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Myosin Va is developmentally regulated and expressed in the human cerebellum from birth to old age

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.

Myosin Va; Human cerebellum; Postnatal development; Aging; Immunohistochemical expression


Introduction

The myosin superfamily is composed of more than 20 structurally distinct classes of myosins, which are widely expressed in eukaryotes 11. Goodson HV, Dawson SC. Multiplying myosins. Proc Natl Acad Sci U S A 2006; 103: 3498-3499, doi: 10.1073/pnas.0600045103.
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). Vertebrate class V myosins are two-headed, actin-based processive motors. They convert the chemical energy of ATP hydrolysis to generate force and movement along actin filaments. Class V myosins are involved in intracellular organelle and mRNA transport and signaling 22. Larson RE. Myosin motors. Vol. 2. In: Lennarz WJ, Lane IMW (Editors), Encyclopedia of biological chemistry. Amsterdam: Elsevier; 2004. p 778-781.,33. Sellers JR, Veigel C. Walking with myosin V. Curr Opin Cell Biol 2006; 18: 68-73, doi: 10.1016/j.ceb.2005.12.014.
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). Three myosin V genes, encoding myosins Va, Vb and Vc that differ mostly in their C-terminal domains, have been described in mammals and display different expression patterns 44. Rodriguez OC, Cheney RE. Human myosin-Vc is a novel class V myosin expressed in epithelial cells. J Cell Sci 2002; 115: 991-1004.. Myosin Va 22. Larson RE. Myosin motors. Vol. 2. In: Lennarz WJ, Lane IMW (Editors), Encyclopedia of biological chemistry. Amsterdam: Elsevier; 2004. p 778-781.,55. Espindola FS, Espreafico EM, Coelho MV, Martins AR, Costa FR, Mooseker MS, et al. Biochemical and immunological characterization of p190-calmodulin complex from vertebrate brain: a novel calmodulin-binding myosin. J Cell Biol 1992; 118: 359-368, doi: 10.1083/jcb.118.2.359.
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,66. Espreafico EM, Cheney RE, Matteoli M, Nascimento AA, De Camilli PV, Larson RE, et al. Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains. J Cell Biol 1992; 119: 1541-1557, doi: 10.1083/jcb.119.6.1541.
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is expressed mainly in neurons, neuroendocrine cells and melanocytes, whereas myosins Vb and Vc are expressed in epithelial cells 77. Desnos C, Huet S, Darchen F. ‘Should I stay or should I go?': myosin V function in organelle trafficking. Biol Cell 2007; 99: 411-423, doi: 10.1042/BC20070021.
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. In neurons and neuroendocrine cells, myosin Va has been associated with distribution, docking and release of secretory granules77. Desnos C, Huet S, Darchen F. ‘Should I stay or should I go?': myosin V function in organelle trafficking. Biol Cell 2007; 99: 411-423, doi: 10.1042/BC20070021.
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, with transport of the endoplasmic reticulum to dendritic spines of Purkinje cells 88. Dekker-Ohno K, Hayasaka S, Takagishi Y, Oda S, Wakasugi N, Mikoshiba K, et al. Endoplasmic reticulum is missing in dendritic spines of Purkinje cells of the ataxic mutant rat. Brain Res 1996; 714: 226-230, doi: 10.1016/0006-8993(95)01560-4.
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, with axonal transport 99. Evans LL, Lee AJ, Bridgman PC, Mooseker MS. Vesicle-associated brain myosin-V can be activated to catalyze actin-based transport. J Cell Sci 1998; 111 (Part 14): 2055-2066., with transport of mRNA and protein translation machinery in neurons 1010. Yoshimura A, Fujii R, Watanabe Y, Okabe S, Fukui K, Takumi T. Myosin-Va facilitates the accumulation of mRNA/protein complex in dendritic spines. Curr Biol 2006; 16: 2345-2351, doi: 10.1016/j.cub.2006.10.024.
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, and with extension of growth cones 1111. Wang FS, Wolenski JS, Cheney RE, Mooseker MS, Jay DG. Function of myosin-V in filopodial extension of neuronal growth cones. Science 1996; 273: 660-663, doi: 10.1126/science.273.5275.660.
https://doi.org/10.1126/science.273.5275...
, and has been localized to synaptic vesicles 1212. Prekeris R, Terrian DM. Brain myosin V is a synaptic vesicle-associated motor protein: evidence for a Ca2+-dependent interaction with the synaptobrevin-synaptophysin complex. J Cell Biol 1997; 137: 1589-1601, doi: 10.1083/jcb.137.7.1589.
https://doi.org/10.1083/jcb.137.7.1589...
.

