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Myosin Va movements in normal and dilute-lethal axons provide support for a dual filament motor complex.

Bridgman PC - J. Cell Biol. (1999)

Bottom Line: In normal neurons, depolymerization of microtubules by nocodazole slowed, but did not stop movement.This suggests that myosin Va-associated organelles become stranded in regions rich in dynamic microtubule endings.Together, these results indicate that myosin Va binds to organelles that are transported in axons along microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. bridgmap@thalamus.wustl.edu

ABSTRACT
To investigate the role that myosin Va plays in axonal transport of organelles, myosin Va-associated organelle movements were monitored in living neurons using microinjected fluorescently labeled antibodies to myosin Va or expression of a green fluorescent protein-myosin Va tail construct. Myosin Va-associated organelles made rapid bi-directional movements in both normal and dilute-lethal (myosin Va ) neurites. In normal neurons, depolymerization of microtubules by nocodazole slowed, but did not stop movement. In contrast, depolymerization of microtubules in dilute-lethal neurons stopped movement. Myosin Va or synaptic vesicle protein 2 (SV2), which partially colocalizes with myosin Va on organelles, did not accumulate in dilute-lethal neuronal cell bodies because of an anterograde bias associated with organelle transport. However, SV2 showed peripheral accumulations in axon regions of dilute-lethal neurons rich in tyrosinated tubulin. This suggests that myosin Va-associated organelles become stranded in regions rich in dynamic microtubule endings. Consistent with these observations, presynaptic terminals of cerebellar granule cells in dilute-lethal mice showed increased cross-sectional area, and had greater numbers of both synaptic and larger SV2 positive vesicles. Together, these results indicate that myosin Va binds to organelles that are transported in axons along microtubules. This is consistent with both actin- and microtubule-based motors being present on these organelles. Although myosin V activity is not necessary for long-range transport in axons, myosin Va activity is necessary for local movement or processing of organelles in regions, such as presynaptic terminals that lack microtubules.

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Rat SCG neurons grown for 3 d in nocodazole (3.3 μg/ml), and then microinjected with anti–myosin Va-Cy3 antibody lack microtubules in cell processes. The distribution of actin (A and D), microinjected myosin Va-Cy3 antibody (B and E), and microtubules (C and F) are shown in two cells (B) used for recording movements of myosin Va-Cy3 antibody spots. The boxed regions in A indicate the areas used for recordings. Although some residual microtubule segments can be observed in the perinuclear region of one noninjected cell (C), they are absent from cell processes that extend peripherally in the injected cells (C and F). D and F are high magnifications of a portion of the cell indicated by the boxed region (right side) in A. Bars: (A–C) 21.5 μm; (B–F) 5 μm.
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Figure 7: Rat SCG neurons grown for 3 d in nocodazole (3.3 μg/ml), and then microinjected with anti–myosin Va-Cy3 antibody lack microtubules in cell processes. The distribution of actin (A and D), microinjected myosin Va-Cy3 antibody (B and E), and microtubules (C and F) are shown in two cells (B) used for recording movements of myosin Va-Cy3 antibody spots. The boxed regions in A indicate the areas used for recordings. Although some residual microtubule segments can be observed in the perinuclear region of one noninjected cell (C), they are absent from cell processes that extend peripherally in the injected cells (C and F). D and F are high magnifications of a portion of the cell indicated by the boxed region (right side) in A. Bars: (A–C) 21.5 μm; (B–F) 5 μm.

Mentions: The above results indicate that motors other than myosin Va must contribute to the movement of myosin Va–associated organelles. The high rates of movement suggest that microtubule motors might be involved. To determine whether microtubules were necessary for the rapid movements of myosin Va–associated organelles, chronic application of nocodazole was used to depolymerize microtubules. Cells were plated and then grown in the continuous presence of the nocodazole (3.3 μg/ml) for 3–6 d (Morris and Hollenbeck 1995). Microtubules were completely eliminated from the broad lamellae and short processes produced by the treated neurons under these conditions (Fig. 7). Despite the difference in morphology, nocodazole-treated cells microinjected with myosin Va-Cy3 antibody showed a similar distribution of fluorescent spots to untreated cells (Fig. 7). Fluorescent spot movements were still apparent in time-lapse recordings, but moved at reduced rates (Fig. 8A and Fig. E). Fluorescent spots (particles) paused and reversed direction more frequently than in untreated cells (compare Fig. 8 A and 2 B). A sizable and significant decrease (t test, P < 0.001) occurred in maximum rate (0.1 ± 0.05 μm/s, n = 12) in the absence of microtubules (compare Fig. 8 E and 2 C).


