Myosin light chain kinase facilitates endocytosis of synaptic vesicles at hippocampal boutons

Abstract

At nerve terminals, endocytosis efficiently recycles vesicle membrane to maintain synaptic transmission under different levels of neuronal activity. Ca2+ and its downstream signal pathways are critical for the activity-dependent regulation of endocytosis. An activity- and Ca2+-dependent kinase, myosin light chain kinase (MLCK) has been reported to regulate vesicle mobilization, vesicle cycling and motility in different synapses, but whether it has a general contribution to regulation of endocytosis at nerve terminals remains unknown. We investigated this issue at rat hippocampal boutons by imaging vesicle endocytosis as the real-time retrieval of vesicular synaptophysin tagged with a pH-sensitive green fluorescence protein. We found that endocytosis induced by 200 action potentials (5 – 40 Hz) was slowed by acute inhibition of MLCK and downregulation of MLCK with RNA interference, while the total amount of vesicle exocytosis and somatic Ca2+ channel current did not change with MLCK downregulation. Acute inhibition of myosin II similarly impaired endocytosis. Furthermore, downregulation of MLCK prevented depolarization-induced phosphorylation of myosin light chain, an effect shared by blockers of Ca2+ channels and calmodulin. These results suggest that MLCK facilitates vesicle endocytosis through activity-dependent phosphorylation of myosin downstream of Ca2+/calmodulin, probably as a widely existing mechanism among synapses. Our study suggests that MLCK is an important activity-dependent regulator of vesicle recycling in hippocampal neurons, which are critical for learning and memory.

Calmodulin Bidirectionally Regulates Evoked and Spontaneous Neurotransmitter Release at Retinal Ribbon Synapses

“…MLCK has been suggested to be involved in regulation of neurotransmitter release, with opposing effects observed at different synapses (Mochida, 1995;Mochida et al, 1994;Polo-Parada et al, 2005;Ryan, 1999;Srinivasan et al, 2008), even though there is a study arguing that MLCK is not a regulator for synaptic vesicle trafficking in hippocampal neurons (Tokuoka and Goda, 2006). In addition, MLCK accelerates vesicle endocytosis at the calyx of Held and hippocampal synapses (Li et al, 2016;Yue and Xu, 2014) and enhances ribbon replenishment in cone photoreceptors (Van Hook et al, 2014). Our findings support the role that MLCK plays in regulating neurotransmitter release.…”

Impairing the function of MLCK, myosin Va or myosin Vb disrupts Rhinovirus B14 replication

“…However, wortmannin can also impair MLCK function by directly binding at or near to the catalytic site of the enzyme in an irreversible manner 37 . Furthermore, MLCK is a calmodulin activated kinase responsible for myosin light chain phosphorylation, thus modulating myosin II binding to F-actin, and is described to be essential in endocytic routes in non-muscular cells 38 . To study whether MLCK might also be involved in RV-B14 entry, cells were pretreated with 10 µM ML9 30 min before infection.…”

High glucose upregulates myosin light chain kinase to induce microfilament cytoskeleton rearrangement in hippocampal neurons

“…In synapses, MLCK and its downstream substrate myosin, regulate different stages of neurotransmitter release ( 30 ), including refilling of readily releasable pools following tetanus stimulation, vesicle mobility ( 31 , 32 ), supply of fast-releasing vesicles ( 33 ) and vesicle mobilization ( 34 ). MLCK is required for activity-dependent functions of myosin in hippocampal neurons ( 35 ). Certain cellular effects of MLCK result from its phosphorylation of MLC ( 36 ).…”