped clarify the fine 3D contractile organization of actin filaments in the lamella, revealing that the primary actin filaments undergoing myosin IIbased contraction were transverse actin arcs running parallel to the top of the cell. As the arcs contracted, they pulled on DSFs, which resisted by pivoting on their attached focal adhesions at the cell bottom, generating 3D forces on the growth substrate. This caused the dorsal membrane of the cell to tilt downward and the lamella to flatten. Removing myosin IIA contractility caused the lamella to lose its flatness, whereas adding myosin IIA to nonmotile cells, which lack a flat lamella, caused cells to create one. Together, our results suggest that myosin II contractile machinery mediates lamella flattening in a process involving counterbalanced contractile and adhesive forces. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions. Proteins destined for the cell surface are first assessed in the endoplasmic reticulum (ER) for proper folding before release into the secretory pathway. This ensures that defective proteins are normally prevented from entering the extracellular environment, where they could be disruptive. We observed that, when ER folding capacity is saturated during stress, misfolded glycosylphosphatidylinositol-anchored proteins dissociate from resident ER chaperones, engage export receptors, and quantitatively leave the ER via vesicular transport to the Golgi. Clearance from the ER commences within minutes of acute ER stress, before the transcriptional component of the unfolded protein response is activated. These aberrant proteins then access the cell surface transiently before destruction in lysosomes. Inhibiting this stress-induced pathway by depleting the ER-export receptors leads to aggregation of the ER-retained misfolded protein. Thus, this rapid response alleviates the elevated burden of misfolded proteins in the ER at the onset of ER stress, promoting protein homeostasis in the ER.