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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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SV2 accumulates in axon terminations of heterozygous and dilute-lethal neurons that are rich in tyrosinated tubulin. (A) The actin distribution is depicted in the terminal portion of an axon from a SCG neuron grown from a heterozygous mouse. (B) Wide areas of the axon (arrowheads in A) show increased staining for SV2 (arrowheads). (C) The areas of SV2 accumulation also correspond to the brightest areas (arrowheads) of tyrosinated tubulin staining. (D) The actin distribution is shown for a terminal portion of a SCG neurite grown from a dilute-lethal mouse. (E) Wide areas of axons (arrowheads in D) show increased bright staining for SV2 (arrowheads). (F) The areas of SV2 accumulation seen in E correspond to bright areas of tyrosinated tubulin staining (arrowheads). Bars, 6 μm.
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Figure 9: SV2 accumulates in axon terminations of heterozygous and dilute-lethal neurons that are rich in tyrosinated tubulin. (A) The actin distribution is depicted in the terminal portion of an axon from a SCG neuron grown from a heterozygous mouse. (B) Wide areas of the axon (arrowheads in A) show increased staining for SV2 (arrowheads). (C) The areas of SV2 accumulation also correspond to the brightest areas (arrowheads) of tyrosinated tubulin staining. (D) The actin distribution is shown for a terminal portion of a SCG neurite grown from a dilute-lethal mouse. (E) Wide areas of axons (arrowheads in D) show increased bright staining for SV2 (arrowheads). (F) The areas of SV2 accumulation seen in E correspond to bright areas of tyrosinated tubulin staining (arrowheads). Bars, 6 μm.

Mentions: The regions showing accumulation of SV2 label also colabeled with a monoclonal antibody to tyrosinated tubulin (Fig. 9). Since tyrosinated tubulin is associated with dynamic plus ends of microtubules, it suggests that these regions contain dynamic microtubule ends. The degree of colocalization was 100%. In other words, the presence of bright SV2 label could predict the occurrence of bright tyrosinated tubulin label and vice versa. To insure that the low density of cells were not affecting the accumulation of SV2, the peak brightness of SV2 label was also compared in neurites of 5-d-old explant cultures. Under these conditions, neurites grow out radially from explants and do not form synapses (outside the body of the explant). Dilute-lethal neurons showed significant increases (P < 0.001) in the peak brightness of staining in varicosities, (dilute-lethal = 6,378 ± 1,289 iu, n = 14 vs. heterozygous = 4,155 ± 400 iu, n = 12).


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

Bridgman PC - J. Cell Biol. (1999)

SV2 accumulates in axon terminations of heterozygous and dilute-lethal neurons that are rich in tyrosinated tubulin. (A) The actin distribution is depicted in the terminal portion of an axon from a SCG neuron grown from a heterozygous mouse. (B) Wide areas of the axon (arrowheads in A) show increased staining for SV2 (arrowheads). (C) The areas of SV2 accumulation also correspond to the brightest areas (arrowheads) of tyrosinated tubulin staining. (D) The actin distribution is shown for a terminal portion of a SCG neurite grown from a dilute-lethal mouse. (E) Wide areas of axons (arrowheads in D) show increased bright staining for SV2 (arrowheads). (F) The areas of SV2 accumulation seen in E correspond to bright areas of tyrosinated tubulin staining (arrowheads). Bars, 6 μm.
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Related In: Results  -  Collection

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Figure 9: SV2 accumulates in axon terminations of heterozygous and dilute-lethal neurons that are rich in tyrosinated tubulin. (A) The actin distribution is depicted in the terminal portion of an axon from a SCG neuron grown from a heterozygous mouse. (B) Wide areas of the axon (arrowheads in A) show increased staining for SV2 (arrowheads). (C) The areas of SV2 accumulation also correspond to the brightest areas (arrowheads) of tyrosinated tubulin staining. (D) The actin distribution is shown for a terminal portion of a SCG neurite grown from a dilute-lethal mouse. (E) Wide areas of axons (arrowheads in D) show increased bright staining for SV2 (arrowheads). (F) The areas of SV2 accumulation seen in E correspond to bright areas of tyrosinated tubulin staining (arrowheads). Bars, 6 μm.
Mentions: The regions showing accumulation of SV2 label also colabeled with a monoclonal antibody to tyrosinated tubulin (Fig. 9). Since tyrosinated tubulin is associated with dynamic plus ends of microtubules, it suggests that these regions contain dynamic microtubule ends. The degree of colocalization was 100%. In other words, the presence of bright SV2 label could predict the occurrence of bright tyrosinated tubulin label and vice versa. To insure that the low density of cells were not affecting the accumulation of SV2, the peak brightness of SV2 label was also compared in neurites of 5-d-old explant cultures. Under these conditions, neurites grow out radially from explants and do not form synapses (outside the body of the explant). Dilute-lethal neurons showed significant increases (P < 0.001) in the peak brightness of staining in varicosities, (dilute-lethal = 6,378 ± 1,289 iu, n = 14 vs. heterozygous = 4,155 ± 400 iu, n = 12).

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