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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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(A) Time-lapse sequence showing a myosin Va-Cy3 antibody-labeled spot (particle) (arrowhead) making anterograde movements along a rat SCG neurite. Only the small relatively dim spots (visible using a 100× 1.4 NA lens) such as the one shown in this sequence, exhibited rapid movements. Large bright spots seen in cell bodies and proximal neurite segments of neurons injected with larger amounts of antibody never made rapid movements. The interval between images is 6 s. (B) The displacement of individual myosin Va-Cy3 antibody spots (particles) at sequential time points is shown. Positive values indicate movement away from the cell body, negative values indicate movement towards the cell body. Movement is saltatory; pauses and direction reversal can occur for varying periods of time. All examples are from rat SCG neurons. (C) The maximum rates of particle movements from untreated neurons (rat, n = 27; mouse, n = 13) show a wide distribution. Bar, 6.4 μm.
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Figure 2: (A) Time-lapse sequence showing a myosin Va-Cy3 antibody-labeled spot (particle) (arrowhead) making anterograde movements along a rat SCG neurite. Only the small relatively dim spots (visible using a 100× 1.4 NA lens) such as the one shown in this sequence, exhibited rapid movements. Large bright spots seen in cell bodies and proximal neurite segments of neurons injected with larger amounts of antibody never made rapid movements. The interval between images is 6 s. (B) The displacement of individual myosin Va-Cy3 antibody spots (particles) at sequential time points is shown. Positive values indicate movement away from the cell body, negative values indicate movement towards the cell body. Movement is saltatory; pauses and direction reversal can occur for varying periods of time. All examples are from rat SCG neurons. (C) The maximum rates of particle movements from untreated neurons (rat, n = 27; mouse, n = 13) show a wide distribution. Bar, 6.4 μm.

Mentions: The rapid movements of individual small bright spots observed in neurites of rat (21 cells) or heterozygous (dv/dl) mouse (nine cells) SCG neurons injected with low concentrations of antibody at reduced pressures were measured and analyzed in several different ways. Most frequently, during a 20-s time period, ∼10% of the spots moved and the remaining were stationary. Movement of spots was bi-directional; spots sometimes paused or transiently reversed direction, but usually made progress either towards or away from the cell body (Fig. 2A and Fig. B). For analysis, movements away from the cell body are designated with positive values, while movements toward the cell body are indicated by negative values. Among the 40 spots selected for analysis, there was an apparent overall bias toward anterograde movement (69%). To determine whether our selection process influenced this result, the direction was scored in three 6-min time-lapse sequences from different cells for every moving spot (n = 53). An anterograde bias (62%) was still detected. An anterograde bias is consistent with previous reports on axonally transported vesicles in cultured neurons (Morris and Hollenbeck 1995; Nakata et al. 1998). However, chi-square analysis using the assumption that transport was equal in both directions ( hypothesis) indicated that the apparent bias was not statistically significant (P > 0.2). The maximum rates of movement achieved by small spots were widely distributed (Fig. 2 C). Because we could not make recordings of spot movement with delays between exposures <5 s, it is not possible to be certain of the reason for the variation in maximum rate. Either the spots achieve highly variable maximum rates or movements between time-lapse intervals are sometimes interrupted by short pauses at irregular intervals. The time-lapse data is clearly influenced by pauses and transient direction reversals, since the average rate of particle movement was considerably less (0.18 ± 0.09 μm/s, n = 24) than the mean maximum rate (0.46 ± 0.28 μm/s, n = 40).


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

Bridgman PC - J. Cell Biol. (1999)

(A) Time-lapse sequence showing a myosin Va-Cy3 antibody-labeled spot (particle) (arrowhead) making anterograde movements along a rat SCG neurite. Only the small relatively dim spots (visible using a 100× 1.4 NA lens) such as the one shown in this sequence, exhibited rapid movements. Large bright spots seen in cell bodies and proximal neurite segments of neurons injected with larger amounts of antibody never made rapid movements. The interval between images is 6 s. (B) The displacement of individual myosin Va-Cy3 antibody spots (particles) at sequential time points is shown. Positive values indicate movement away from the cell body, negative values indicate movement towards the cell body. Movement is saltatory; pauses and direction reversal can occur for varying periods of time. All examples are from rat SCG neurons. (C) The maximum rates of particle movements from untreated neurons (rat, n = 27; mouse, n = 13) show a wide distribution. Bar, 6.4 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169472&req=5

Figure 2: (A) Time-lapse sequence showing a myosin Va-Cy3 antibody-labeled spot (particle) (arrowhead) making anterograde movements along a rat SCG neurite. Only the small relatively dim spots (visible using a 100× 1.4 NA lens) such as the one shown in this sequence, exhibited rapid movements. Large bright spots seen in cell bodies and proximal neurite segments of neurons injected with larger amounts of antibody never made rapid movements. The interval between images is 6 s. (B) The displacement of individual myosin Va-Cy3 antibody spots (particles) at sequential time points is shown. Positive values indicate movement away from the cell body, negative values indicate movement towards the cell body. Movement is saltatory; pauses and direction reversal can occur for varying periods of time. All examples are from rat SCG neurons. (C) The maximum rates of particle movements from untreated neurons (rat, n = 27; mouse, n = 13) show a wide distribution. Bar, 6.4 μm.
Mentions: The rapid movements of individual small bright spots observed in neurites of rat (21 cells) or heterozygous (dv/dl) mouse (nine cells) SCG neurons injected with low concentrations of antibody at reduced pressures were measured and analyzed in several different ways. Most frequently, during a 20-s time period, ∼10% of the spots moved and the remaining were stationary. Movement of spots was bi-directional; spots sometimes paused or transiently reversed direction, but usually made progress either towards or away from the cell body (Fig. 2A and Fig. B). For analysis, movements away from the cell body are designated with positive values, while movements toward the cell body are indicated by negative values. Among the 40 spots selected for analysis, there was an apparent overall bias toward anterograde movement (69%). To determine whether our selection process influenced this result, the direction was scored in three 6-min time-lapse sequences from different cells for every moving spot (n = 53). An anterograde bias (62%) was still detected. An anterograde bias is consistent with previous reports on axonally transported vesicles in cultured neurons (Morris and Hollenbeck 1995; Nakata et al. 1998). However, chi-square analysis using the assumption that transport was equal in both directions ( hypothesis) indicated that the apparent bias was not statistically significant (P > 0.2). The maximum rates of movement achieved by small spots were widely distributed (Fig. 2 C). Because we could not make recordings of spot movement with delays between exposures <5 s, it is not possible to be certain of the reason for the variation in maximum rate. Either the spots achieve highly variable maximum rates or movements between time-lapse intervals are sometimes interrupted by short pauses at irregular intervals. The time-lapse data is clearly influenced by pauses and transient direction reversals, since the average rate of particle movement was considerably less (0.18 ± 0.09 μm/s, n = 24) than the mean maximum rate (0.46 ± 0.28 μm/s, n = 40).

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