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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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GFP-myosin Va-t fluorescence is correctly targeted in neurons as indicated by colocalization with immunofluorescence staining for myosin Va or SV2. The DIL-2 antibody to myosin Va used for staining was made to amino acids 910–1106 of the myosin Va heavy chain (Wu et al. 1997). This overlapped by two amino acids with the sequence used for the GFP-myosin Va-t fusion protein. To remove a cross-reacting epitope resulting from this overlap, the antiserum was preincubated with a purified GST-myosin Va-t fusion protein coupled to agarose beads. The beads were pelleted by centrifugation and the supernatant used for immunofluorescence staining. (A) GFP-myosin Va-t fluorescence in a neurite from a heterozygous mouse. Arrowheads indicate bright spots of staining along the length of the neurite. (B) Immunofluorescence image of the same area as in A using the preabsorbed DIL-2 antibody. Bright spots of fluorescence (arrowheads) in the neurite correspond with the bright spots seen in A. The neurite lies along the surface of nonneuronal cells that also show bright spots of antibody stain. (C) GFP-myosin Va-t fluorescence in a neurite from a dilute-lethal mouse. (D) Immunofluorescence image of the same area as in C using the preabsorbed DIL-2 antibody. The fluorescence does not colocalize, indicating that the preabsorbed antibody does not cross-react with the GFP-myosin Va-t fusion protein. The few bright spots observed are nonspecific staining. (E) GFP-myosin Va-t fluorescence in a neurite from a heterozygous mouse. (F) Immunofluorescence image of the same area as in E using a mAb to SV2. The brightest spots in the two images show colocalization (arrowheads). Bar, 1.5 μm.
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Figure 5: GFP-myosin Va-t fluorescence is correctly targeted in neurons as indicated by colocalization with immunofluorescence staining for myosin Va or SV2. The DIL-2 antibody to myosin Va used for staining was made to amino acids 910–1106 of the myosin Va heavy chain (Wu et al. 1997). This overlapped by two amino acids with the sequence used for the GFP-myosin Va-t fusion protein. To remove a cross-reacting epitope resulting from this overlap, the antiserum was preincubated with a purified GST-myosin Va-t fusion protein coupled to agarose beads. The beads were pelleted by centrifugation and the supernatant used for immunofluorescence staining. (A) GFP-myosin Va-t fluorescence in a neurite from a heterozygous mouse. Arrowheads indicate bright spots of staining along the length of the neurite. (B) Immunofluorescence image of the same area as in A using the preabsorbed DIL-2 antibody. Bright spots of fluorescence (arrowheads) in the neurite correspond with the bright spots seen in A. The neurite lies along the surface of nonneuronal cells that also show bright spots of antibody stain. (C) GFP-myosin Va-t fluorescence in a neurite from a dilute-lethal mouse. (D) Immunofluorescence image of the same area as in C using the preabsorbed DIL-2 antibody. The fluorescence does not colocalize, indicating that the preabsorbed antibody does not cross-react with the GFP-myosin Va-t fusion protein. The few bright spots observed are nonspecific staining. (E) GFP-myosin Va-t fluorescence in a neurite from a heterozygous mouse. (F) Immunofluorescence image of the same area as in E using a mAb to SV2. The brightest spots in the two images show colocalization (arrowheads). Bar, 1.5 μm.

