Expression of the Dominant-Negative Tail of Myosin Va Enhances Exocytosis of Large Dense Core Vesicles in Neurons
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Regulated exocytosis of secretory vesicles is a fundamental process in neurotransmission and the release of hormones and growth factors. The F-actin-binding motor protein myosin Va was recently shown to be involved in exocytosis of peptide-containing large dense core vesicles of neuroendocrine cells. It has not previously been discussed whether it plays a similar role in neurons. We performed live-cell imaging of cultured hippocampal neurons to measure the exocytosis of large dense core vesicles containing fluorescently labelled neuropeptide Y. To address the role of myosin Va in this process, neurons were transfected with the dominant-negative tail domain of myosin Va (myosinVa-tail). Under control conditions, about 0.75% of the labelled large dense core vesicles underwent exocytosis during 5 min of stimulation. This value was doubled to 1.80% of the vesicles when myosinVa-tail was expressed. Depolymerization of F-actin using latrunculin B resulted in a similar increase in exocytosis in both control and myosinVa-tail expressing cells. Interestingly, the increase in exocytosis caused by myosinVa-tail expression was completely abolished in the presence of KN-62, an inhibitor of calcium–calmodulin-dependent kinase II. We suggest that myosinVa-tail causes the liberation of large dense core vesicles from the actin cytoskeleton, leading to an increase in exocytosis in the cultured hippocampal neurons.
KeywordsNeuropeptide Y Hippocampal neurons Large dense core vesicles Exocytosis Myosin Va
Enhanced green fluorescent protein
Large dense core vesicles
Region of interest
Calcium- and calmodulin-dependent kinase II
Brain-derived neurotrophic factor
The imaging was performed at the Molecular Imaging Center (FUGE, Norwegian Research Council), University of Bergen. The authors are grateful to W. Almers for providing NPY-EGFP and NPY-mRFP, to J. A. Hammer III for providing myosinVa-tail-mCherry, to C. Kaether providing synaptophysin-EGFP and to the University of Bergen for financial support including research fellowships. H.-H. G. acknowledges grants from the Meltzer Foundation.
Time-lapse recording of a neuron at 18 DIV transfected with NPY-EGFP (green) and mCherry (not shown). One frame was taken every two seconds for ten minutes. Cells were kept in a low- K+-buffer (2,5mM K+) for five minutes (−300s - 0s) followed by a high- K+-buffer (60 mM K+) to depolarize the plasma membrane for another five minutes (0s - 300s).The arrow points to a vesicle (a) that disappears at 214s (= 3,5 min) of depolarization. Please see figure 1 B1 for the intensity profile of vesicle (a).
Control experiment with a neuron transfected with synaptophysin-GFP. The cell was imaged as in movie 1. Note that synaptophysin-GFP signals do not disappear.
The time-lapse recording of a neuron transfected with NPY-EGFP and myosinVa-tail-mCherry.The movie shows NPY-EGFP fluorescence. Arrows point to the vesicles that disappear during plasma membrane depolarization (60 mM K+; 0s - 300s).
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