Biomechanical Role of Epsin in Influenza A Virus Entry.

Influenza A virus (IAV) utilizes clathrin-mediated endocytosis for cellular entry. Membrane-bending protein epsin is a cargo-specific adaptor for IAV entry. Epsin interacts with ubiquitinated surface receptors bound to IAVs via its ubiquitin interacting motifs (UIMs). Recently, epsin was shown to have membrane tension sensitivity via its amphiphilic H0 helix. We hypothesize this feature is important as IAV membrane binding would bend the membrane and clinical isolates of IAVs contain filamentous IAVs that may involve more membrane bending. However, it is not known if IAV internalization might also depend on epsin's H0 helix. We found that CALM, a structurally similar protein to epsin lacking UIMs shows weaker recruitment to IAV-containing clathrin-coated structures (CCSs) compared to epsin. Removal of the ENTH domain of epsin containing the N-terminus H0 helix, which detects changes in membrane curvature and membrane tension, or mutations in the ENTH domain preventing the formation of H0 helix reduce the ability of epsin to be recruited to IAV-containing CCSs, thereby reducing the internalization of spherical IAVs. However, internalization of IAVs competent in filamentous particle formation is not affected by the inhibition of H0 helix formation in the ENTH domain of epsin. Together, these findings support the hypothesis that epsin plays a biomechanical role in IAV entry.

Complimentary action of structured and unstructured domains of epsin supports clathrin-mediated endocytosis at high tension.

Membrane tension plays an inhibitory role in clathrin-mediated endocytosis (CME) by impeding the transition of flat plasma membrane to hemispherical clathrin-coated structures (CCSs). Membrane tension also impedes the transition of hemispherical domes to omega-shaped CCSs. However, CME is not completely halted in cells under high tension conditions. Here we find that epsin, a membrane bending protein which inserts its N-terminus H0 helix into lipid bilayer, supports flat-to-dome transition of a CCS and stabilizes its curvature at high tension. This discovery is supported by molecular dynamic simulation of the epsin N-terminal homology (ENTH) domain that becomes more structured when embedded in a lipid bilayer. In addition, epsin has an intrinsically disordered protein (IDP) C-terminus domain which induces membrane curvature via steric repulsion. Insertion of H0 helix into lipid bilayer is not sufficient for stable epsin recruitment. Epsin's binding to adaptor protein 2 and clathrin is critical for epsin's association with CCSs under high tension conditions, supporting the importance of multivalent interactions in CCSs. Together, our results support a model where the ENTH and unstructured IDP region of epsin have complementary roles to ensure CME initiation and CCS maturation are unimpeded under high tension environments.

Mechanical Regulation of Endocytosis: New Insights and Recent Advances.

Endocytosis is a mechanosensitive process. It involves remodeling of the plasma membrane from a flat shape to a budded morphology, often at the sub-micrometer scale. This remodeling process is energy-intensive and is influenced by mechanical factors such as membrane tension, membrane rigidity, and physical properties of cargo and extracellular surroundings. The cellular responses to a variety of mechanical factors by distinct endocytic pathways are important for cells to counteract rapid and extreme disruptions in the mechanohomeostasis of cells. Recent advances in microscopy and mechanical manipulation at the cellular scale have led to new discoveries of mechanoregulation of endocytosis by the aforementioned factors. While factors such as membrane tension and membrane rigidity are generally shown to inhibit endocytosis, other mechanical stimuli have complex relationships with endocytic pathways. At this juncture, it is now possible to utilize experimental techniques to interrogate theoretical predictions on mechanoregulation of endocytosis in cells and even living organisms.

The Machado-Joseph disease-associated form of ataxin-3 impacts dynamics of clathrin-coated pits.

Expansion above a certain threshold in the polyglutamine (polyQ) tract of ataxin-3 is the main cause of neurodegeneration in Machado-Joseph disease. Ataxin-3 contains an N-terminal catalytic domain, called Josephin domain, and a highly aggregation-prone C-terminal domain containing the polyQ tract. Recent work has shown that protein aggregation inhibits clathrin-mediated endocytosis (CME). However, the effects of polyQ expansion in ataxin-3 on CME have not been investigated. We hypothesize that the expansion of the polyQ tract in ataxin-3 could impact CME. Here, we report that both the wild-type and the expanded ataxin-3 reduce transferrin internalization and expanded ataxin-3 impacts dynamics of clathrin-coated pits (CCPs) by reducing CCP nucleation and increasing short-lived abortive CCPs. Since endocytosis plays a central role in regulating receptor uptake and cargo release, our work highlights a potential mechanism linking protein aggregation to cellular dysregulation.

Integrated quantification based on confocal imaging: cell crowding modulates heterogeneity in GPCR-mediated calcium oscillation.

Results from single cell imaging, facilitated by high resolution microscopy, have demonstrated cell-to-cell variability within the same cell population in contexts ranging from cell growth to cell migration. Recent studies suggest that such variability conveys important information about diseased states. However, manual analysis and interpretation of heterogeneous calcium oscillation based on time-lapsed images, as practiced today, is tedious, and essentially infeasible for large datasets. As a practical alternative, we present an integrated platform that includes calcium imaging using confocal microscope, algorithmic cell segmentation, and statistical analysis. Automated quantification of cell crowding via cell segmentation and statistical analysis of cell-to-cell variability on a representative dataset indicates that the heterogeneity in GPCR (G-protein coupled receptor) mediated calcium oscillation is a function of cell crowding.