The movement of melanosomes, the best studied cargo of mammalian myosin Va, depends on the formation of an organelle-specific transport complex, composed of myosin Va, Rab-27a and melanophilin (for a review, see Ref. 77. Desnos C, Huet S, Darchen F. ‘Should I stay or should I go?': myosin V function in organelle trafficking. Biol Cell 2007; 99: 411-423, doi: 10.1042/BC20070021.
https://doi.org/10.1042/BC20070021...
). Mutations in the human myosin Va gene (MyoVa) result in the Griscelli syndrome type I, a recessive disease characterized by partial albinism, hypotonia, mental retardation, epilepsy, and ataxia 1313. Pastural E, Barrat FJ, Dufourcq-Lagelouse R, Certain S, Sanal O, Jabado N, et al. Griscelli disease maps to chromosome 15q21 and is associated with mutations in the myosin-Va gene. Nat Genet 1997; 16: 289-292, doi: 10.1038/ng0797-289.
https://doi.org/10.1038/ng0797-289...
). A similar phenotype has also been observed in the dilute MyoVa mouse mutant 1414. Mercer JA, Seperack PK, Strobel MC, Copeland NG, Jenkins NA. Novel myosin heavy chain encoded by murine dilute coat colour locus. Nature 1991; 349: 709-713, doi: 10.1038/349709a0.
https://doi.org/10.1038/349709a0...
. However, mutations in the other components of the human transport complex have not been associated with primary neurological defects 1515. Menasche G, Pastural E, Feldmann J, Certain S, Ersoy F, Dupuis S, et al. Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome. Nat Genet 2000; 25: 173-176, doi: 10.1038/76024.
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,1616. Menasche G, Ho CH, Sanal O, Feldmann J, Tezcan I, Ersoy F, et al. Griscelli syndrome restricted to hypopigmentation results from a melanophilin defect (GS3) or a MYO5A F-exon deletion (GS1). J Clin Invest 2003; 112: 450-456.. Thus, myosin Va clearly plays an important role in both normal and pathological CNS physiology. However, very little is known about the expression of myosin Va in the human nervous system from development to senescence.

The cerebellum is a useful model for the study of myosin Va expression because it is a relatively simple adult trilaminar structure that contains only a few neuronal cell types. During the first postnatal year there is a fourth layer, which is a secondary cerebellar proliferative matrix, the external germinative layer (EGL). The EGL generates new prospective granule cells that migrate on Bergman glia processes towards their final destination, the granule cell layer. Therefore, we have studied the expression of immunoreactive myosin Va in the postnatal developing, adult and aging human cerebellum.

Material and Methods

Tissue characterization and processing

Human nervous tissue was obtained from autopsies performed in the Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, according to protocols approved by the local Ethics Committee. The brains did not show any evidence of disease, as demonstrated by systematic neuropathologic examination. Twenty-nine autopsy cases ranging from the first postnatal day to the 98th year of life were studied. For each case the age, causa mortis, and postmortem interval are given in Table 1. Cerebella were fixed in 10% (v/v) formalin for 1 to 4 weeks. Fixed tissues were dehydrated in ethanol, cleared in xylene and embedded in paraffin. Five-micrometer sections were cut, mounted on gelatin-chrome alum-coated microscope slides, and dried at 58°C for 2 h.

