Organization and Dynamics of Endomembrane Pathways and Organelles

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

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.