Myosin VA Research Articles

Immunohistochemical localization of myosin va in the adult rat brain

Brain myosin Va (MVa) is a molecular motor associated with plastic changes during development. MVa has previously been detected in the cell body and in dendrites of neuronal cells in culture, in cells of the guinea-pig cochlea, as well as in cerebellar cells. Adult Wistar rats ( n=14), 250–300 g, were perfused with standard methods for immunohistochemistry, using a polyclonal, affinity-purified rabbit antibody against MVa tail domain. Anti-MVa antibody specifically stained neuronal nuclei from forebrain to cerebellar regions, and more intensely sensory nuclei. Differences in MVa immunoreactivity were detected between brain nuclei, ranging from very intense to weak staining. The analysis of MVa and glial fibrillary acidic protein staining in adjacent brain sections demonstrated a clear-cut neuronal labeling rather than an astroglial staining. The studies presented here represent a comprehensive map of MVa regional distribution in the CNS of the adult rat and may contribute to the basic understanding of its role in brain function and plasticity, particularly in relationship to phenomena that involve molecular motors, such as neurite outgrowth, organelle transport and neurotransmitter-vesicle cycling. It is important to highlight that this is a pioneer immunohistochemical study on the distribution of MVa on the whole brain of adult rats, a first step toward the understanding of its function in the CNS.

Myosin Va is locally synthesized following nerve injury.

The presence of Myosin Va (an actin-based molecular motor) in the peripheral nervous system was examined and its subcellular distribution within the axons of the sciatic nerve was demonstrated via immunocytochemistry. Myosin Va (M-Va) in the nerve was detected by using SDS-PAGE and Western blot techniques with a polyclonal antibody specifically raised against the M-Va globular tail domain. In addition, purification of M-Va from the rat sciatic nerve prior to immunoblotting yielded a M-Va standard band. Likewise, optical immunocytochemical procedures revealed the presence of M-Va, particularly in the cortical axoplasmic territory, but also in the Schwann cell soma. The above experiments were carried out both on intact as well as on severed sciatic nerves with similar results. The proximal stumps of severed sciatic nerves (from 0 to 72 h after injury) were labelled in vivo with (35)S-methionine. SDS-PAGE autoradiography of the immunoabsorbed M-Va from the radiolabelled homogenized nerve tissue showed a significant increment of the radioactive intensity of M-Va heavy chain band through time. Moreover, a significant increment of transcripts coding for M-Va heavy chain was detected through time using RT-PCR after nerve injury and compared to intact nerves. This data suggest that M-Va is up-regulated in a time-dependent manner. The latter suggests a possible involvement of M-Va in nerve regeneration processes.

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

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 null) 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.

Molecular genetic dissection of mouse unconventional myosin-VA: head region mutations.

The mouse dilute (d) locus encodes unconventional myosin-VA (MyoVA). Mice carrying null alleles of dilute have a lightened coat color and die from a neurological disorder resembling ataxia and opisthotonus within three weeks of birth. Immunological and ultrastructural studies suggest that MyoVA is involved in the transport of melanosomes in melanocytes and smooth endoplasmic reticulum in cerebellar Purkinje cells. In studies described here, we have used an RT-PCR-based sequencing approach to identify the mutations responsible for 17 viable dilute alleles that vary in their effects on coat color and the nervous system. Seven of these mutations mapped to the MyoVA motor domain and are reported here. Crystallographic modeling and mutant expression studies were used to predict how these mutations might affect motor domain function and to attempt to correlate these effects with the mutant phenotype.