CAP2 deficiency delays myofibril actin cytoskeleton differentiation and disturbs skeletal muscle architecture and function.

Actin filaments (F-actin) are key components of sarcomeres, the basic contractile units of skeletal muscle myofibrils. A crucial step during myofibril differentiation is the sequential exchange of α-actin isoforms from smooth muscle (α-SMA) and cardiac (α-CAA) to skeletal muscle α-actin (α-SKA) that, in mice, occurs during early postnatal life. This "α-actin switch" requires the coordinated activity of actin regulators because it is vital that sarcomere structure and function are maintained during differentiation. The molecular machinery that controls the α-actin switch, however, remains enigmatic. Cyclase-associated proteins (CAP) are a family of actin regulators with largely unknown physiological functions. We here report a function for CAP2 in regulating the α-actin exchange during myofibril differentiation. This α-actin switch was delayed in systemic CAP2 mutant mice, and myofibrils remained in an undifferentiated stage at the onset of the often excessive voluntary movements in postnatal mice. The delay in the α-actin switch coincided with the onset of motor function deficits and histopathological changes including a high frequency of type IIB ring fibers. Our data suggest that subtle disturbances of postnatal F-actin remodeling are sufficient for predisposing muscle fibers to form ring fibers. Cofilin2, a putative CAP2 interaction partner, has been recently implicated in myofibril actin cytoskeleton differentiation, and the myopathies in cofilin2 and CAP2 mutant mice showed striking similarities. We therefore propose a model in which CAP2 and cofilin2 cooperate in actin regulation during myofibril differentiation.

Cortactin interacts with αDystrobrevin-1 and regulates murine neuromuscular junction morphology.

Neuromuscular junctions transmit signals from the nervous system to skeletal muscles, triggering their contraction, and their proper organization is essential for breathing and voluntary movements. αDystrobrevin-1 is a cytoplasmic component of the dystrophin-glycoprotein complex and has pivotal functions in regulating the integrity of muscle fibers and neuromuscular junctions. Previous studies identified that αDystrobrevin-1 functions in the organization of the neuromuscular junction and that its phosphorylation in the C-terminus is required in this process. Our proteomic screen identified several putative αDystrobrevin-1 interactors recruited to the Y730 site in phosphorylated and unphosphorylated states. Amongst various actin-modulating proteins, we identified the Arp2/3 complex regulator cortactin. We showed that similarly to αDystrobrevin-1, cortactin is strongly enriched at the neuromuscular postsynaptic machinery and obtained results suggesting that these two proteins interact in cell homogenates and at the neuromuscular junctions. Analysis of synaptic morphology in cortactin knockout mice showed abnormalities in the slow-twitching soleus muscle and not in the fast-twitching tibialis anterior. However, muscle strength examination did not reveal apparent deficits in knockout animals.

Arhgef5 Binds α-Dystrobrevin 1 and Regulates Neuromuscular Junction Integrity.

The neuromuscular junctions (NMJs) connect muscle fibers with motor neurons and enable the coordinated contraction of skeletal muscles. The dystrophin-associated glycoprotein complex (DGC) is an essential component of the postsynaptic machinery of the NMJ and is important for the maintenance of NMJ structural integrity. To identify novel proteins that are important for NMJ organization, we performed a mass spectrometry-based screen for interactors of α-dystrobrevin 1 (aDB1), one of the components of the DGC. The guanidine nucleotide exchange factor (GEF) Arhgef5 was found to be one of the aDB1 binding partners that is recruited to Tyr-713 in a phospho-dependent manner. We show here that Arhgef5 localizes to the NMJ and that its genetic depletion in the muscle causes the fragmentation of the synapses in conditional knockout mice. Arhgef5 loss in vivo is associated with a reduction in the levels of active GTP-bound RhoA and Cdc42 GTPases, highlighting the importance of actin dynamics regulation for the maintenance of NMJ integrity.