A role for non-muscle Myosin II in force generation of muscle progenitor cells characterized by traction force microscopy

Abstract

In most tissues, the majority of forces at a subcellular level are driven by the contraction of the molecular motor Non-Muscle Myosin II (NMII). Force generation relies on a conformational movement of the NMII head while bound to actin. This process is fuelled by A TP ( Adenosine TriPhosphate) hydrolysis. This movement produces a linear displacement of the actin filament to which NMII is bound, doing mechanical work. Such work is essential in the evolution of the cells and tissues, and it has profound ramifications in physiology and pathology. Due to the critical role of force generation in development and homeostasis, this is a tightly controlled event. A major checkpoint is the phosphorylation of the RLC (Regulatory Light Chain) of NMII in serine aminoacid at position 19 (Ser19). Phospho-Ser19 RLC catalyzes ATP hydrolysis, powering movement and work generation, whereas dephosphorylated RLC inhibits ATP hydrolysis, thereby preventing force generation. Forces are particularly important in muscle remodelling. Progenitor muscle cells initially produce only NMII, progressively expressing Muscle Myosin II (MMII) as they differentiate into functional muscle fibers. Strikingly, the role of NMII in muscle cells remains relatively unexplored, with few reports addressing the possible interplay between NMII and MMII. In this work, we intend to perform an initial characterization of the role of NMII in the generation of mechanical forces by pre-muscle cells. This study is divided in two parts. In the first part, we have validated a model of musde differentiation using a cell line of undifferentiated myoblasts (Sol 8). We have demonstrated their ability to form twitching (fast) fibers by phase contrast microscopy and the appearance of molecular differentiation markers, namely MMII and metavinculin. Using vVestern blot and immunofluorescence microscopy, we prove that chemical treatments used in other cell lines to ma.nipulate the function of NMII are also useful to control NMII function in these cells. Finally, we aimed at characterizing the ability of these cells to generate NMII-dependent forces and work on a controlled substratum. This was done using a form of qua.ntitative microscopy named Traction Force Microscopy (TFM). The inhibition of the Rho Dependent Kinase using the specific inhibitor Y-27632 and the inhibition of Myosin Light Chain Kinase (MLCK) by ML-7 had a detectable decrease in the traction forces exerted by the cells. Interestingly, a well-documented activator of NMII, Calyculin A, had not a quantifiable traction increase by this technique. In summary, this work provides initial evidences of the role of NMII in force generation by pre-muscle cells and lays out important questions regarding the specific functions and interplay of NMII and MMII during muscle differentiation and repair.