title: Subcellular localization of GFP-myosin-V in live mouse melanocytes.
abstract: Class-V myosins are two-headed actin-based mechanoenzymes that function in the transport and subcellular localization of organelles and possibly in the outgrowth of cellular processes. To determine which domains of myosin-V are involved in intracellular localization of this motor protein, we have expressed fusions of the green fluorescent protein with segments from two distinct myosin-V heavy chains. The expression patterns of constructs encoding four different domains of chick brain myosin-Va were compared to a single construct encoding the globular tail region of mouse myosin-Vb. In transfected mouse melanocytes, expression of the NH(2)-terminal head (catalytic domain) of chick brain myosin-Va codistributed with actin filaments throughout the cytoplasm. A similar construct encoding the myosin-Va head with the associated neck (light chain binding sites), also codistributed with actin filaments. The GFP-head-neck peptide was also highly concentrated in the tips of filopodia in B16 melanocytes wild type for myosin-Va (MYO5a gene), but was concentrated throughout the entire filopodia of S91-6 melanocytes derived from dilute mice with mutations in the MYO5a gene. Evidence is also presented that the globular tail of myosin-Va, but not myosin-Vb, targets this motor molecule to the centrosome as confirmed by colocalization in cells stained with antibodies to (gamma)-tubulin. Expression of the GFP-myosin-Va globular tail causes displacement of endogenous myosin-V from centrosomes as visualized by immunolabeling with antibodies to the head domain of myosin-V. Treatment with the microtubule-disrupting drug nocodazole markedly reduces myosin-V staining at the centrosome. In contrast, there was no detectable diminution of myosin-V staining at the centrosome in cells treated with the actin filament-disrupting drug cytochalasin D. Thus, while localization of the myosin-V motor domain to actin-rich regions is consistent with conventional models of actomyosin-based motility, localization to the centrosome occurs in the complete absence of the myosin-V motor domain and is dependent on intact microtubules.
authors.affiliation: Department of Molecular Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA.
authors.forename: V
authors.initials: V
authors.lastname: Tsakraklides
authors.forename: K
authors.initials: K
authors.lastname: Krogh
authors.forename: L
authors.initials: L
authors.lastname: Wang
authors.forename: J C
authors.initials: JC
authors.lastname: Bizario
authors.forename: R E
authors.initials: RE
authors.lastname: Larson
authors.forename: E M
authors.initials: EM
authors.lastname: Espreafico
authors.forename: J S
authors.initials: JS
authors.lastname: Wolenski
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects; D000199:Actins / Q000378:metabolism; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018385:Centrosome / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D015638:Cytochalasin D / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006200:Hair Color / Q000235:genetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008544:Melanocytes / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D008546:Melanoma, Experimental / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008817:Mice, Mutant Strains; D008870:Microtubules / Q000187:drug effects; D020409:Molecular Motor Proteins; D024701:Myosin Type V; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D011554:Pseudopodia / Q000378:metabolism / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000199:Actins; D002148:Calmodulin-Binding Proteins; D008164:Luminescent Proteins; D020409:Molecular Motor Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C078829:chicken brain myosin-V p190; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D; D024701:Myosin Type V; D015739:Nocodazole