Preparation of anti-myosin Va antibody

Polyclonal antibodies against the head domain of chicken myosin Va were raised in rabbits by inoculation with a recombinant protein corresponding to amino acid residues 5-572 of chicken myosin Va 66. Espreafico EM, Cheney RE, Matteoli M, Nascimento AA, De Camilli PV, Larson RE, et al. Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains. J Cell Biol 1992; 119: 1541-1557, doi: 10.1083/jcb.119.6.1541.
https://doi.org/10.1083/jcb.119.6.1541...
,1717. Costa MC, Mani F, Santoro W Jr, Espreafico EM, Larson RE. Brain myosin-V, a calmodulin-carrying myosin, binds to calmodulin-dependent protein kinase II and activates its kinase activity. J Biol Chem 1999; 274: 15811-15819, doi: 10.1074/jbc.274.22.15811.
https://doi.org/10.1074/jbc.274.22.15811...
. These antibodies were affinity-purified using purified chicken brain myosin Va 1818. Cheney RE. Purification and assay of myosin V. Methods Enzymol 1998; 298: 3-18, doi: 10.1016/S0076-6879(98)98003-X.
https://doi.org/10.1016/S0076-6879(98)98...
immobilized on polyvinylidene difluoride membranes. The membranes were washed with 50 mM Tris-HCl buffer, pH 7.5, containing 0.9% (w/v) NaCl (TBS), and the antibody was then eluted with 100 mM triethylamine, pH 12.2. The pH of the eluate was adjusted to 8 with 1.0 M Tris-HCl buffer, pH 8.0. The eluate was dialyzed against TBS, and concentrated to 1 mL, at 4°C. The final concentration of affinity-purified anti-myosin Va antibody was 0.5 mg/mL.

Immunohistochemistry

The immunohistochemical protocol used here has been described in detail by Martins et al. 1919. Martins AR, Dias MM, Vasconcelos TM, Caldo H, Costa MC, Chimelli L, et al. Microwave-stimulated recovery of myosin-V immunoreactivity from formalin-fixed, paraffin-embedded human CNS. J Neurosci Methods 1999; 92: 25-29, doi: 10.1016/S0165-0270(99)00090-4.
https://doi.org/10.1016/S0165-0270(99)00...
,2020. Martins AR, Zanella CA, Zucchi FC, Dombroski TC, Costa ET, Guethe LM, et al. Immunolocalization of nitric oxide synthase isoforms in human archival and rat tissues, and cultured cells. J Neurosci Methods 2011; 198: 16-22, doi: 10.1016/j.jneumeth.2011.02.024.
https://doi.org/10.1016/j.jneumeth.2011....
. Briefly, myosin Va antigenicity in dewaxed and hydrated human cerebellar tissue sections was microwave-retrieved in 10 mM sodium citrate buffer, pH 6, for 20 min at 900 W. Myosin Va detection in cerebellar sections was carried out by incubating sections overnight with an affinity-purified rabbit anti-myosin Va antibody diluted 1:50 (v/v) in 20 mM sodium phosphate buffer, pH 7.4, containing 0.45 M NaCl, 0.3% (w/v) Triton X-100, 5% (w/v) defatted dry milk, and 15% (v/v) normal donkey serum (blocking buffer). Endogenous biotin in tissue sections was blocked using a biotin blocking system (Vector, USA). Anti-myosin Va antibody was detected using a biotinylated swine anti-rabbit IgG (Dako, USA) diluted 1:100 (v/v) in blocking buffer, and the Elite ABC kit (Vector). Peroxidase was detected using a 2000-fold dilution of a 30 volumes hydrogen peroxide solution (Merck, Germany) and 0.66 mg/mL 3,3′-diaminebenzidine tetrahydrochloride as the chromogen, for 12 min. The reaction was stopped with water. All incubations were carried out at room temperature. Immunohistochemical controls were obtained by omitting the primary antibody and by preadsorbing the primary antibody with polyvinylidene difluoride membranes to which recombinant myosin Va was blotted. Tissue sections were examined and photographed using an Olympus light microscope, model BX-60 (Japan). All objectives (4X/0.16, 10X/0.40, 20X/0.70, 40X/0.85, 100X/1.35) were UPlanApo (Olympus).

