The Role of mDia1/3 Formins and the Actin Cytoskeleton in Synaptic Vesicle Endocytosis

Neurotransmission, essential for sensory perception, motor control, cognition, and behavior, occurs at synapses, where neurotransmitters are released from the presynaptic neuron into the synaptic cleft, triggering responses at the postsynaptic cell. At chemical synapses, neurotransmitter release involves the fusion of synaptic vesicles (SVs) with the presynaptic membrane, necessitating compensatory membrane retrieval, termed synaptic vesicle endocytosis, to reform and refill SVs for subsequent fusion cycles. Despite the recognized role of the Actin cytoskeleton in synaptic vesicle endocytosis, the precise mechanisms governing Actin polymerization and its function within presynaptic nerve terminals remain poorly understood. Here, we delineate the pivotal role of Actin regulatory diaphanous-related formins mDia1/3 and small Rho GTPases, RhoA/B and Rac1, in orchestrating synaptic vesicle recycling at rodent central synapses. Employing optical recordings of presynaptic membrane dynamics, ultrastructural and proteomic analyses, in combination with genetic/pharmacological manipulations, we demonstrate that mDia1/3 localize to the presynaptic membrane, proximal to the endocytic machinery, and govern the formation of presynaptic filamentous Actin structures (F-Actin). Loss of F-Actin due to perturbation of mDia1/3 results in significant alterations in presynaptic architecture, impacting plasma membrane homeostasis. Furthermore, our findings highlight that in the absence of mDia1/3, downregulation of RhoA and activation of Rac1 drive a compensatory response to mitigate the disruption of formin-mediated Actin dynamics. Besides modulating Rho GTPase signaling, we find that mDia1/3 negatively regulate the complex signaling network mediated by the mechanistic target of rapamycin complex 2 (mTORC2). We identify mTORC2 activation to be inversely coupled to the kinetics of SV recycling, likely through the modulation of cytoskeletal dynamics. In conclusion, our study elucidates that the endocytic cytoskeleton is governed by ...