The formation, maintenance, and regeneration of skeletal muscle tissue depend on the interplay between muscle progenitor cells, known as myoblasts, and other local cell populations. Successful myogenesis requires precise regulation of multiple cellular processes, culminating in the formation of myofibers, multinuclear contractile syncytia from myoblasts. Proliferating myoblasts migrate to the muscle formation region, adhere to each other, express terminal differentiation markers, and eventually fuse their cytoplasmic contents. These processes are characterized by dynamic alterations in cytoskeletal architecture. The cytoskeleton comprises polymerizing proteins such as microfilaments, microtubules, intermediate filaments, and septins, forming an interconnected network of filaments. While the roles of actin, tubulin, and intermediate filaments are well studied, septins have only recently begun to emerge in the myogenic context. In my thesis, I investigated the expression and organization of septin paralogues in dividing and differentiating myoblasts, describing septins as a necessary component of the myoblast cytoskeleton. I propose the existence of septin octamers in myoblasts consisting of Septin2-7-11 and 9, with Septin9 undergoing substantial changes in expression. Septin9 mRNA is induced as quiescent cells transition to activated progenitors in regenerating mouse muscle and is downregulated by the end of the regenerative process. Septin9 mRNA and protein expression were also reduced during in vitro differentiation in C2C12 and primary myoblasts. Cycling myoblasts incorporate Septin9 into actin-decorating septin filaments that form primarily near the nucleus, in close proximity to the plasma membrane. However, these filaments undergo substantial reorganization after myogenic differentiation is induced. I observed that a fraction of Myogenin-positive cells exhibits short, curved septin rods and filament remnants, with no co-localization with actin filaments. This reorganization potentially coincides with the accumulation of muscle specific actin and myosin proteins. These findings were confirmed via live-cell microscopy using an endogenously tagged eGFP-Septin9 C2C12 cell line. Furthermore, nascent myotubes showed septin filaments extending across the perinuclear area in the basal plane, while the rest of the sarcoplasm contained filamentous remnants and rings. Mature myotubes, which exhibit sarcomeric actin organization, were mostly devoid of septin structures. SiRNA-mediated depletion of Septin9 resulted in the disruption of septin filaments and in a premature transition from proliferating to committed transcriptional signature in C2C12 and primary myoblasts, as evident from RNASeq analysis. Additionally, Septin9-deficient myoblasts showed signs of premature cell cycle exit, increased apoptosis, and a precocious onset of myogenic differentiation during in vitro differentiation. Taken together, my findings establish Septin9 as a critical regulator of myoblast differentiation during the initial commitment phase. We propose that filamentous septin structures are part of a temporal regulatory mechanism governing myogenic differentiation, with their reorganization marking the progress of unfolding myoblast differentiation.