Gel electrophoresis and immunoblotting

A fragment of human left temporal lobe from a 20-year-old female patient was resected to provide access for the resection of a cavernoma during a temporal craniotomy. We have used a surgical biopsy sample of cerebral cortex instead of a cerebellar one because both cortices express myosin Va, and because of difficulties in obtaining appropriate cerebellar tissue at autopsy. The procedures below were carried out according to protocols approved by the local Ethics Committee. The tissue was frozen and stored in liquid nitrogen until use. All subsequent procedures were performed on ice. The tissue was homogenized in 20 mM Tris-HCl buffer, pH 7.5, containing 10 mM EDTA, 0.3 mM PMSF, 1 mM benzamidine and 3 µM aprotinin. The total homogenate (10 mg protein/mL) was diluted 4-fold with 0.26 M Tris-HCl buffer, pH 6.8, containing 7.3% (w/v) sodium dodecyl sulfate, 16.6% (w/v) sucrose, 3.5 M β-mercaptoethanol and 0.005% (w/v) bromophenol blue, and boiled for 4 min. The material was frozen and stored at -80°C until the time for SDS-PAGE and Western blotting.

SDS-PAGE was carried out on 8% gradient minislab gels using a discontinuous system 2121. Laemmli UK, Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol 1973; 80: 575-599, doi: 10.1016/0022-2836(73)90198-8.
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. Western blotting to nitrocellulose membranes (Hybond-C extra, Amersham, USA) was performed according to Towbin et al. 2222. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979; 76: 4350-4354, doi: 10.1073/pnas.76.9.4350.
https://doi.org/10.1073/pnas.76.9.4350...
. Molecular mass standards used were: 205-kDa rabbit muscle myosin, 116-kDa E. coli β-galactosidase, 97.4-kDa rabbit muscle phosphorylase b, 66-kDa bovine serum albumin, and 45-kDa egg albumin (Sigma, USA).

Protein determination

Protein was measured by the method of Lowry et al. 2323. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275. using bovine serum albumin as the standard.

Results

The detection of myosin Va was specific since the affinity-purified rabbit anti-myosin Va antibody used here labeled a single-intense band corresponding to 200 kDa in Western blots of a human cerebral cortex (Figure 1A). As immunohistochemical specificity controls, anti-myosin Va antibody was diluted 1:25 (v/v) in blocking buffer and incubated with purified myosin Va bound to polyvinylidene membranes for up to 250 min, at room temperature. Aliquots of non-adsorbed material were collected at 0, 50, and 250 min, and used to immunostain adult (data not shown) and developing (Figure 1) adjacent cerebellar sections. The 0-min aliquot (non-adsorbed antibody) stained all three cerebellar layers, i.e., molecular, Purkinje and granular layers (Figure 1B), but the 50- (Figure 1C) or 250-min (Figure 1D) aliquots showed a decrease or a complete loss of the immunostaining pattern, respectively.

Figure 1
Specificity of anti-myosin Va antibody and immunohistochemical controls.A, Western blotting of a homogenate (20 µg per lane) of human cerebral cortex shows intense staining of a 200-kDa region, corresponding to myosin Va heavy chain (lane 1). The control lane (lane 2) was probed only with the secondary antibody. Immunohistochemical controls were obtained by pre-adsorbing anti-myosin Va antibody with myosin Va for 0 (B), 50 (C) and 250 (D) min before performing the assay on adjacent cerebellar sections. Nomarski optics. Magnification bars: 100 µm.

Myosin Va was expressed in molecular, Purkinje and granular cell layers in human cerebella from the 10th postnatal day to 98 years (Figure 2A, K, N). The molecular layer (MOL) exhibited a radial-like staining at all ages (Figure 2B, K, N) that was more pronounced during the first postnatal year (Figure 2N). Stellate cells in the outer MOL and interneurons in the inner MOL were also stained at all ages (Figure 2C, K, M). Anti-myosin Va antibody strongly decorated dendrites, and structures resembling cytoplasmic vesicles and the subcortical region in Purkinje cells [Figure 2A, D, K, L, M, N (inset)]. Structures resembling climbing fibers were stained along Purkinje cell dendrites of adult and elderly specimens (Figure 2M). A thin rim of granule cell cytoplasm was strongly stained at all ages (Figure 2E). In the dentate nucleus, myosin Va showed a strong punctate expression in the neuropile, perikarya (Figure 2G, H), nucleolus (Figure 2H, inset) and neuronal processes (Figure 2I, J), some of which were extended for hundreds of micrometers (Figure 2I). Omitting the anti-myosin Va antibody from the tissue section incubation led to no staining (Figure 2F).

Figure 2
Expression of myosin Va in the human cerebellum from birth to old age.Panels A-J, adult; panels K-M, elderly; panels N-P, postnatal development.A, Myosin Va was expressed in all three adult cerebellar layers, the molecular layer (MOL), Purkinje and granule cell layers (GL). B, The MOL exhibited a radial-like pattern upon staining by anti-myosin Va antibody. C, Arrowheads indicate stained interneurons in the MOL. D, A coarse punctate expression of myosin Va is evident in the cytoplasm and primary dendrites of Purkinje cells. E, Myosin Va is seen in the thin rim of cytoplasm of granule cells. F, Control section obtained by omitting the anti-Myo Va antibody during staining.G, Low magnification shows strong staining of neuronal soma and fibers throughout a cerebellar dentate nucleus. H, Dentate neurons and neuropile are strongly stained; the arrow indicates a neuron with a stained nucleolus, shown in the inset. I, A long dentate neuronal process is strongly stained (arrowheads).J, High magnification shows strong staining in the cytoplasm and processes of a dentate neuron. K, The staining pattern of the cerebellar cortex of an aged person is similar to that of an adult; arrow indicates outer MOL-situated interneurons, probably stellate cells; arrowheads indicate unidentified interneurons in the inner MOL. L, A higher magnification of the cerebellar cortex of an elderly person; staining intensity of adult and elderly cerebellum is about the same; M, High magnification of the Purkinje cell dendrites of a 98-year-old person shows strong labeling of discrete points along the dendrite, and also apparent overlaying structures that resemble climbing fibers (arrowhead); the arrow indicates an interneuron.N, anti-myosin Va weakly stains cells in the external granular layer (EGL), but strongly stains the MOL of a 10-day-old infant. The inset shows granular staining of the subcortical, perinuclear and dendritic regions of a Purkinje cell. O, High magnification of a 10-day-old infant cerebellum shows more intense staining of prospective granule cells in the premigratory zone (MZ) of EGL than those in the proliferative zone (PZ). Also note the staining of radial-like fibers (arrowheads) that penetrate the EGL up to the pial surface (PS).P, Cells in the MOL exhibiting a migratory profile (arrow) moderately expressed myosin Va (see inset). Magnification bars: A, H, L, N 50 µm; B, C, F, I, J, K, 100 µm; D, E, M, O, P, 20 µm; G, 200 µm.

The EGL, a germinative cerebellar matrix that persists throughout the first postnatal year 2424. Rakic P. Principles of neural cell migration. Experientia 1990; 46: 882-891, doi: 10.1007/BF01939380.
https://doi.org/10.1007/BF01939380...
, was lightly stained (Figure 2N). The prospective granule cells in the EGL proliferative zone (PZ) were not stained, but those in the premigratory zone (MZ) were well stained by the anti-myosin Va antibody (Figure 2O). Fibers exhibiting a radial-like pattern were also stained (Figure 2O, arrowheads). Granule cells exhibiting a migratory profile in the MOL expressed myosin Va in their subcortical region and processes (Figure 2P, arrow and inset).

Discussion

Using an affinity purified antibody against myosin Va to determine its expression in the human cerebellum, we showed that i) myosin Va was expressed in the cytoplasm of interneurons in the MOL, Purkinje, granule and dentate nucleus neurons from birth to advanced age; ii) myosin Va was expressed in structures resembling cytoplasmic granules and perinuclear and subcortical regions of Purkinje and dentate neurons; iii) no significant differences in this pattern of expression of cerebellar myosin Va described above were observed in this age series; iv) prospective granule cells in the MZ of the external granular layer of infants moderately expressed myosin Va, whereas those in the PZ were lightly or not detectably stained; v) the subcortical region of cells with a migratory profile in the molecular layer were moderately stained, as were fibers in the molecular layer that penetrated into the EGL.

Myosin Va is highly expressed in the human brain 1919. Martins AR, Dias MM, Vasconcelos TM, Caldo H, Costa MC, Chimelli L, et al. Microwave-stimulated recovery of myosin-V immunoreactivity from formalin-fixed, paraffin-embedded human CNS. J Neurosci Methods 1999; 92: 25-29, doi: 10.1016/S0165-0270(99)00090-4.
https://doi.org/10.1016/S0165-0270(99)00...
, as well as in neurons of most other eukaryotes such as rats 2525. Tilelli CQ, Martins AR, Larson RE, Garcia-Cairasco N. Immunohistochemical localization of myosin Va in the adult rat brain. Neuroscience 2003; 121: 573-586, doi: 10.1016/S0306-4522(03)00546-3.
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, chickens 2626. de Azevedo A, Lundardi LO, Larson RE. Immunolocalization of myosin Va in the developing nervous system of embryonic chicks. Anat Embriol 2004; 208: 395-402. and squid 2727. Cohen DL. Squid p196, a new member of the myosin-V class of motor proteins, is associated with motile axoplasmic organelles. Brain Res 2001; 890: 233-245, doi: 10.1016/S0006-8993(00)03165-6.
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. Taken together, these demonstrations of the ubiquitous localization of myosin Va to neurons and nervous tissue over a broad phylogenetic range underscore the importance of this molecular motor to fundamental neuronal processes. In the mouse, myosin Va is encoded by the dilute gene 1414. Mercer JA, Seperack PK, Strobel MC, Copeland NG, Jenkins NA. Novel myosin heavy chain encoded by murine dilute coat colour locus. Nature 1991; 349: 709-713, doi: 10.1038/349709a0.
https://doi.org/10.1038/349709a0...
, where it has been shown to function in the transport and/or tethering of organelles, such as melanosomes within the dendritic processes of melanocytes 2828. Nascimento AA, Amaral RG, Bizario JC, Larson RE, Espreafico EM. Subcellular localization of myosin-V in the B16 melanoma cells, a wild-type cell line for the dilute gene. Mol Biol Cell 1997; 8: 1971-1988.,2929. Wu X, Bowers B, Wei Q, Kocher B, Hammer JA III. Myosin V associates with melanosomes in mouse melanocytes: evidence that myosin V is an organelle motor. J Cell Sci 1997; 110 (Part 7): 847-859., and synaptic 1212. Prekeris R, Terrian DM. Brain myosin V is a synaptic vesicle-associated motor protein: evidence for a Ca2+-dependent interaction with the synaptobrevin-synaptophysin complex. J Cell Biol 1997; 137: 1589-1601, doi: 10.1083/jcb.137.7.1589.
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and secretory pancreatic acinar vesicles 3030. Abu-Hamdah R, Cho WJ, Horber JK, Jena BP. Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: a study using photonic force microscopy. Ultramicroscopy 2006; 106: 670-673, doi: 10.1016/j.ultramic.2006.01.013.
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. Also, the transport and/or positioning of smooth endoplasmic reticulum within the dendritic spines of Purkinje cells 88. Dekker-Ohno K, Hayasaka S, Takagishi Y, Oda S, Wakasugi N, Mikoshiba K, et al. Endoplasmic reticulum is missing in dendritic spines of Purkinje cells of the ataxic mutant rat. Brain Res 1996; 714: 226-230, doi: 10.1016/0006-8993(95)01560-4.
https://doi.org/10.1016/0006-8993(95)015...
, as well as the insertion of AMPA receptors in spines during synaptic plasticity 3131. Correia SS, Bassani S, Brown TC, Lise MF, Backos DS, El-Husseini A, et al. Motor protein-dependent transport of AMPA receptors into spines during long-term potentiation. Nat Neurosci 2008; 11: 457-466, doi: 10.1038/nn2063.
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, require myosin Va. The immunolocalization of myosin Va in cerebellar neurons and neuronal processes shown here can be related to activities in Purkinje cells and dentate neurons.

