Structural studies of septin hetero-complexes by electron microscopy

• Grant number: 17/05665-1
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: July 01, 2017
• End date: July 31, 2020
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Richard Charles Garratt
• Grantee: Samuel Leite Guimarães
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Associated research grant: 14/15546-1 - Septins: comparative studies and the correlation between structure and function, AP.TEM
• Associated scholarship(s): 18/20816-9 - Single-particle Cryo-EM analysis of septin heterocomplexes, BE.EP.PD

Abstract

Septins are filament forming GTP binding proteins which were first described as being associated with cytokinesis in yeast. Besides their role in cell division, many additional functions have been attributed to septins in many different organisms after the identification of their binding partners. Nevertheless, despite the existence of dozens of theoretically possible hetero-filaments, only one crystal structure at low resolution has been reported to date. The present proposal aims to explore septin complexes with the initial focus being on the octameric hetero-complex formed by septins 3, 5, 7 and 8 due to its physiological relevance given that all such septins are expressed in the central nervous system. Furthermore, new hetero-complexes from the model organism Ciona intestinalis will be co-expressed. The correct folding and stability of such complexes will be studied by circular dichroism. Electron microscopy will be used to determine the oligomeric state and homogeneity of the complexes obtained. It is worthy of note that the use of single particle crio-electron microscopy has grown rapidly over recent years and has become an attractive alternative to crystallography particularly for cases where crystals are not readily obtained. Despite the fact that this project is limited, at least initially, to the use of negative stain, in the future it is expected to evolve towards the use of crio-EM. Furthermore, the complexes obtained will also be subjected to crystallization trials with a view to obtaining their structures by X-ray diffraction methods. The results generated during this project should help to elucidate the molecular mechanism of oligomerization and to produce structural data useful in the understanding of the cellular events in which spetins are involved. (AU)