Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB187

Poster Communications

The role of septins in skeletal muscle

M. Gonczi1, B. Dienes1, P. Szentesi1, K. Cseri1, N. Balogh1, J. Fodor1, L. Csernoch1

1. Department of Physiology, University of Debrecen, Faculty of Medicine, Debrecen, Hungary.

Septins are 30-65 kDa, evolutionarily highly conserved GTP-binding proteins that now deserve recognition as the fourth cytoskeletal component. The encoded 13 septins in humans are either ubiquitous or tissue specific, and they are classified into four homology groups (SEPT2, SEPT3, SEPT6 and SEPT7). Septins control different cellular processes by polymerizing into hetero-oligomeric protein complexes that can further form filaments, bundles or rings. All septin filaments include SEPT7, which is the only member of its group and it occupies the ends of hexameric building blocks which assemble into non-polarized filaments. The function of the ubiquitously expressed SEPT7 is diverse, it has a role in actin dynamics, axon growth, chromosome segregation, cytokinesis and dendrite formation, but also regulates cell shape, membrane trafficking, microtubule regulation, cell motility and DNA repair. Septin assemblies have been shown to act as scaffolds at the plasma membrane to regulate the distribution of membrane-bound proteins. So far there are limited information about the expression and function of septins in skeletal muscle. We have identified several septin isoforms at mRNA and protein levels both in skeletal muscle samples from mice and human, and in cultured C2C12 cell line. We observed ontogenesis- and differentiation-dependent septin 7 expression in mice and C2C12 cells, respectively. To provide more information about the role of septin 7 in skeletal muscle either gene expression was modified using shRNA gene silencing in vitro (Lipofectamine evaluated cell transfection) and in vivo (electroporation of intact muscles). Because SEPT7 knockout is embryonically lethal, we are in the process of generating an in vitro SEPT7 knockout cell line with Crispr-Cas9 technique and an in vivo skeletal muscle-specific septin 7 knockout mouse using the Cre/lox system. In cultured C2C12 cell line stable septin 7 knockdown (KD) clones were generated and marked changes in cell shape and size were observed. The average area and perimeter of the cells increased in septin 7 KD clones, and cells appeared more circular/round following the gene silencing. In control cells the presence of septin 7 was present as a long, filamentous structure throughout the cytoplasm mostly co-localized with actin filaments, while in septin 7 KD cells this well organized structure was broken, the immunpositivity showed pointwise pattern indicating an shRNA-induced inhibition of translation and/or protein degradation. In enzymatically isolated skeletal muscle fibres marked septin 7 immunostaining was detected representing strong co-localization with RyR1. In conclusion, septin 7 has a crucial role in skeletal muscle physiology, and it could have a potential function in muscle regeneration and/or in different muscle diseases.

Where applicable, experiments conform with Society ethical requirements