Myosin Light Chain Kinase Facilitates Endocytosis of Synaptic Vesicles at Hippocampal Boutons

J Neurochem. 2016 Jul;138(1):60-73. doi: 10.1111/jnc.13635. Epub 2016 May 16.

Abstract

At nerve terminals, endocytosis efficiently recycles vesicle membrane to maintain synaptic transmission under different levels of neuronal activity. Ca(2+) and its downstream signal pathways are critical for the activity-dependent regulation of endocytosis. An activity- and Ca(2+) -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 down-regulation of MLCK with RNA interference, while the total amount of vesicle exocytosis and somatic Ca(2+) channel current did not change with MLCK down-regulation. Acute inhibition of myosin II similarly impaired endocytosis. Furthermore, down-regulation of MLCK prevented depolarization-induced phosphorylation of myosin light chain, an effect shared by blockers of Ca(2+) channels and calmodulin. These results suggest that MLCK facilitates vesicle endocytosis through activity-dependent phosphorylation of myosin downstream of Ca(2+) /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. The kinetics of vesicle membrane endocytosis at nerve terminals has long been known to depend on activity and Ca(2+) . This study provides evidence suggesting that myosin light chain kinase increases endocytosis efficiency at hippocampal neurons by mediating Ca(2+) /calmodulin-dependent phosphorylation of myosin. The authors propose that this signal cascade may serve as a common pathway contributing to the activity-dependent regulation of vesicle endocytosis at synapses.

Keywords: endocytosis; hippocampus; myosin light chain kinase; vesicle cycling.

MeSH terms

  • Androstadienes / pharmacology
  • Animals
  • Animals, Newborn
  • Cells, Cultured
  • Down-Regulation / drug effects
  • Down-Regulation / genetics
  • Endocytosis / drug effects
  • Endocytosis / physiology*
  • Enzyme Inhibitors / pharmacology
  • Exocytosis / drug effects
  • Exocytosis / physiology
  • Hippocampus / cytology*
  • Membrane Potentials / drug effects
  • Membrane Potentials / genetics
  • Myosin-Light-Chain Kinase / genetics
  • Myosin-Light-Chain Kinase / metabolism*
  • Neurons / cytology*
  • Neurons / drug effects
  • Neurons / physiology*
  • Patch-Clamp Techniques
  • Phosphorylation / drug effects
  • Phosphorylation / genetics
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Rats
  • Synaptic Vesicles / drug effects
  • Synaptic Vesicles / physiology*
  • Transfection
  • Wortmannin

Substances

  • Androstadienes
  • Enzyme Inhibitors
  • RNA, Small Interfering
  • Myosin-Light-Chain Kinase
  • Wortmannin