Non-canonical septins interactions: analysis of the interaction via G interface of SEPT3 and group II septins

Author(s)

Paola Lanzoni

• Document type: Master's Dissertation
• Press: São Carlos.
• Institution: Universidade de São Paulo (USP). Instituto de Física de São Carlos (IFSC/BT)
• Defense date: 2017-05-26

Examining board members

Richard Charles Garratt; José Luiz de Souza Lopes; Wanius Jose Garcia da Silva

• Advisor: Richard Charles Garratt

Abstract

The septins are accepted to be the fourth cytoskeleton component of the eukaryotic cells, after actin, myosin and intermediate filaments. They are filament forming proteins that are organized in fibers and rings, having a structural role in the cell. Humans express 13 septins, which are divided into 4 different groups according to their primary structure: group I (SEPT3, SEPT9, SEPT12); group II (SEPT6, SEPT8, SEPT10, SEPT11, SEPT14); group III (SEPT1, SEPT2, SEPT4, SEPT5) e group IV (SEPT7). SEPT13 was later characterized as a SEPT7 pseudogene. The best characterized filament is built up from SEPT2-SEPT6-SEPT7-SEPT9 (in this exact sequence), and is used as a basis for the description of the so-called canonical arrangement, which accepts that septins from the same group can occupy the same position within the filament. However yeast two-hybrid assays identified several unexpected interactions such as SEPT9-SEPT6 and SEPT9-SEPT8, raising the possibility that these could also exist in vivo. Furthermore, studies have shown the existence of interactions between group I and group II, and especially in the SEPT11-SEPT12, the interaction dissolves when a mutation in the G interface is inserted. The present work investigates the interaction between SEPT3, a group I septin, with all of those from group II. This interaction was studied through co-expression and co-purification methods using metal affinity chromatography, where only the SEPT3 contained the six histidines extention. This initial analysis showed that SEPT3 did not co-purify with all group II members, clearly pointing to variability in the affinity within group. Using this approach SEPT6, SEPT10 e SEPT14 showed no interaction with SEPT3, whilst SEPT8 and SEPT11 co-purified with SEPT3, but not in stoichiometric concentrations. For the SEPT3-SEPT8 and SEPT3-SEPT11 complexes, a second purification stage was performed using size exclusion chromatography, where a broad peak was observed corresponding to a molecular mass value which was intermediate between a dimer and a monomer. The same complexes, when evaluated by multiple angle light scattering revealed a variation in the molecular mass across the peak as it eluted. Such variation was compatible with elution of dimers at the beginning and monomers at the end. Studies for the SEPT3-SEPT8 interaction via analytical ultracentrifugation suggested a trend to associate in high protein concentration, consistent with the dissociation constant found by microscale thermophoresis, which was of the order of ten micromolar. The results raise questions concerning the physiological relevance of these complexes and reinforce the importance of further studies on the non-canonical assembly of septin complexes for cellular development. (AU)

• FAPESP's process: 14/11772-7 - Non-canonical septins interactions: analysis of the interaction via G interface of SEPT3 and group II septins
• Grantee: Paola Lanzoni
• Support Opportunities: Scholarships in Brazil - Master