Myosin Va movements in normal and dilute-lethal axons provide support for a dual filament motor complex.

Bridgman PC - J. Cell Biol. (1999)

Rat SCG neurons grown for 3 d in nocodazole (3.3 μg/ml), and then microinjected with anti–myosin Va-Cy3 antibody lack microtubules in cell processes. The distribution of actin (A and D), microinjected myosin Va-Cy3 antibody (B and E), and microtubules (C and F) are shown in two cells (B) used for recording movements of myosin Va-Cy3 antibody spots. The boxed regions in A indicate the areas used for recordings. Although some residual microtubule segments can be observed in the perinuclear region of one noninjected cell (C), they are absent from cell processes that extend peripherally in the injected cells (C and F). D and F are high magnifications of a portion of the cell indicated by the boxed region (right side) in A. Bars: (A–C) 21.5 μm; (B–F) 5 μm.
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Related In: Results  -  Collection

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Figure 7: Rat SCG neurons grown for 3 d in nocodazole (3.3 μg/ml), and then microinjected with anti–myosin Va-Cy3 antibody lack microtubules in cell processes. The distribution of actin (A and D), microinjected myosin Va-Cy3 antibody (B and E), and microtubules (C and F) are shown in two cells (B) used for recording movements of myosin Va-Cy3 antibody spots. The boxed regions in A indicate the areas used for recordings. Although some residual microtubule segments can be observed in the perinuclear region of one noninjected cell (C), they are absent from cell processes that extend peripherally in the injected cells (C and F). D and F are high magnifications of a portion of the cell indicated by the boxed region (right side) in A. Bars: (A–C) 21.5 μm; (B–F) 5 μm.
Mentions: The above results indicate that motors other than myosin Va must contribute to the movement of myosin Va–associated organelles. The high rates of movement suggest that microtubule motors might be involved. To determine whether microtubules were necessary for the rapid movements of myosin Va–associated organelles, chronic application of nocodazole was used to depolymerize microtubules. Cells were plated and then grown in the continuous presence of the nocodazole (3.3 μg/ml) for 3–6 d (Morris and Hollenbeck 1995). Microtubules were completely eliminated from the broad lamellae and short processes produced by the treated neurons under these conditions (Fig. 7). Despite the difference in morphology, nocodazole-treated cells microinjected with myosin Va-Cy3 antibody showed a similar distribution of fluorescent spots to untreated cells (Fig. 7). Fluorescent spot movements were still apparent in time-lapse recordings, but moved at reduced rates (Fig. 8A and Fig. E). Fluorescent spots (particles) paused and reversed direction more frequently than in untreated cells (compare Fig. 8 A and 2 B). A sizable and significant decrease (t test, P < 0.001) occurred in maximum rate (0.1 ± 0.05 μm/s, n = 12) in the absence of microtubules (compare Fig. 8 E and 2 C).

Bottom Line: In normal neurons, depolymerization of microtubules by nocodazole slowed, but did not stop movement.This suggests that myosin Va-associated organelles become stranded in regions rich in dynamic microtubule endings.Together, these results indicate that myosin Va binds to organelles that are transported in axons along microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. bridgmap@thalamus.wustl.edu

ABSTRACT
To investigate the role that myosin Va plays in axonal transport of organelles, myosin Va-associated organelle movements were monitored in living neurons using microinjected fluorescently labeled antibodies to myosin Va or expression of a green fluorescent protein-myosin Va tail construct. Myosin Va-associated organelles made rapid bi-directional movements in both normal and dilute-lethal (myosin Va ) neurites. In normal neurons, depolymerization of microtubules by nocodazole slowed, but did not stop movement. In contrast, depolymerization of microtubules in dilute-lethal neurons stopped movement. Myosin Va or synaptic vesicle protein 2 (SV2), which partially colocalizes with myosin Va on organelles, did not accumulate in dilute-lethal neuronal cell bodies because of an anterograde bias associated with organelle transport. However, SV2 showed peripheral accumulations in axon regions of dilute-lethal neurons rich in tyrosinated tubulin. This suggests that myosin Va-associated organelles become stranded in regions rich in dynamic microtubule endings. Consistent with these observations, presynaptic terminals of cerebellar granule cells in dilute-lethal mice showed increased cross-sectional area, and had greater numbers of both synaptic and larger SV2 positive vesicles. Together, these results indicate that myosin Va binds to organelles that are transported in axons along microtubules. This is consistent with both actin- and microtubule-based motors being present on these organelles. Although myosin V activity is not necessary for long-range transport in axons, myosin Va activity is necessary for local movement or processing of organelles in regions, such as presynaptic terminals that lack microtubules.

Show MeSH
Related in: MedlinePlus