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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) Cross-sectional synaptic profiles of parallel fiber boutons and Purkinje cell dendritic spines from the molecular layer of a P18 heterozygous mouse. Parallel fiber boutons are small and relatively uniform in size. Each bouton contacts a single spine. Purkinje cell spines contain smooth endoplasmic reticulum (arrowheads). (B) Cross-sectional profile of a single large parallel fiber bouton making contact with three (1, 2, and 3) Purkinje cell spines from the molecular layer of a P18 dilute-lethal mouse. Spines lack smooth endoplasmic reticulum. (C) The distribution of SV2 antibody label (12 nm colloidal gold) in a parallel fiber bouton from a heterozygous mouse. Many of the synaptic vesicles (arrowheads) are labeled. Some larger vesicles (arrows) also show label in this oblique section. (D) The distribution of SV2 antibody label in a parallel fiber bouton from a dilute-lethal mouse. Synaptic vesicles (arrowheads) are labeled. (Inset) From another synaptic terminal showing SV2 antibody label on several larger vesicles (arrows). (E) A nonterminal region of a parallel fiber also shows SV2 antibody labeling of large vesicles in the molecular layer of a dilute-lethal mouse. Bars: (A and B) 470 nm; (C–E) 315 nm.
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Figure 10: (A) Cross-sectional synaptic profiles of parallel fiber boutons and Purkinje cell dendritic spines from the molecular layer of a P18 heterozygous mouse. Parallel fiber boutons are small and relatively uniform in size. Each bouton contacts a single spine. Purkinje cell spines contain smooth endoplasmic reticulum (arrowheads). (B) Cross-sectional profile of a single large parallel fiber bouton making contact with three (1, 2, and 3) Purkinje cell spines from the molecular layer of a P18 dilute-lethal mouse. Spines lack smooth endoplasmic reticulum. (C) The distribution of SV2 antibody label (12 nm colloidal gold) in a parallel fiber bouton from a heterozygous mouse. Many of the synaptic vesicles (arrowheads) are labeled. Some larger vesicles (arrows) also show label in this oblique section. (D) The distribution of SV2 antibody label in a parallel fiber bouton from a dilute-lethal mouse. Synaptic vesicles (arrowheads) are labeled. (Inset) From another synaptic terminal showing SV2 antibody label on several larger vesicles (arrows). (E) A nonterminal region of a parallel fiber also shows SV2 antibody labeling of large vesicles in the molecular layer of a dilute-lethal mouse. Bars: (A and B) 470 nm; (C–E) 315 nm.

Mentions: Third, the terminal area and density of synaptic vesicles in synapses from heterozygous and dilute-lethal mouse littermates were compared in two different presynaptic terminals. The presynaptic terminals of P12 Purkinje cells did not show significant differences (P > 0.5) in terminal cross-sectional area (heterozygous area = 1.1 ± 0.24 μm2, n = 6, dilute-lethal area = 1.3 ± 0.67 μm2, n = 5) or synaptic vesicle density (heterozygous = 259 ± 89 vesicles/μm2, dilute-lethal = 310 ± 114 vesicles/μm2). In contrast, the presynaptic terminals of granule cells appeared to have a much larger average terminal cross-sectional area in dilute-lethal mice at all ages that were inspected (P10, P12, P14, P15, P18) (Fig. 10). At P18, the difference was more than fivefold (heterozygous area = 0.76 ± 0.36 μm2, n = 21 vs. dilute-lethal area = 4.36 ± 3.6 μm2, n = 12, difference was significant, P < 0.001). Synaptic vesicle density was somewhat lower in these enlarged dilute-lethal terminals (P18 heterozygous = 25 ± 8 vesicle/μm2, vs. P18 dilute-lethal = 18 ± 12 vesicles/μm2, P = 0.05). At P10 the difference in presynaptic terminal area was not as great (heterozygous area = 0.41 ± 0.26 μm2, n = 37 vs. dilute-lethal area = 0.96 ± 0.61 μm2, n = 22), but the difference was still significant (P < 0.001). P10 is before the onset of seizures in dilute-lethal mice (our unpublished observations), indicating that the increase in presynaptic terminal area is not a secondary effect of the seizures. The greater area indicates that the presynaptic terminal volume of granule cells will be increased in dilute-lethal mice. Because of the increased volume, the total number of synaptic vesicles in these terminals will be greater.


