Myosin Light Chain Kinase Accelerates Vesicle Endocytosis at the Calyx of Held Synapse

Abstract

Neuronal activity triggers endocytosis at synaptic terminals to retrieve efficiently the exocytosed vesicle membrane, ensuring the membrane homeostasis of active zones and the continuous supply of releasable vesicles. The kinetics of endocytosis depends on Ca 2ϩ and calmodulin which, as a versatile signal pathway, can activate a broad spectrum of downstream targets, including myosin light chain kinase (MLCK). MLCK is known to regulate vesicle trafficking and synaptic transmission, but whether this kinase regulates vesicle endocytosis at synapses remains elusive. We investigated this issue at the rat calyx of Held synapse, where previous studies using whole-cell membrane capacitance measurement have characterized two common forms of Ca 2ϩ /calmodulin-dependent endocytosis, i.e., slow clathrin-dependent endocytosis and rapid endocytosis. Acute inhibition of MLCK with pharmacological agents was found to slow down the kinetics of both slow and rapid forms of endocytosis at calyces. Similar impairment of endocytosis occurred when blocking myosin II, a motor protein that can be phosphorylated upon MLCK activation. The inhibition of endocytosis was not accompanied by a change in Ca 2ϩ channel current. Combined inhibition of MLCK and calmodulin did not induce synergistic inhibition of endocytosis. Together, our results suggest that activation of MLCK accelerates both slow and rapid forms of vesicle endocytosis at nerve terminals, likely by functioning downstream of Ca 2ϩ /calmodulin.

Synaptic Vesicle Endocytosis Occurs on Multiple Timescales and Is Mediated by Formin-Dependent Actin Assembly

“…Inhibition of myosin II activity by para-nitroblebbistatin caused a significant delay in SV endocytosis resulting in a more than 2-fold increase in the time constant for endocytosis and reacidification ( Figures 3B and 3C). These results are consistent with previous morphological observations in blebbistatin-treated neurons and in myosin II KO mice (Chandrasekar et al, 2013;Yue and Xu, 2014) as well as with the reported role of myosin II in endocytic membrane fission (Flores et al, 2014).…”

“…Our own data together with prior results favor a model in which the internalization step of SV endocytosis is driven by formin/ mDia1-mediated assembly of actin (Ganguly et al, 2015), which has recently been shown to be required for SV endocytosis . Linear actin filaments anchored to the membrane by formin/mDia1 provide a substrate for membrane fission by dynamin (Armbruster et al, 2013;Ferguson et al, 2007;Raimondi et al, 2011) and endophilin Llobet et al, 2011;Milosevic et al, 2011) that act in conjunction with myosin II motors (Chandrasekar et al, 2013;Flores et al, 2014;Yue and Xu, 2014) to catalyze membrane fission. This pathway bears similarity to CIE in yeast, which requires the assembly of linear actin filaments by the formin Bni1 but not N-WASP/Arp2/3-dependent branched actin networks (Prosser et al, 2011).…”

Promotion of endocytosis efficiency through an ATP‐independent mechanism at rat calyx of Held terminals

“…; Yamashita et al . ; Yao & Sakaba, ), myosin light chain kinase/myosin (Yue & Xu, ) and actin polymerization (Wu et al . ), which rely on ATP hydrolysis for functions.…”

Non-muscle myosin II in disease: mechanisms and therapeutic opportunities

“…Likewise, the Rho GTPase Rac regulates the formation of dendritic spines and synapses through its effector kinase, PAK, and subsequent RLC phosphorylation ( Zhang et al, 2005 ). Finally, there is some evidence that Ca 2+ /calmodulin-activated MLCK regulates both pre-synaptic vesicle trafficking ( Polo-Parada et al, 2005 ; Tokuoka and Goda, 2006 ; Yue and Xu, 2014 ), as well as post-synaptic NMDA receptor currents ( Lei et al, 2001 ). Thus, myosin kinases are vitally important regulators of synaptic strengthening through changes in synapse morphology and molecular composition, which underlie excitatory neurotransmission.…”