Structural studies of septin complexes analyzed by transmission electron microscopy

Author(s)

Déborah Cezar Mendonça

• Document type: Doctoral Thesis
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2023-05-19

Examining board members

Richard Charles Garratt; Andre Luis Berteli Ambrosio; Shaker Chuck Farah; Germán Gustavo Sgro; Napoleão Fonseca Valadares

• Advisor: Richard Charles Garratt; Rodrigo Villares Portugal

Abstract

Septins are cytoskeletal GTPases capable of associating to form heterocomplexes, usually hexamers or octamers, which polymerize into filaments that subsequently organize into more complex structures such as rings and networks. They are involved in a number of important intracellular processes including cell division, vesicle trafficking, exocytosis, among others. In humans, changes in the expression pattern of these proteins or the presence of mutations are related to diseases, such as some types of cancer and neurological disorders such as Parkinson\'s and Alzheimer\'s diseases. However, some mechanical aspects of these proteins are still not fully understood, including how heterocomplexes assemble correctly. In this work, the complexes of human and urochordate C. intestinalis septins were studied using transmission electron microscopy. For both, the order of the hexameric complexes (composed of septins 2, 6 and 7) was determined, concluding that the SEPT2 of both organisms is located at the end of the complex (2-6-7-7-6-2). Specifically for the human complex, this result proves that the order of the hexamer previously accepted in the literature was inverted, and the correction of this information made it possible to correlate the human complex with complexes from other organisms, in addition to explaining how hexamers and octamers can be part of the same filament. To obtain structural details of the complexes, the structures of the two hexamers were solved by Cryo-EM, with a resolution of 3.6 Å for the human complex and 3.3 Å for the C. intestinalis complex. The structural analysis of the human complex allowed the understanding of the molecular details of the interface formed between SEPT6 and SEPT7 with a higher resolution compared to the previous crystallographic structure. The NC interface features a cavity that is closed at its base by the α0 helices of the two subunits, which include a polybasic region. They are kept buried within the interface through the interaction of these basic residues with the elbow formed between the α5 and α6 helices of the neighboring subunit. Observing the space left by the cavity and the important residues to stabilize the α0 helix in its position help to understand how the NC interface is capable of undergoing a conformational change. Additionally, it was possible to observe a flexibility of the complex as a whole in a specific direction, which may be related to the way that the septin complexes interact with membranes. In the case of C. intestinalis, the structural information of the three septins present in the complex is unprecedented and shows that the structure of the septins and the way they interact at the molecular level are evolutionarily conserved. (AU)

• FAPESP's process: 18/20209-5 - Structural studies on the assembly of septin complexes by transmission electron microscopy and single particle analysis
• Grantee: Deborah Cezar Mendonça
• Support Opportunities: Scholarships in Brazil - Doctorate