Dysferlin is a transmembrane C-2 domain-containing protein involved in vesicle trafficking and membrane remodeling in skeletal muscle cells. However, the mechanism by which dysferlin regulates these cellular processes remains unclear. Since actin dynamics is critical for vesicle trafficking and membrane remodeling, we studied the role of dysferlin in Ca²⁺-induced G-actin incorporation into filaments in four different immortalized myoblast cell lines (DYSF2, DYSF3, AB320, and ER) derived from patients harboring mutations in the dysferlin gene. As compared with immortalized myoblasts obtained from a control subject, dysferlin expression and G-actin incorporation were significantly decreased in myoblasts from dysferlinopathy patients. Stable knockdown of dysferlin with specific shRNA in control myoblasts also significantly reduced G-actin incorporation. The impaired G-actin incorporation was restored by the expression of full-length dysferlin as well as dysferlin N-terminal or C-terminal regions, both of which contain three C2 domains. DYSF3 myoblasts also exhibited altered distribution of annexin A2, a dysferlin partner involved in actin remodeling. However, dysferlin N-terminal and C-terminal regions appeared to not fully restore such annexin A2 mislocation. Then, our results suggest that dysferlin regulates actin remodeling by a mechanism that does to not involve annexin A2.

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 μM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.

Background/Objectives: Dysferlinopathies are inherited muscular dystrophies caused by DYSF mutations that lead to progressive muscle degeneration. Proteomic analyses of patient muscle reveal altered abundance of proteins involved in regulated exocytosis and energy metabolism, reflecting complex molecular remodeling within these pathways. However, whether dysferlin deficiency impairs GLUT4 exocytosis remains unknown. Methods: Immortalized human myoblasts from a healthy control (C25) and a dysferlinopathy patient (DYSF2) were used. Cytosolic Ca²⁺ was measured using Fura-2 and Fluo-4; submembrane Ca²⁺ dynamics were recorded with membrane-targeted sensor pN1-Lck-GCaMP3. Single exocytosis events induced by 10 µM ionomycin or 100 nM insulin were tracked with IRAP-pHluorin and total internal reflection fluorescence microscopy (TIRFM). GLUT4 puncta near the plasma membrane were quantified using immunofluorescence and visualized by TIRFM. Dysferlin-3HA was expressed to test rescue. Results: Both cell lines showed comparable cytosolic and submembrane Ca²⁺ responses to ionomycin. However, the frequency of IRAP-pHluorin exocytosis events evoked by both ionomycin and insulin was significantly reduced in DYSF2 myoblasts. Expression of dysferlin-3HA restored insulin-induced exocytosis in DYSF2 cells to control levels but failed to fully rescue ionomycin-induced events. Insulin increased the trafficking of GLUT4 puncta near the plasma membrane in C25 cells, whereas DYSF2 cells lacked this insulin-stimulated response. Conclusions: In the patient-derived cell line examined here, dysferlin deficiency impaired stimulus-evoked exocytosis and insulin-induced GLUT4 translocation without altering Ca²⁺ dynamics. The inability of dysferlin-3HA to significantly rescue ionomycin-induced exocytosis points to additional downstream defects, likely secondary proteomic alterations, that constrain full functional recovery.

Objective: Autosomal dominant centronuclear myopathy (AD-CNM) is a neuromuscular congenital disease caused by mutations in the DNM2 gene that encodes dynamin-2 (DNM2). The main clinical features of AD-CNM are progressive weakness and atrophy of skeletal muscles. However, cognitive defects have also been reported, suggesting that AD-CNM-causing mutations in DNM2 might also affect central nervous system (CNS). We recently demonstrated that defects in excitatory synaptic transmission occur in the brain of transgenic knock-in (KI) mice harboring the DNM2 p.R465W mutation, the most common causing AD-CNM. As DNM2 regulates the trafficking of glutamate-AMPA receptors (AMPARs), major mediators of excitatory synaptic transmission in mammals, it is feasible that the synaptic availability of AMPAR is affected in the context of AD-CNM. The main objective of this work was to evaluate the impact of the p.R465W DNM2 mutation on the GluA1-AMPAR-subunit synaptic availability in the brain of KI mice. Methods: We addressed an experimental quantitative study. By using subcellular fractionation and western blot we quantified the expression of GluA1 and synaptic proteins in hippocampal total homogenates and postsynaptic densities (PSDs) in the brain of WT and KI mice. By total internal reflection microscopy (TIRFM) we also analyzed the arrival and residence time of GluA1 into the plasma membrane of hippocampal cultured neurons. Results: Although we did not observe significant differences in the GluA1 expression in hippocampal total homogenates, it was significantly reduced in the PSDs of KI compared to wild-type (WT) brains. Moreover, the residence time of GluA1 in the surface membranes of KI hippocampal neurons was significantly reduced compared to WT neurons. Conclusion: These data strongly suggest that the p.R465W mutation in DNM2 perturbs synaptic GluA1-availability in hippocampal neurons, likely leading to defects in excitatory synaptic transmission.