The axon initial segment (AIS) has a key role in the establishment of neuronal polarity, in the generation of action potentials, and as a site of activity-dependent homeostatic plasticity. These AIS functions depend on a complex of voltage-gated sodium channels and an ankyrin G and beta-IV spectrin cytoskeletal scaffold that is enriched in the proximal axon. Recent studies indicate this complex is tethered, via beta-IV spectrin, to submembranous actin rings that are regularly arrayed along the axon. A key unresolved question is why this beta-IV spectrin-ankyrin G-ion channel complex is specifically concentrated in the proximal but not distal actin cytoskeleton. Here we show that phosphorylated myosin light chain (pMLC), which controls actomyosin organization and contractility, is enriched in the proximal axon and has an essential role in demarcating the AIS. pMLC accumulates in the developing AIS coincident with the arrival of ankyrin G and incorporates into the mature, detergent-resistant AIS complex. We further show that a balance of kinase and phosphatase activities regulates the assembly of this pMLC scaffold and thereby of the AIS. Inhibition of myosin light chain kinase in developing neurons markedly reduces the accumulation of pMLC in the proximal axon and the levels of all key AIS components. Conversely, knockout of myosin light chain phosphatase causes ectopic accumulation of pMLC, and other AIS components, in the distal axon. Additionally, we find that MLC is rapidly lost upon depolarization; this activity-dependent change is calcium-dependent and precedes remodeling of the AIS. Genetic ablation of myosin phosphatase does not protect the AIS from activity-dependent remodeling but results in ectopic recruitment of AIS components to the distal axon. Finally, using STORM microscopy, we resolved pMLC as a helical spiral in the AIS and as regularly spaced punctae more distally. Upon depolarization, these helices collapse into small punctae throughout the AIS. Together, these results suggest that MLC phosphorylation, by regulating actomyosin, drives the assembly and delineates the distribution of an ankyrin G-based AIS complex in the proximal axon.
|Advisor:||Salzer, James L.|
|Commitee:||Burden, Steven J., Polleux, Franck, Skolnik, Edward Y., Tsien, Richard W.|
|School:||New York University|
|Department:||Basic Medical Science|
|School Location:||United States -- New York|
|Source:||DAI-B 78/05(E), Dissertation Abstracts International|
|Subjects:||Neurosciences, Cellular biology|
|Keywords:||Actin, Axon, Axon initial segment, MLC, Myosin, Phosphatase|
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