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

Bridgman PC - J. Cell Biol. (1999)

(A) Cross-sectional synaptic profiles of parallel fiber boutons and Purkinje cell dendritic spines from the molecular layer of a P18 heterozygous mouse. Parallel fiber boutons are small and relatively uniform in size. Each bouton contacts a single spine. Purkinje cell spines contain smooth endoplasmic reticulum (arrowheads). (B) Cross-sectional profile of a single large parallel fiber bouton making contact with three (1, 2, and 3) Purkinje cell spines from the molecular layer of a P18 dilute-lethal mouse. Spines lack smooth endoplasmic reticulum. (C) The distribution of SV2 antibody label (12 nm colloidal gold) in a parallel fiber bouton from a heterozygous mouse. Many of the synaptic vesicles (arrowheads) are labeled. Some larger vesicles (arrows) also show label in this oblique section. (D) The distribution of SV2 antibody label in a parallel fiber bouton from a dilute-lethal mouse. Synaptic vesicles (arrowheads) are labeled. (Inset) From another synaptic terminal showing SV2 antibody label on several larger vesicles (arrows). (E) A nonterminal region of a parallel fiber also shows SV2 antibody labeling of large vesicles in the molecular layer of a dilute-lethal mouse. Bars: (A and B) 470 nm; (C–E) 315 nm.
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Related In: Results  -  Collection

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Figure 10: (A) Cross-sectional synaptic profiles of parallel fiber boutons and Purkinje cell dendritic spines from the molecular layer of a P18 heterozygous mouse. Parallel fiber boutons are small and relatively uniform in size. Each bouton contacts a single spine. Purkinje cell spines contain smooth endoplasmic reticulum (arrowheads). (B) Cross-sectional profile of a single large parallel fiber bouton making contact with three (1, 2, and 3) Purkinje cell spines from the molecular layer of a P18 dilute-lethal mouse. Spines lack smooth endoplasmic reticulum. (C) The distribution of SV2 antibody label (12 nm colloidal gold) in a parallel fiber bouton from a heterozygous mouse. Many of the synaptic vesicles (arrowheads) are labeled. Some larger vesicles (arrows) also show label in this oblique section. (D) The distribution of SV2 antibody label in a parallel fiber bouton from a dilute-lethal mouse. Synaptic vesicles (arrowheads) are labeled. (Inset) From another synaptic terminal showing SV2 antibody label on several larger vesicles (arrows). (E) A nonterminal region of a parallel fiber also shows SV2 antibody labeling of large vesicles in the molecular layer of a dilute-lethal mouse. Bars: (A and B) 470 nm; (C–E) 315 nm.
Mentions: Third, the terminal area and density of synaptic vesicles in synapses from heterozygous and dilute-lethal mouse littermates were compared in two different presynaptic terminals. The presynaptic terminals of P12 Purkinje cells did not show significant differences (P > 0.5) in terminal cross-sectional area (heterozygous area = 1.1 ± 0.24 μm2, n = 6, dilute-lethal area = 1.3 ± 0.67 μm2, n = 5) or synaptic vesicle density (heterozygous = 259 ± 89 vesicles/μm2, dilute-lethal = 310 ± 114 vesicles/μm2). In contrast, the presynaptic terminals of granule cells appeared to have a much larger average terminal cross-sectional area in dilute-lethal mice at all ages that were inspected (P10, P12, P14, P15, P18) (Fig. 10). At P18, the difference was more than fivefold (heterozygous area = 0.76 ± 0.36 μm2, n = 21 vs. dilute-lethal area = 4.36 ± 3.6 μm2, n = 12, difference was significant, P < 0.001). Synaptic vesicle density was somewhat lower in these enlarged dilute-lethal terminals (P18 heterozygous = 25 ± 8 vesicle/μm2, vs. P18 dilute-lethal = 18 ± 12 vesicles/μm2, P = 0.05). At P10 the difference in presynaptic terminal area was not as great (heterozygous area = 0.41 ± 0.26 μm2, n = 37 vs. dilute-lethal area = 0.96 ± 0.61 μm2, n = 22), but the difference was still significant (P < 0.001). P10 is before the onset of seizures in dilute-lethal mice (our unpublished observations), indicating that the increase in presynaptic terminal area is not a secondary effect of the seizures. The greater area indicates that the presynaptic terminal volume of granule cells will be increased in dilute-lethal mice. Because of the increased volume, the total number of synaptic vesicles in these terminals will be greater.

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