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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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MT dynamics are necessary for PDBu-mediated MT invasion of the P-domain. (A–H) Effects of a 30-min treatment with 100 nM nocodazole (A–D) or 1 μM taxol (E–H) on structure, F-actin, and MTs. Note central domain retraction in both nocodazole (dotted lines, A and B, and taxol, E and F, and accompanying MT retraction, D and H (dotted line indicates proximal P-domain boundary). C- and P-domains are clearly separated after either treatment, yet P-domain actin structure remains intact (C and G) at the 30-min time point. (I) Low doses of nocodazole or taxol inhibit PDBu-mediated MT invasion of the P-domain. Growth cones were treated for 30 min with the doses of nocodazole indicated, and then 1 μM PDBu + Nocodazole for an additional 30 min. Cells were fixed, stained for MTs and F-actin, and MT density was then assessed as in Fig. 3. Note that 10 nM nocodazole inhibited the PDBu-mediated MT invasion of the P-domain. *P < 0.001, Student's t test. Scale bar, 5 μm.
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Figure 5: MT dynamics are necessary for PDBu-mediated MT invasion of the P-domain. (A–H) Effects of a 30-min treatment with 100 nM nocodazole (A–D) or 1 μM taxol (E–H) on structure, F-actin, and MTs. Note central domain retraction in both nocodazole (dotted lines, A and B, and taxol, E and F, and accompanying MT retraction, D and H (dotted line indicates proximal P-domain boundary). C- and P-domains are clearly separated after either treatment, yet P-domain actin structure remains intact (C and G) at the 30-min time point. (I) Low doses of nocodazole or taxol inhibit PDBu-mediated MT invasion of the P-domain. Growth cones were treated for 30 min with the doses of nocodazole indicated, and then 1 μM PDBu + Nocodazole for an additional 30 min. Cells were fixed, stained for MTs and F-actin, and MT density was then assessed as in Fig. 3. Note that 10 nM nocodazole inhibited the PDBu-mediated MT invasion of the P-domain. *P < 0.001, Student's t test. Scale bar, 5 μm.

Mentions: To address whether MT assembly was involved, PDBu effects were assessed under conditions where MT dynamics were damped with low concentrations of nocodazole or taxol. We specifically chose conditions previously shown to inhibit plus-end MT assembly without significantly reducing polymer mass (Bamburg et al. 1986; Tanaka et al. 1995; Rochlin et al. 1996) or, alternatively, to stabilize MTs (Letourneau et al. 1987; Jordan and Wilson 1998b). Fig. 5A–D, illustrates the effects of exposure to 100 nM nocodazole on growth-cone structure and the cytoskeleton. Similar effects were observed after exposure to 1 μM taxol (Fig. 5, E–H). Note retraction of the C-domain (Fig. 5A and Fig. B, Fig. E and Fig. F, dotted lines) and depletion of MTs from the T zone (D and H) observed after a 30-min exposure to either nocodazole or taxol. Interestingly, despite essentially complete separation of C and P cytoplasmic domains and the underlying microtubule retraction, retrograde F-actin flow continued unabated (data not shown) and P-domain F-actin remained relatively unperturbed in both cases. These results suggest that dynamic MTs are not necessary for maintenance of retrograde F-actin flow, but are a prerequisite for MT advance out of the C-domain into more distal regions. In agreement with these results, persistent growth-cone motility and absence of P-domain MTs were reported after treatment of Xenopus neurons with nanomolar levels of vinblastine (Tanaka et al. 1995). Note also the relatively high level of diffuse tubulin labeling in nocodazole (Fig. 5 D) when compared with taxol-treated growth cones (Fig. 5 H), where diffuse tubulin labeling is low, as expected from the decrease in critical tubulin concentration.


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)

MT dynamics are necessary for PDBu-mediated MT invasion of the P-domain. (A–H) Effects of a 30-min treatment with 100 nM nocodazole (A–D) or 1 μM taxol (E–H) on structure, F-actin, and MTs. Note central domain retraction in both nocodazole (dotted lines, A and B, and taxol, E and F, and accompanying MT retraction, D and H (dotted line indicates proximal P-domain boundary). C- and P-domains are clearly separated after either treatment, yet P-domain actin structure remains intact (C and G) at the 30-min time point. (I) Low doses of nocodazole or taxol inhibit PDBu-mediated MT invasion of the P-domain. Growth cones were treated for 30 min with the doses of nocodazole indicated, and then 1 μM PDBu + Nocodazole for an additional 30 min. Cells were fixed, stained for MTs and F-actin, and MT density was then assessed as in Fig. 3. Note that 10 nM nocodazole inhibited the PDBu-mediated MT invasion of the P-domain. *P < 0.001, Student's t test. Scale bar, 5 μm.
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Figure 5: MT dynamics are necessary for PDBu-mediated MT invasion of the P-domain. (A–H) Effects of a 30-min treatment with 100 nM nocodazole (A–D) or 1 μM taxol (E–H) on structure, F-actin, and MTs. Note central domain retraction in both nocodazole (dotted lines, A and B, and taxol, E and F, and accompanying MT retraction, D and H (dotted line indicates proximal P-domain boundary). C- and P-domains are clearly separated after either treatment, yet P-domain actin structure remains intact (C and G) at the 30-min time point. (I) Low doses of nocodazole or taxol inhibit PDBu-mediated MT invasion of the P-domain. Growth cones were treated for 30 min with the doses of nocodazole indicated, and then 1 μM PDBu + Nocodazole for an additional 30 min. Cells were fixed, stained for MTs and F-actin, and MT density was then assessed as in Fig. 3. Note that 10 nM nocodazole inhibited the PDBu-mediated MT invasion of the P-domain. *P < 0.001, Student's t test. Scale bar, 5 μm.
Mentions: To address whether MT assembly was involved, PDBu effects were assessed under conditions where MT dynamics were damped with low concentrations of nocodazole or taxol. We specifically chose conditions previously shown to inhibit plus-end MT assembly without significantly reducing polymer mass (Bamburg et al. 1986; Tanaka et al. 1995; Rochlin et al. 1996) or, alternatively, to stabilize MTs (Letourneau et al. 1987; Jordan and Wilson 1998b). Fig. 5A–D, illustrates the effects of exposure to 100 nM nocodazole on growth-cone structure and the cytoskeleton. Similar effects were observed after exposure to 1 μM taxol (Fig. 5, E–H). Note retraction of the C-domain (Fig. 5A and Fig. B, Fig. E and Fig. F, dotted lines) and depletion of MTs from the T zone (D and H) observed after a 30-min exposure to either nocodazole or taxol. Interestingly, despite essentially complete separation of C and P cytoplasmic domains and the underlying microtubule retraction, retrograde F-actin flow continued unabated (data not shown) and P-domain F-actin remained relatively unperturbed in both cases. These results suggest that dynamic MTs are not necessary for maintenance of retrograde F-actin flow, but are a prerequisite for MT advance out of the C-domain into more distal regions. In agreement with these results, persistent growth-cone motility and absence of P-domain MTs were reported after treatment of Xenopus neurons with nanomolar levels of vinblastine (Tanaka et al. 1995). Note also the relatively high level of diffuse tubulin labeling in nocodazole (Fig. 5 D) when compared with taxol-treated growth cones (Fig. 5 H), where diffuse tubulin labeling is low, as expected from the decrease in critical tubulin concentration.

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