Mentions: A different method of marking myosin Va–associated organelles was necessary to: (a) control for the possible effects of surface antibody binding on myosin Va function, and (b) allow labeling of myosin Va–associated structures in dilute-lethal neurons. There is evidence that the globular tail portion of the myosin Va protein is responsible for its putative interaction with organelle surfaces (Cheney et al. 1993). Expression of a GFP–myosin Va construct containing this domain would be expected to produce a fusion protein capable of binding to organelles. In melanocytes, cultured cells and yeast expression of similar constructs have been shown to target the tail truncate to the full-length myosin V's normal location (Bizario et al. 1998; Wu et al. 1998; Reck-Peterson et al. 1999). At appropriate expression levels, this would provide a marker for individual organelles that normally associate with native myosin Va. To determine if a rat GFP–myosin Va tail construct could act as an appropriate marker, we expressed it (designated as GFP–myosin Va-t) in cultured SCG neurons. We then used immunofluorescence with a myosin Va antibody (Dil-2, gift of John Hammer) that was treated so that it lacked reaction with the expressed fusion protein to determine if it was correctly targeted in mouse neurons. Most heterozygous cell bodies appeared bright and fluorescence could be detected at low magnification in all neurites emerging from the cell body. At higher magnification, the fluorescence consisted mainly of bright spots of varying size and intensity. In cell bodies, bright irregular shaped spots were frequently seen in the perinuclear region and near the plasma membrane. Large bright spots were also seen in proximal portions of some neurites, but most neurites contained only small spots. Terminal varicosities in older cultures (>2 d) contained many small dim spots and an occasional bright spot of fluorescence. Nearly complete overlap in the distribution of GFP–myosin Va-t and myosin Va immunofluorescence label was observed in thin neurites (Fig. 5A and Fig. B). Similar overlap was seen in cell bodies, growth cones, or terminal, but the increased thickness made it more difficult to determine the precision of the colocalization. The exception was very bright large GFP spots (possibly vacuoles) located in a few cell bodies that increased in size and number with time in culture. Similar large very bright spots were seen in most nonneuronal cells. The large very bright spots showed weak or no colocalization with myosin Va antibody staining (not shown). We assume that the very bright spots in these cell bodies (and nonneuronal cells) represent sequestered and/or degraded GFP–myosin Va-t fusion protein. Cells showing such large bright spots were not used for analysis of spot movement or cell body brightness. Dilute-lethal neurons showed a similar distribution of the GFP–myosin Va-t. However, no specific DIL-2 antibody staining was seen in the cells expressing GFP–myosin Va-t, indicating that the antibody did not recognize the expressed myosin Va tail fusion protein (Fig. 5C and Fig. D).


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

Bridgman PC - J. Cell Biol. (1999)

