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Protein kinase C activation promotes microtubule advance in neuronal growth cones by increasing average microtubule growth lifetimes.

Kabir N, Schaefer AW, Nakhost A, Sossin WS, Forscher P - J. Cell Biol. (2001)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Yale University, New Haven, Connecticut 06520-8103, USA.

ABSTRACT
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.

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Quantification of the MT invasion data. Bag cells were treated with various drugs as indicated for 30 min, and then fixed and stained for MTs and F-actin. MT density was calculated by counting the number of MTs crossing >75% of the P-domain and normalizing by area. *P < 0.001, Student's t test.
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Figure 3: Quantification of the MT invasion data. Bag cells were treated with various drugs as indicated for 30 min, and then fixed and stained for MTs and F-actin. MT density was calculated by counting the number of MTs crossing >75% of the P-domain and normalizing by area. *P < 0.001, Student's t test.

Mentions: MT density in the P-domain was measured by counting the number of microtubules spanning 75% of the P-domain as defined by distal and proximal borders of F-actin bundle labeling (see Fig. 3). Data was normalized between growth cones by GC area and expressed as the number of MT/100 μm2; i.e., MT density.


Protein kinase C activation promotes microtubule advance in neuronal growth cones by increasing average microtubule growth lifetimes.

Kabir N, Schaefer AW, Nakhost A, Sossin WS, Forscher P - J. Cell Biol. (2001)

Quantification of the MT invasion data. Bag cells were treated with various drugs as indicated for 30 min, and then fixed and stained for MTs and F-actin. MT density was calculated by counting the number of MTs crossing >75% of the P-domain and normalizing by area. *P < 0.001, Student's t test.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198821&req=5

Figure 3: Quantification of the MT invasion data. Bag cells were treated with various drugs as indicated for 30 min, and then fixed and stained for MTs and F-actin. MT density was calculated by counting the number of MTs crossing >75% of the P-domain and normalizing by area. *P < 0.001, Student's t test.
Mentions: MT density in the P-domain was measured by counting the number of microtubules spanning 75% of the P-domain as defined by distal and proximal borders of F-actin bundle labeling (see Fig. 3). Data was normalized between growth cones by GC area and expressed as the number of MT/100 μm2; i.e., MT density.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Yale University, New Haven, Connecticut 06520-8103, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus