GTPase domain driven dimerization of SEPT7 is dispensable for the critical role of septins in fibroblast cytokinesis.

Megha Abbey,Alexey Kotlyarov,Axel Schambach,Roopsee Anand,Juri Lafera,Andreas Kispert,Matthias Gaestel,Cosima Hakim,Manuel H Taft,Manoj B Menon,Volkhard Kaever

doi:10.1038/srep20007

Megha Abbey, Alexey Kotlyarov + Show 9 more

Open Access

https://doi.org/10.1038/srep20007

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Abstract

Septin 7 (SEPT7) has been described to be essential for successful completion of cytokinesis in mouse fibroblasts, and Sept7-deficiency in fibroblasts constitutively results in multinucleated cells which stop proliferation. Using Sept7flox/floxfibroblasts we generated a cellular system, where the cytokinetic defects of Cre-mediated deletion of the Sept7 gene can be rescued by ectopically expressed doxycycline-inducible wild type SEPT7. Using this system, we analyzed the ability of SEPT7-mutants with alterations in their GTPase domain-dependent dimerization to prevent multinucleation and rescue proliferation. Although biochemical analysis of the mutants demonstrates differences in homo- and/or hetero-polymerization, in GTP-binding and/or GTPase activities, all analyzed mutants were able to rescue the cytokinesis phenotype of Sept7flox/floxfibroblasts associated with Cre-mediated deletion of endogenous Sept7. These findings indicate that the ability of septins to assemble into well-defined SEPT7-dimerization dependent native filaments is dispensable for cytokinesis in fibroblasts and opens the way to search for other mechanisms of the involvement of SEPT7 in cytokinesis.

Highlights

SEPT6 was GTP-bound, leading to the conclusion that SEPT6 family proteins are GTPase-deficient[6,7]
We utilized this model to understand the role played by the well-characterized G-G interface of SEPT7 in fibroblast cytokinesis
The observation that the SEPT7 mutations, despite their alterations in filament assembly and oligomerization (S63N), could rescue the proliferation defects of the SEPT7 knockout fibroblasts indicates that the native-like filament assembly of septins could be dispensable for cytokinesis

Summary

Introduction

SEPT6 was GTP-bound, leading to the conclusion that SEPT6 family proteins are GTPase-deficient[6,7]. Later studies established the presence of an octameric septin assembly with SEPT9 associated at the terminal positions of the canonical hexamer[8]. Higher order assembly of septin filaments are facilitated by the association of these oligomers to the cell membrane[9]. These biophysical studies proposed a model where the position occupied by each septin subunit can be replaced by other group members. Consistent with the pivotal role of SEPT7 in septin assembly, the deletion of Sept[7] in fibroblasts was associated with co-depletion of other core septin subunits. We utilized Sept7-deficient fibroblasts to generate a rescue model for analysis of the effect of different GTPase domain mutations of SEPT7 in fibroblast cytokinesis. We show that neither native septin filaments nor the G-G homodimerization interface of SEPT7 is necessary for SEPT7-dependent completion of fibroblast cytokinesis

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Conclusion