The human cerebellum continues its development, produces granule cells in the EGL, and prospective granule cells continue to migrate from the EGL to the granular layer during the first 12-18 postnatal months 3232. Rakic P, Sidman RL. Histogenesis of cortical layers in human cerebellum, particularly the lamina dissecans. J Comp Neurol 1970; 139: 473-500, doi: 10.1002/cne.901390407.
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. These are among the reasons that make the postnatal cerebellum a classical choice for developmental studies. Particularly for human studies, the prenatal cerebellum is obtained after abortion, and its morphology is usually not well preserved. Using the postnatal human cerebellum, we have demonstrated a differential expression of myosin Va within the EGL, where prospective granule cells in the MZ expressed myosin Va, whereas those cells in the PZ showed faint or no staining by anti-myosin Va antibody. The prospective granule cells in the MZ are indeed in a migratory state, moving in the direction of the long axis of the cerebellar folia 3333. Komuro H, Yacubova E, Yacubova E, Rakic P. Mode and tempo of tangential cell migration in the cerebellar external granular layer. J Neurosci 2001; 21: 527-540.,3434. Komuro H, Rakic P. Dynamics of granule cell migration: a confocal microscopic study in acute cerebellar slice preparations. J Neurosci 1995; 15: 1110-1120.. Moreover, cells exhibiting a migratory profile in the MOL 3434. Komuro H, Rakic P. Dynamics of granule cell migration: a confocal microscopic study in acute cerebellar slice preparations. J Neurosci 1995; 15: 1110-1120.,3535. Rivas RJ, Hatten ME. Motility and cytoskeletal organization of migrating cerebellar granule neurons. J Neurosci 1995; 15: 981-989. clearly expressed myosin Va in the subcortical region, leading and trailing processes, and were apposed to radial glial fibers. Granule cell migration in the developing cerebellum occurs within a crowded terrain, and requires the ability of exerting forces to perform the calcium-dependent 3636. Komuro H, Rakic P. Intracellular Ca2+fluctuations modulate the rate of neuronal migration. Neuron 1996; 17: 275-285, doi: 10.1016/S0896-6273(00)80159-2.
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, saltatory3434. Komuro H, Rakic P. Dynamics of granule cell migration: a confocal microscopic study in acute cerebellar slice preparations. J Neurosci 1995; 15: 1110-1120. movement towards their final destination. It is noteworthy that the structure and enzymatic activity of myosin Va also depends on calcium (for a review, see Ref. 3737. Trybus KM. Myosin V from head to tail. Cell Mol Life Sci 2008; 65: 1378-1389, doi: 10.1007/s00018-008-7507-6.
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. Moreover, the movement of the leading process is similar to that of a growth cone, for which a role for myosin Va has been proposed 1111. Wang FS, Wolenski JS, Cheney RE, Mooseker MS, Jay DG. Function of myosin-V in filopodial extension of neuronal growth cones. Science 1996; 273: 660-663, doi: 10.1126/science.273.5275.660.
https://doi.org/10.1126/science.273.5275...
. Therefore, the results presented here suggest that myosin Va expression is developmentally regulated, and can play a role in cerebellar granule cell migration.

Myosin Va was differentially expressed during the proliferation, differentiation and migration of the granule cell, and can thus be considered to be developmentally regulated during this period. In addition, it was expressed in most neuron types of the human cerebellum, both within their soma and processes. The striking similarity of the myosin Va localization pattern shown here for children, adults and elderly individuals, together with the association of mutations in thedilute gene with severe neurological disturbances and lethality in both mice and humans 1313. Pastural E, Barrat FJ, Dufourcq-Lagelouse R, Certain S, Sanal O, Jabado N, et al. Griscelli disease maps to chromosome 15q21 and is associated with mutations in the myosin-Va gene. Nat Genet 1997; 16: 289-292, doi: 10.1038/ng0797-289.
https://doi.org/10.1038/ng0797-289...
,3838. Pastural E, Ersoy F, Yalman N, Wulffraat N, Grillo E, Ozkinay F, et al. Two genes are responsible for Griscelli syndrome at the same 15q21 locus. Genomics 2000; 63: 299-306, doi: 10.1006/geno.1999.6081.
https://doi.org/10.1006/geno.1999.6081...
, suggest that myosin Va expression is required for cerebellar function from early postnatal life to senescence.

Research supported by FAPESP, FAEPA, CAPES, and CNPq.

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Publication Dates

  • Publication in this collection
    Feb 2013

History

  • Received
    26 Aug 2012
  • Accepted
    3 Dec 2012
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