Myosin-II negatively regulates minor process extension and the temporal development of neuronal polarity.
The earliest stage in the development of neuronal polarity is characterized by extension of undifferentiated "minor processes" (MPs), which subsequently differentiate into the axon and dendrites. We investigated the role of the myosin II motor protein in MP extension using forebrain and hippocampal neuron cultures. Chronic treatment of neurons with the myosin II ATPase inhibitor blebbistatin increased MP length, which was also seen in myosin IIB knockouts. Through live-cell imaging, we demonstrate that myosin II inhibition triggers rapid minor process extension to a maximum length range. Myosin II activity is determined by phosphorylation of its regulatory light chains (rMLC) and mediated by myosin light chain kinase (MLCK) or RhoA-kinase (ROCK). Pharmacological inhibition of MLCK or ROCK increased MP length moderately, with combined inhibition of these kinases resulting in an additive increase in MP length similar to the effect of direct inhibition of myosin II. Selective inhibition of RhoA signaling upstream of ROCK, with cell-permeable C3 transferase, increased both the length and number of MPs. To determine whether myosin II affected development of neuronal polarity, MP differentiation was examined in cultures treated with direct or indirect myosin II inhibitors. Significantly, inhibition of myosin II, MLCK, or ROCK accelerated the development of neuronal polarity. Increased myosin II activity, through constitutively active MLCK or RhoA, decreased both the length and number of MPs and, consequently, delayed or abolished the development of neuronal polarity. Together, these data indicate that myosin II negatively regulates MP extension, and the developmental time course for axonogenesis.
Kollins
K M
KM
Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, USA. katherine.kollins@drexelmed.edu
Hu
J
J
Bridgman
P C
PC
Huang
Y Q
YQ
Gallo
G
G
D000818:Animals; D048708:Cell Growth Processes / Q000187:drug effects / Q000502:physiology; D016764:Cell Polarity / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002642:Chick Embryo; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018994:Myosin Light Chains / Q000378:metabolism; D024462:Myosin Type II / Q000037:antagonists & inhibitors / Q000502:physiology*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009218:Myosins / Q000037:antagonists & inhibitors; D055495:Neurogenesis / Q000187:drug effects / Q000502:physiology*; D009473:Neuronal Plasticity / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation; D016548:Prosencephalon / Q000166:cytology; D013997:Time Factors; D014162:Transfection / Q000379:methods; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D004791:Enzyme Inhibitors; D006576:Heterocyclic Compounds, 4 or More Rings; D007527:Isoenzymes; D018994:Myosin Light Chains; C472645:blebbistatin; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase; D008666:Metalloendopeptidases; C079298:myosinase II; D024462:Myosin Type II; D009218:Myosins