Li Lin, Wu Xiaomei, Yue Hai-Yuan, Zhu Yong-Chuan, Xu Jianhua
Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Augusta, Georgia, USA.
Department of Neurochemistry, Institute of Nautical Medicine, Nantong University, Nantong, Jiangsu, China.
J Neurochem. 2016 Jul;138(1):60-73. doi: 10.1111/jnc.13635. Epub 2016 May 16.
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.
在神经末梢，内吞作用有效地回收囊泡膜，以在不同水平的神经元活动下维持突触传递。钙离子（Ca²⁺）及其下游信号通路对于内吞作用的活动依赖性调节至关重要。据报道，一种依赖于活动和Ca²⁺的激酶，肌球蛋白轻链激酶（MLCK），可调节不同突触中的囊泡动员、囊泡循环和运动，但它是否对神经末梢内吞作用的调节有普遍贡献仍不清楚。我们通过将囊泡内吞作用成像为用pH敏感的绿色荧光蛋白标记的囊泡突触素的实时回收，在大鼠海马小体中研究了这个问题。我们发现，急性抑制MLCK以及用RNA干扰下调MLCK后，由200次动作电位（5 - 40Hz）诱导的内吞作用减慢，而囊泡外排总量和体细胞Ca²⁺通道电流在MLCK下调时并未改变。急性抑制肌球蛋白II同样损害内吞作用。此外，下调MLCK可阻止去极化诱导的肌球蛋白轻链磷酸化，这一效应与Ca²⁺通道阻滞剂和钙调蛋白相同。这些结果表明，MLCK通过Ca²⁺/钙调蛋白下游的肌球蛋白的活动依赖性磷酸化促进囊泡内吞作用，这可能是突触中广泛存在的一种机制。我们的研究表明，MLCK是海马神经元中囊泡循环的重要活动依赖性调节因子，而海马神经元对学习和记忆至关重要。长期以来已知神经末梢囊泡膜内吞作用的动力学依赖于活动和Ca²⁺。这项研究提供了证据表明，肌球蛋白轻链激酶通过介导肌球蛋白的Ca²⁺/钙调蛋白依赖性磷酸化来提高海马神经元的内吞效率。作者提出，这种信号级联可能是突触中囊泡内吞作用的活动依赖性调节的共同途径。
