The Role of Actin Turnover in Retrograde Actin Network Flow in Neuronal Growth Cones

Protein kinase C (PKC) can dramatically alter cell structure and motility via effects on actin filament networks. In neurons, PKC activation has been implicated in repulsive guidance responses and inhibition of axon regeneration; however, the cytoskeletal mechanisms underlying these effects are not well understood. Here we investigate the acute effects of PKC activation on actin network structure and dynamics in large Aplysia neuronal growth cones. We provide evidence of a novel two-tiered mechanism of PKC action: 1) PKC activity enhances myosin II regulatory light chain phosphorylation and C-kinase–potentiated protein phosphatase inhibitor phosphorylation. These effects are correlated with increased contractility in the central cytoplasmic domain. 2) PKC activation results in significant reduction of P-domain actin network density accompanied by Arp2/3 complex delocalization from the leading edge and increased rates of retrograde actin network flow. Our results show that PKC activation strongly affects both actin polymerization and myosin II contractility. This synergistic mode of action is relevant to understanding the pleiotropic reported effects of PKC on neuronal growth and regeneration.

Download Full-text

Glycogen synthase kinase 3beta phosphorylation of microtubule-associated protein 1B regulates the stability of microtubules in growth cones

Journal of Cell Science ◽

10.1242/jcs.112.19.3373 ◽

1999 ◽

Vol 112 (19) ◽

pp. 3373-3384 ◽

Cited By ~ 14

Author(s):

R.G. Goold ◽

R. Owen ◽

P.R. Gordon-Weeks

Keyword(s):

Growth Cone ◽

Glycogen Synthase ◽

Growth Cones ◽

Microtubule Dynamics ◽

Neuronal Growth ◽

Transfected Cells ◽

Cos Cells ◽

Glycogen Synthase Kinase 3Beta ◽

Neuronal Growth Cones

We have recently shown that glycogen synthase kinase 3beta (GSK3beta) phosphorylates the microtubule-associated protein (MAP) 1B in an in vitro kinase assay and in cultured cerebellar granule cells. Mapping studies identified a region of MAP1B high in serine-proline motifs that is phosphorylated by GSK3beta. Here we show that COS cells, transiently transfected with both MAP1B and GSK3beta, express high levels of the phosphorylated isoform of MAP1B (MAP1B-P) generated by GSK3beta. To investigate effects of MAP1B-P on microtubule dynamics, double transfected cells were labelled with antibodies to tyrosinated and detyrosinated tubulin markers for stable and unstable microtubules. This showed that high levels of MAP1B-P expression are associated with the loss of a population of detyrosinated microtubules in these cells. Transfection with MAP1B protected microtubules in COS cells against nocodazole depolymerisation, confirming previous studies. However, this protective effect was greatly reduced in cells containing high levels of MAP1B-P following transfection with both MAP1B and GSK3beta. Since we also found that MAP1B binds to tyrosinated, but not to detyrosinated, microtubules in transfected cells, we propose that MAP1B-P prevents tubulin detyrosination and subsequent conversion of unstable to stable microtubules and that this involves binding of MAP1B-P to unstable microtubules. The highest levels of MAP1B-P are found in neuronal growth cones and therefore our findings suggest that a primary role of MAP1B-P in growing axons may be to maintain growth cone microtubules in a dynamically unstable state, a known requirement of growth cone microtubules during pathfinding. To test this prediction, we reduced the levels of MAP1B-P in neuronal growth cones of dorsal root ganglion cells in culture by inhibiting GSK3beta with lithium. In confirmation of the proposed role of MAP1B-P in maintaining microtubule dynamics we found that lithium treatment dramatically increased the numbers of stable (detyrosinated) microtubules in the growth cones of these neurons.

Download Full-text