ABSTRACT
Myosin Va functions as a processive, actin-based motor molecule highly enriched in the nervous system, which transports and/or tethers organelles, vesicles, and mRNA and protein translation machinery. Mutation of myosin Va leads to Griscelli disease that is associated with severe neurological deficits and a short life span. Despite playing a critical role in development, the expression of myosin Va in the central nervous system throughout the human life span has not been reported. To address this issue, the cerebellar expression of myosin Va from newborns to elderly humans was studied by immunohistochemistry using an affinity-purified anti-myosin Va antibody. Myosin Va was expressed at all ages from the 10th postnatal day to the 98th year of life, in molecular, Purkinje and granular cerebellar layers. Cerebellar myosin Va expression did not differ essentially in localization or intensity from childhood to old age, except during the postnatal developmental period. Structures resembling granules and climbing fibers in Purkinje cells were deeply stained. In dentate neurons, long processes were deeply stained by anti-myosin Va, as were punctate nuclear structures. During the first postnatal year, myosin Va was differentially expressed in the external granular layer (EGL). In the EGL, proliferating prospective granule cells were not stained by anti-myosin Va antibody. In contrast, premigratory granule cells in the EGL stained moderately. Granule cells exhibiting a migratory profile in the molecular layer were also moderately stained. In conclusion, neuronal myosin Va is developmentally regulated, and appears to be required for cerebellar function from early postnatal life to senescence.
Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Male , Young Adult , Cerebellum/metabolism , Myosin Type V/metabolism , Age Factors , Cadaver , Electrophoresis, Agar Gel , Immunoblotting , ImmunohistochemistryABSTRACT
Myosin Va is an actin-based, processive molecular motor protein highly enriched in the nervous tissue of vertebrates. It has been associated with processes of cellular motility, which include organelle transport and neurite outgrowth. The in vivo expression of myosin Va protein in the developing nervous system of mammals has not yet been reported. We describe here the immunolocalization of myosin Va in the developing rat hippocampus. Coronal sections of the embryonic and postnatal rat hippocampus were probed with an affinity-purified, polyclonal anti-myosin Va antibody. Myosin Va was localized in the cytoplasm of granule cells in the dentate gyrus and of pyramidal cells in Ammon's horn formation. Myosin Va expression changed during development, being higher in differentiating rather than already differentiated granule and pyramidal cells. Some of these cells presented a typical migratory profile, while others resembled neurons that were in the process of differentiation. Myosin Va was also transiently expressed in fibers present in the fimbria. Myosin Va was not detected in germinative matrices of the hippocampus proper or of the dentate gyrus. In conclusion, myosin Va expression in both granule and pyramidal cells showed both position and time dependency during hippocampal development, indicating that this motor protein is under developmental regulation.
Subject(s)
Animals , Female , Rats , Hippocampus/embryology , Hippocampus/metabolism , Myosin Type V/analysis , Dentate Gyrus/embryology , Dentate Gyrus/metabolism , Immunohistochemistry , Myosin Type V/metabolism , Pyramidal Cells/embryology , Pyramidal Cells/metabolism , Rats, WistarSubject(s)
Animals , Adenosine Triphosphate/metabolism , Calmodulin-Binding Proteins/chemistry , Nerve Tissue Proteins/chemistry , Binding Sites , Calmodulin-Binding Proteins/metabolism , Calmodulin-Binding Proteins/ultrastructure , Molecular Structure , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/ultrastructure , Sequence Analysis, DNAABSTRACT
the subcellular localization in brain of an unconventional, calmodulin-binding myosin (myosin-V) found in neurons, astrocytes and other secretory cells of vertebrates has been investigated by probing Western blots of synaptic fractions from rat cerebral cortex with affinity-purified polyclonal antibodies against myosin-V. Myosin-V was detected in intact synaptosomes and in lysed synaptosomes associated with a particulate fration. Our data suggest a role for brain myosin-V in membrane-cytoskeleton function in the synaptic region
Subject(s)
Animals , Rats , Cerebral Cortex/chemistry , Nerve Tissue Proteins/chemistry , Synaptosomes/chemistry , Blotting, WesternABSTRACT
1. Myosin-V from vertebrate brain is a novel molecular motor with a myosin-like head domain, a calmodulin-binding neck region and a unique tail domain of unknown function. Previous studies showed brain myosin-V to be a phosphoprotein substrate for Ca2+/calmodulin-dependent protein kinase associated with actomyosin. In the present study we describe the preparation of a specific actin-cytoskeletal fraction which is enriched in brain myosin-V. 2. We show that Ca2+/calmodulin-dependent protein kinase activity is also associated with this preparation and phosphorylates brain myosin-V. 3. Calpain, a Ca(2+)-dependent protease, generates a M(r) 80,000 fragment from the COOH terminal region of brain myosin-V containing most or all of the phosphorylation sites. 4. These results suggest that the unique tail domain of this novel myosin is subject to Ca2+ control via phosphorylation by kinase activity associated with the actin cytoskeleton