GFP-myosin Va-t fluorescence is correctly targeted in neurons as indicated by colocalization with immunofluorescence staining for myosin Va or SV2. The DIL-2 antibody to myosin Va used for staining was made to amino acids 910–1106 of the myosin Va heavy chain (Wu et al. 1997). This overlapped by two amino acids with the sequence used for the GFP-myosin Va-t fusion protein. To remove a cross-reacting epitope resulting from this overlap, the antiserum was preincubated with a purified GST-myosin Va-t fusion protein coupled to agarose beads. The beads were pelleted by centrifugation and the supernatant used for immunofluorescence staining. (A) GFP-myosin Va-t fluorescence in a neurite from a heterozygous mouse. Arrowheads indicate bright spots of staining along the length of the neurite. (B) Immunofluorescence image of the same area as in A using the preabsorbed DIL-2 antibody. Bright spots of fluorescence (arrowheads) in the neurite correspond with the bright spots seen in A. The neurite lies along the surface of nonneuronal cells that also show bright spots of antibody stain. (C) GFP-myosin Va-t fluorescence in a neurite from a dilute-lethal mouse. (D) Immunofluorescence image of the same area as in C using the preabsorbed DIL-2 antibody. The fluorescence does not colocalize, indicating that the preabsorbed antibody does not cross-react with the GFP-myosin Va-t fusion protein. The few bright spots observed are nonspecific staining. (E) GFP-myosin Va-t fluorescence in a neurite from a heterozygous mouse. (F) Immunofluorescence image of the same area as in E using a mAb to SV2. The brightest spots in the two images show colocalization (arrowheads). Bar, 1.5 μm.
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Figure 5: GFP-myosin Va-t fluorescence is correctly targeted in neurons as indicated by colocalization with immunofluorescence staining for myosin Va or SV2. The DIL-2 antibody to myosin Va used for staining was made to amino acids 910–1106 of the myosin Va heavy chain (Wu et al. 1997). This overlapped by two amino acids with the sequence used for the GFP-myosin Va-t fusion protein. To remove a cross-reacting epitope resulting from this overlap, the antiserum was preincubated with a purified GST-myosin Va-t fusion protein coupled to agarose beads. The beads were pelleted by centrifugation and the supernatant used for immunofluorescence staining. (A) GFP-myosin Va-t fluorescence in a neurite from a heterozygous mouse. Arrowheads indicate bright spots of staining along the length of the neurite. (B) Immunofluorescence image of the same area as in A using the preabsorbed DIL-2 antibody. Bright spots of fluorescence (arrowheads) in the neurite correspond with the bright spots seen in A. The neurite lies along the surface of nonneuronal cells that also show bright spots of antibody stain. (C) GFP-myosin Va-t fluorescence in a neurite from a dilute-lethal mouse. (D) Immunofluorescence image of the same area as in C using the preabsorbed DIL-2 antibody. The fluorescence does not colocalize, indicating that the preabsorbed antibody does not cross-react with the GFP-myosin Va-t fusion protein. The few bright spots observed are nonspecific staining. (E) GFP-myosin Va-t fluorescence in a neurite from a heterozygous mouse. (F) Immunofluorescence image of the same area as in E using a mAb to SV2. The brightest spots in the two images show colocalization (arrowheads). Bar, 1.5 μm.
Mentions: A different method of marking myosin Va–associated organelles was necessary to: (a) control for the possible effects of surface antibody binding on myosin Va function, and (b) allow labeling of myosin Va–associated structures in dilute-lethal neurons. There is evidence that the globular tail portion of the myosin Va protein is responsible for its putative interaction with organelle surfaces (Cheney et al. 1993). Expression of a GFP–myosin Va construct containing this domain would be expected to produce a fusion protein capable of binding to organelles. In melanocytes, cultured cells and yeast expression of similar constructs have been shown to target the tail truncate to the full-length myosin V's normal location (Bizario et al. 1998; Wu et al. 1998; Reck-Peterson et al. 1999). At appropriate expression levels, this would provide a marker for individual organelles that normally associate with native myosin Va. To determine if a rat GFP–myosin Va tail construct could act as an appropriate marker, we expressed it (designated as GFP–myosin Va-t) in cultured SCG neurons. We then used immunofluorescence with a myosin Va antibody (Dil-2, gift of John Hammer) that was treated so that it lacked reaction with the expressed fusion protein to determine if it was correctly targeted in mouse neurons. Most heterozygous cell bodies appeared bright and fluorescence could be detected at low magnification in all neurites emerging from the cell body. At higher magnification, the fluorescence consisted mainly of bright spots of varying size and intensity. In cell bodies, bright irregular shaped spots were frequently seen in the perinuclear region and near the plasma membrane. Large bright spots were also seen in proximal portions of some neurites, but most neurites contained only small spots. Terminal varicosities in older cultures (>2 d) contained many small dim spots and an occasional bright spot of fluorescence. Nearly complete overlap in the distribution of GFP–myosin Va-t and myosin Va immunofluorescence label was observed in thin neurites (Fig. 5A and Fig. B). Similar overlap was seen in cell bodies, growth cones, or terminal, but the increased thickness made it more difficult to determine the precision of the colocalization. The exception was very bright large GFP spots (possibly vacuoles) located in a few cell bodies that increased in size and number with time in culture. Similar large very bright spots were seen in most nonneuronal cells. The large very bright spots showed weak or no colocalization with myosin Va antibody staining (not shown). We assume that the very bright spots in these cell bodies (and nonneuronal cells) represent sequestered and/or degraded GFP–myosin Va-t fusion protein. Cells showing such large bright spots were not used for analysis of spot movement or cell body brightness. Dilute-lethal neurons showed a similar distribution of the GFP–myosin Va-t. However, no specific DIL-2 antibody staining was seen in the cells expressing GFP–myosin Va-t, indicating that the antibody did not recognize the expressed myosin Va tail fusion protein (Fig. 5C and Fig. D).

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