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© The Rockefeller University Press,
0021-9525/2001//1033 $5.00
The Journal of Cell Biology, Volume 152, Number 5,
, 2001 1033-1044
Original Article |
Protein Kinase C Activation Promotes Microtubule Advance in Neuronal Growth Cones by Increasing Average Microtubule Growth Lifetimes
paul.forscher{at}yale.edu
We describe a novel mechanism for protein kinase C regulation of axonal microtubule invasion of growth cones. Activation of PKC by phorbol esters resulted in a rapid, robust advance of distal microtubules (MTs) into the F-actin rich peripheral domain of growth cones, where they are normally excluded. In contrast, inhibition of PKC activity by bisindolylmaleimide and related compounds had no perceptible effect on growth cone motility, but completely blocked phorbol ester effects. Significantly, MT advance occurred despite continued retrograde F-actin flow—a process that normally inhibits MT advance. Polymer assembly was necessary for PKC-mediated MT advance since it was highly sensitive to a range of antagonists at concentrations that specifically interfere with microtubule dynamics. Biochemical evidence is presented that PKC activation promotes formation of a highly dynamic MT pool. Direct assessment of microtubule dynamics and translocation using the fluorescent speckle microscopy microtubule marking technique indicates PKC activation results in a nearly twofold increase in the typical lifetime of a MT growth episode, accompanied by a 1.7-fold increase and twofold decrease in rescue and catastrophe frequencies, respectively. No significant effects on instantaneous microtubule growth, shortening, or sliding rates (in either anterograde or retrograde directions) were observed. MTs also spent a greater percentage of time undergoing retrograde transport after PKC activation, despite overall MT advance. These results suggest that regulation of MT assembly by PKC may be an important factor in determining neurite outgrowth and regrowth rates and may play a role in other cellular processes dependent on directed MT advance.
Key Words: protein kinase C • microtubules • axon outgrowth • actin • growth cone
© 2001 The Rockefeller University Press
The online version of this paper contains supplemental material.Drs. Kabir and Schaefer contributed equally to this work and should be considered co-first authors.
Abbreviations used in this paper: Bis, bisindolylmaleimide; FSM, fluorescent speckle microscopy; GC, growth cone; MAP, microtubule-associated protein; MT, microtubules; OAG, 1-oleoyl-2-acetyl-sn-glycerol; P (or C)-domain, peripheral (or central) domain; PDBu, phorbol dibutyrate; T zone, transition zone; TPA, 12-O-tetradecanoylphorbol-13-acetate; VE-DIC, video-enhanced differential interference microscopy.
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