Visualization of synaptic vesicle movement in intact synaptic boutons using fluorescence fluctuation spectroscopy.
Not much is known about the mobility of synaptic vesicles inside small synapses of the central nervous system, reflecting a lack of methods for visualizing these dynamics. We adapted confocal spot detection with fluctuation analysis to monitor the mobility of fluorescently labeled synaptic vesicles inside individual boutons of cultured hippocampal neurons. Using Monte Carlo simulations we were able to propose a simple quantitative model that can describe vesicle mobility in small hippocampal boutons under resting conditions and different pharmacological treatments. We find that vesicle mobility in a time window of 20 s can be well described by caged diffusion (D approximately 5 x 10(-5) microm(2)/s, cage sizes of approximately 50 nm). Mobility can be upregulated by phosphatase blockage and increased further by actin disruption in a dose-dependent manner. Inhibition of the myosin light chain kinase slows down vesicle mobility 10-fold, whereas other kinases like protein kinase C (PKC), A (PKA), and calmodulin kinase II (caMKII) do not affect mobility in unstimulated boutons.
Jordan
Randolf
R
Max-Planck Institute for Biophysical Chemistry, Department of Membrane Biophysics, Am Fassberg 11, 37077 Goettingen, Germany.
Lemke
Edward A
EA
Klingauf
Jurgen
J
D000199:Actins / Q000737:chemistry; D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry; D003599:Cytoskeleton / Q000378:metabolism; D004058:Diffusion; D004305:Dose-Response Relationship, Drug; D006624:Hippocampus / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence; D009010:Monte Carlo Method; D009068:Movement; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009218:Myosins / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D011493:Protein Kinase C / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence / Q000379:methods*; D019311:Staurosporine / Q000494:pharmacology; D013569:Synapses / Q000473:pathology*; D013997:Time Factors; D014404:Tubulin / Q000737:chemistry; D015854:Up-Regulation
D000199:Actins; D014404:Tubulin; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D010744:Phosphoric Monoester Hydrolases; D009218:Myosins; D019311:Staurosporine