Single-particle and sub-tomogram analysis of septin filaments

• Grant number: 21/10247-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: January 01, 2022
• End date: December 23, 2022
• Field of knowledge: Biological Sciences - Biophysics
• Principal Investigator: Richard Charles Garratt
• Grantee: Deborah Cezar Mendonça
• Supervisor: Bruno Klaholz
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Institution abroad: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), France
• Associated to the scholarship: 18/20209-5 - Structural studies on the assembly of septin complexes by transmission electron microscopy and single particle analysis, BP.DR

Abstract

Septins are GTP binding proteins considered to be a novel component of the cytoskeleton. These proteins interact with each other to form filamentous heterocomplexes and higher order structures which are important for cytokinesis, plasma membrane compartmentalization and a variety of other cellular processes. The oligomers themselves can vary in size and composition from species to species but always present a specific subunit arrangement. Isolated septin complexes can be observed in high salt buffer in vitro, while filament formation occurs at low salt concentrations. Even though there exists a growing interest in this important cytoskeletal component, many aspects of septin polymerization, bundling and function remain unclear. In an attempt to shed light on these questions, it is important to elucidate the 3D structure of their heterofilaments. Although crystallographic approaches have been successful in studying the separate components of the system, there has been difficulty in obtaining high resolution structures of the full complexes. Recently, we were able to provide a complete model for a human septin hexameric complex, by Cryo-EM, with a global resolution of ~3.6Å. Furthermore, we observed that the hexamer shows a tendency to bend at its centre which is possibly related to the ability of septin filaments to recognize and even induce membrane curvature. Our next objective is to expand our knowledge on septins assembly, principally by exploring the regions responsible for the interactions between filaments. These interfaces are believed to be stabilized by the formation of coiled-coils from the septin C-domains. We intend to use a combination of single-particle and sub-tomogram analysis to elucidate how this interface is formed. Altogether, this should give us a more complete understanding of the septin structure-function relationship. (AU)