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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 MT assembly versus translocation dynamics. Data compiled from four control and four PDBu-treated growth cones. DIC and fluorescence images were collected at 10-s intervals for 240 s. A total of 41 control and 37 PDBu MTs were analyzed. (A) PDBu treatment significantly increases the length of time MTs spend in growth phase, *P < 0.00004, paired student's t test. (B) Percentage of time MTs undergo retrograde or anterograde translocation and time not moving under control conditions and after PDBu. The actual time MTs undergo retrograde translocation increases significantly (P < 0.01, paired Student's t test) in control (27 ± 3 s) versus PDBu treatment (50 ± 6 s).
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Figure 8: Quantification of MT assembly versus translocation dynamics. Data compiled from four control and four PDBu-treated growth cones. DIC and fluorescence images were collected at 10-s intervals for 240 s. A total of 41 control and 37 PDBu MTs were analyzed. (A) PDBu treatment significantly increases the length of time MTs spend in growth phase, *P < 0.00004, paired student's t test. (B) Percentage of time MTs undergo retrograde or anterograde translocation and time not moving under control conditions and after PDBu. The actual time MTs undergo retrograde translocation increases significantly (P < 0.01, paired Student's t test) in control (27 ± 3 s) versus PDBu treatment (50 ± 6 s).

Mentions: To directly address the mechanism of the PKC effects described above, we assessed microtubule dynamics before and after treatment with PDBu using FSM. This relatively new microtubule marking technique (Waterman-Storer et al. 1998; Waterman-Storer and Salmon 1998) permits rapid assessment of the critical dynamic parameters of MT behavior, including assembly, disassembly, and translocation (sliding) rates as well as MT lifetimes in each state. Fig. 7 shows DIC-MT overlays illustrating the distribution of MTs before (A) and after (B) PDBu exposure with respect to growth-cone structure. Fig. 7 C is another growth cone in PDBu illustrating extended undbundled MTs typically observed after PDBu treatment. Note the spatial overlap between MTs and actin cables that extend into filopodia (Fig. 7 C, arrowheads), as observed in fixed cells (compare Fig. 4C and Fig. D). Representative MT behavior under control conditions is illustrated in the time-lapse image montages shown in Fig. 7D and Fig. E. MTs underwent stochastic bursts of growth and shortening (green arrowheads indicate the penultimate plus-end speckle) superimposed on relatively short MT translocations in both directions, as indicated by speckles maintained within the body of MTs over time (red arrowheads). Under control conditions, microtubule growth, shrinkage, and sliding activity occurred within the transition zone with only occasional MT excursions into the P-domain. In contrast, after PDBu exposure, MTs often extended deep into the P-domain and occasionally reached the leading edge of the growth cone. This extension resulted from an increase in MT growth lifetimes as shown in the MT growth lifetime histograms in Fig. 7H and Fig. I, under control conditions and after PDBu treatment. Note the presence of MT growth lifetimes in excess of 80 s in PDBu (Fig. 7 I) and the nearly twofold increase in mean MT growth lifetime (Fig. 8 A). An example of a prolonged MT growth lifetime in PDBu is shown in Fig. 7 F. The increase in growth lifetimes in PDBu was accompanied by a 1.7-fold increase in the MT rescue frequency and an approximately twofold decrease in catastrophe frequency (Table ). Rates of MT growth and shortening increased slightly after PDBu treatment (Table ), but these changes were not statistically significant. Anterograde and retrograde translocation rates remained unchanged (Table ).


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 MT assembly versus translocation dynamics. Data compiled from four control and four PDBu-treated growth cones. DIC and fluorescence images were collected at 10-s intervals for 240 s. A total of 41 control and 37 PDBu MTs were analyzed. (A) PDBu treatment significantly increases the length of time MTs spend in growth phase, *P < 0.00004, paired student's t test. (B) Percentage of time MTs undergo retrograde or anterograde translocation and time not moving under control conditions and after PDBu. The actual time MTs undergo retrograde translocation increases significantly (P < 0.01, paired Student's t test) in control (27 ± 3 s) versus PDBu treatment (50 ± 6 s).
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Figure 8: Quantification of MT assembly versus translocation dynamics. Data compiled from four control and four PDBu-treated growth cones. DIC and fluorescence images were collected at 10-s intervals for 240 s. A total of 41 control and 37 PDBu MTs were analyzed. (A) PDBu treatment significantly increases the length of time MTs spend in growth phase, *P < 0.00004, paired student's t test. (B) Percentage of time MTs undergo retrograde or anterograde translocation and time not moving under control conditions and after PDBu. The actual time MTs undergo retrograde translocation increases significantly (P < 0.01, paired Student's t test) in control (27 ± 3 s) versus PDBu treatment (50 ± 6 s).
Mentions: To directly address the mechanism of the PKC effects described above, we assessed microtubule dynamics before and after treatment with PDBu using FSM. This relatively new microtubule marking technique (Waterman-Storer et al. 1998; Waterman-Storer and Salmon 1998) permits rapid assessment of the critical dynamic parameters of MT behavior, including assembly, disassembly, and translocation (sliding) rates as well as MT lifetimes in each state. Fig. 7 shows DIC-MT overlays illustrating the distribution of MTs before (A) and after (B) PDBu exposure with respect to growth-cone structure. Fig. 7 C is another growth cone in PDBu illustrating extended undbundled MTs typically observed after PDBu treatment. Note the spatial overlap between MTs and actin cables that extend into filopodia (Fig. 7 C, arrowheads), as observed in fixed cells (compare Fig. 4C and Fig. D). Representative MT behavior under control conditions is illustrated in the time-lapse image montages shown in Fig. 7D and Fig. E. MTs underwent stochastic bursts of growth and shortening (green arrowheads indicate the penultimate plus-end speckle) superimposed on relatively short MT translocations in both directions, as indicated by speckles maintained within the body of MTs over time (red arrowheads). Under control conditions, microtubule growth, shrinkage, and sliding activity occurred within the transition zone with only occasional MT excursions into the P-domain. In contrast, after PDBu exposure, MTs often extended deep into the P-domain and occasionally reached the leading edge of the growth cone. This extension resulted from an increase in MT growth lifetimes as shown in the MT growth lifetime histograms in Fig. 7H and Fig. I, under control conditions and after PDBu treatment. Note the presence of MT growth lifetimes in excess of 80 s in PDBu (Fig. 7 I) and the nearly twofold increase in mean MT growth lifetime (Fig. 8 A). An example of a prolonged MT growth lifetime in PDBu is shown in Fig. 7 F. The increase in growth lifetimes in PDBu was accompanied by a 1.7-fold increase in the MT rescue frequency and an approximately twofold decrease in catastrophe frequency (Table ). Rates of MT growth and shortening increased slightly after PDBu treatment (Table ), but these changes were not statistically significant. Anterograde and retrograde translocation rates remained unchanged (Table ).

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