. The separation of membranes into discrete compartments through the process of membrane fission is essential for diverse cellular processes ranging from cell division to viral entry. While the specialized fission machine dynamin is well-known to induce fission through constriction of membrane tubes, recent evidence shows that other proteins drive fission by previously unknown mechanisms. In particular, the epsin 1 N-terminal homology (ENTH) domain is a potent driver of membrane curvature (Ford et al., Nature 2002), and has recently been shown to play a role in membrane fission. Specifically, a recent report proposed that insertion of a wedge-like amphipathic helix by ENTH curves and destabilizes membranes, as evidenced by decreasing membrane fission ability among ENTH mutants with decreasing helix hydrophobicity (Boucrot et al., Cell 2012). However, our group recently showed that collisions among dense, membrane-bound ENTH proteins generate steric pressure, which drives membrane bending in the absence of helix insertions (Stachowiak et al., Nature Cell Biology 2012). These results prompted us to ask: is steric pressure also responsible for membrane fission by ENTH? In my preliminary studies, I found that ENTH mutants with reduced helix hydrophobicity are capable of driving fission to a similar degree as wild-type ENTH when bound to the membrane at comparable density. Interestingly, I also found that full-length epsin, which contains a bulky, intrinsically-disordered C-terminal domain, drives fission more potently than the ENTH domain alone. These results imply that, while helix insertions are important for binding proteins tightly to membrane surfaces, helices are not required for fission. However, once bound to the membrane surface at sufficient density, bulky molecules of arbitrary structure can create steric pressure that increases membrane curvature until fission occurs. Taken together, my findings reveal a novel mechanism for membrane fission. The objective of the proposed research is to quantitatively compare this new mechanism with other key mechanisms of membrane fission. The first specific aim will delineate the specific roles of wedge-like helix insertion and protein crowding in driving membrane fission. The second specific aim will examine how dynamin works cooperatively with helix insertion and protein crowding to drive robust fission. The third specific aim will utilize quantitative imaging of live cells to examine how helix insertion and protein crowding modulate fission dynamics in a physiological context. This work will create innovative biophysical tools for the simultaneous study of membrane fission and protein-lipid interactions both in vitro and in live cells. The overall outcome of this research will be a deeper understanding of the physical mechanisms of membrane fission, including the novel mechanism of membrane fission by protein crowding. The bold hypothesis described here asserts that any membrane-bound protein can contribute to fission, an idea that will influence understanding of diverse membrane compartmentalizing processes, including endocytosis, cell division, and viral entry.
The proposed research is relevant to public health because defects in the formation of membrane compartments via membrane fission underlie debilitating diseases from neurological disorders to diabetes, and because viral and bacterial pathogens rely on membrane fission to enter cells. Therefore, the proposed research on the physical mechanisms of membrane fission is relevant to the part of NIH's mission that seeks to develop fundamental knowledge that will help to reduce the burdens of human disability and disease. Ultimately, it is envisioned that increased fundamental understanding of membrane fission will enable its clinical manipulation, providing new approaches for treating diseases that arise from disruptions in membrane fission and preventing its exploitation by pathogens.
| Snead, Wilton T; Zeno, Wade F; Kago, Grace et al. (2018) BAR scaffolds drive membrane fission by crowding disordered domains. J Cell Biol : |
| Snead, Wilton T; Stachowiak, Jeanne C (2018) A Tethered Vesicle Assay for High-Throughput Quantification of Membrane Fission. Methods Enzymol 611:559-582 |
| Snead, Wilton T; Stachowiak, Jeanne C (2018) Structure Versus Stochasticity-The Role of Molecular Crowding and Intrinsic Disorder in Membrane Fission. J Mol Biol 430:2293-2308 |
| Snead, Wilton T; Hayden, Carl C; Gadok, Avinash K et al. (2017) Membrane fission by protein crowding. Proc Natl Acad Sci U S A 114:E3258-E3267 |
