Autosomal dominant Centronuclear myopathy (CNM) is a rare neuromuscular disorder due to mutations in the DNM2 gene coding for Dynamin 2 (DNM2). We have demonstrated that the ubiquitous clathrin heavy chain (CHC) and DNM2, well characterized for their role in endocytosis of clathrin-coated vesicles, regulate the formation of large clathrin-coated plaques. These structures are localized at plasma membrane (PM) adhesion sites known as costameres where they associate with branched actin filaments. Clathrin plaques are absolutely required for organizing the actin cytoskeleton at the costameres and for subsequent formation of the contractile apparatus. Both in vitro and in vivo depletion of clathrin disrupts actin scaffolding, costamere formation and the capacity of muscle tissue to generate mechanical force. This project aims at understanding the articulated mechanisms of clathrin-coated plaque regulation and to identify the signalling cascades initiated at these structures which contribute to transduce mecanical stimuli into biochemical responses. We hypothesize that CHC and DNM2 sense mechanical forces through interactions with actin binding proteins (Hip1, Hip1R, cortactin), and adjust actin scaffolding. We will use both in vitro and in vivo approaches coupled to high resolution electron microscopy, intravital imaging and biochemical analysis in order to decipher the contribution of CHC, DNM2, and several actin binding proteins to plaque assembly and costamere organization. We will test the possibility that CHC and DNM2 phosphorylation by the Src and FAK kinases induces assembly of clathrin-coated plaques at the plasma membrane and we will analyze the effect of stretching and contractions on clathrin-coated plaque abundance. In light of our identification of DNM2 gene mutations as the cause for autosomal dominant centronuclear myopathy (CNM), we will address the effect of CNM causing DNM2 mutants on clathrin-coated plaque assembly and costamere integrity. Experiments will be conducted on several experimental models including DNM2 knock-in/knock-out mice and myogenic cells from CNM patients. Our proposal will establish a novel unconventional role for endocytosis proteins in cellular architecture and mechanical force transduction. This project may also prove relevant to additional tissues and to the pathophysiology of diseases such as CNM, other neuromuscular disorders where costamere integrity is compromised and cancer where abnormal clathrin plaque assembly could perturb adhesion leading to aberrant cellular migration.