Subcellular Localization of Myosin-V in the B16 Melanoma Cells, a Wild-type Cell Line for the<i>dilute</i>Gene

Nascimento, A A C; Amaral, Rita Goreti; Bizario, João C. S.; Larson, Roy Edward; Espreáfico, Enilza Maria

doi:10.1091/mbc.8.10.1971

Cited by 91 publications

(88 citation statements)

References 48 publications

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“…Second, in some cases there are proteins present on the cargo that inactivate M-V, possibly limiting the activity (16). Third, M-V may be localized on one part of the cargo, resulting in many M-Vs that are out of reach of an AF (17,18). Finally, the 1 7 2 motor hypothesis is consistent with p s , switching from Ϸ50% to Ϸ0%.…”

Section: Discussionmentioning

confidence: 99%

Intracellular actin-based transport: How far you go depends on how often you switch

Snider

Lin

Zahedi

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Intracellular molecular motor-driven transport is essential for such diverse processes as mitosis, neuronal function, and mitochondrial transport. Whereas there have been in vitro studies of how motors function at the single-molecule level, and in vivo studies of the structure of filamentary networks, studies of how the motors effectively use the networks for transportation have been lacking. We investigate how the combined system of myosin-V motors plus actin filaments is used to transport pigment granules in Xenopus melanophores. Experimentally, we characterize both the actin filament network, and how this transport is altered in response to external signals. We then develop a theoretical formalism to explain these changes. We show that cells regulate transport by controlling how often granules switch from one filament to another, rather than by altering individual motor activity at the single-molecule level, or by relying on structural changes in the network.I ntracellular transport of various cargos is essential for proper cell function, yet the principles regulating cargo motion are still not well understood. Whereas thermal diffusion is sufficient for the transportation of many small molecules, e.g., ATP or acetyl CoA, larger cargos require an active transit system. Such intracellular transport is present in all eukaryotic cells and is realized by a system of polymerized filaments [actin filaments (AFs) and microtubules (MTs)] and molecular motors [myosin-V (M-V), kinesin, dynein, etc.], not unlike roadways and trucks. The general model (1) is that the orderly MT filaments provide long-distance transport from the periphery to the nucleus or vice versa, in a fairly linear manner. In conjunction, AFs provide local transport from the MT superhighways to the remainder of the cell (2). Whereas there have been careful studies of single-motor properties as well as of the structure of the filamentary networks, there are deficiencies in our understanding of how those components work together to provide efficient, reliable transport. In this paper, we show that cells can change the way they transport cargo predominantly by changing the way the cargos use the network, rather than by changing individual motor properties, or by relying on changes to the structure of the filamentary networks.To make general transport into a tractable problem, we simplify it considerably and study it within the context of the Xenopus melanophore model system. The skin cells of this system are adapted for color camouflage by either moving pigment granules to the vicinity of the center of the cell in a process called aggregation, or by distributing them uniformly throughout the cell in a process known as dispersion (1). Because these cells are very nearly two-dimensional and the halfmicrometer pigment granule cargos are easily discerned, they are an ideal system in which to observe active transport with single-particle tracking. Because MTs are long, radially arranged filaments, there is less uncertainty about how MT-based transport works ...

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Section: Discussionmentioning

confidence: 99%

Intracellular actin-based transport: How far you go depends on how often you switch

Snider

Lin

Zahedi

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…Immunofluorescence localisation of myosin Va has shown that as well as being associated with melanosomes the protein is found in regions of cellular protrusions and in the tips of the dendrites of all melanocyte and melanoma cell lines examined ( Fig. 3 ; Edgar et al 1996 ;Nascimento et al 1997 ;Wu et al 1997). Immunoelectron microscopy confirms that in melanocytes a significant proportion of the labelling is not on melanosomes (Nascimento et al 1997).…”

Section: mentioning

confidence: 96%

“…3 ; Edgar et al 1996 ;Nascimento et al 1997 ;Wu et al 1997). Immunoelectron microscopy confirms that in melanocytes a significant proportion of the labelling is not on melanosomes (Nascimento et al 1997). Myosin Va is present in most other types of mammalian cell where it must associate with material other than melanosomes : it is found for example in the distal processes and growth cones of a number of neuronal cell types (Espindola et al 1992 ;Davies et al 1995 ;Wang et al 1996) where it colocalises with Factin and in the distal ends of the microvilli of the intestinal brush border enterocytes (Heintzelman et al 1994).…”

Section: mentioning

confidence: 99%

Inhibition of dendrite formation in mouse melanocytes transiently transfected with antisense DNA to myosin Va

Edgar¹,

Bennett²

1999

Journal of Anatomy

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In mice a molecular motor of the myosin V class (designated myosin Va) is known to be the product of the dilute locus, where a mutation prevents melanosome transport in melanocytes. There is conflicting evidence about whether it has a role in dendrite outgrowth. We investigated its role by transiently transfecting antisense oligonucleotides to inhibit its expression in a melanocyte cell line. We demonstrated mRNA and protein expression of myosin Va in 3 mouse melanocyte lines and 1 human melanoma cell line, using RT-PCR and immunoblotting. Two splice variants were found in human cells whilst only the longer transcript, containing an additional exon, was present in mouse melanocyte lines. The shorter variant was detected in other mouse tissues. Myosin Va protein levels were similar in 3 melanocyte lines with differing amounts of pigmentation, indicating that expression of myosin Va is not tightly coupled to expression of melanin. Immunocytochemistry showed 2 types of myosin Va localisation. A punctate pattern of staining concentrated in the perinuclear region was indicative of organelle association, and the observation of occasional linear punctate staining aligned with F-actin bundles supported the idea that myosin Va has a role in transporting melanosomes along actin filaments. Staining was also intense at tips of dendrites and at sites of dendrite-cell contact, consistent with a possible role in dendrite growth. Transient transfection of antisense phosphorothioate oligodeoxynucleotides targeted against myosin Va mRNA reduced expression of myosin Va protein in cultured mouse melan-a melanocytes by over 70 % 20 h after transfection whereas a control (shuffled sequence) oligonucleotide did not. Upon trypsinisation and replating these cells the capacity of the transfected cells to extend new dendrites was reduced in the cells containing the specific antisense oligonucleotides but unaffected by the control oligonucleotide. Image analysis confirmed that the effect of transfection on morphology was statistically significant (P 0.01). In contrast when cells were not trypsinised and replated following transfection so that previously existing dendrites could persist, the normal dendritic morphology continued to be observed. We conclude that in addition to its involvement in melanosome transport, myosin Va has a role in the extension of new dendrites by melanocytes but not in maintenance of pre-existing dendrites.

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“…MyoVa is highly expressed in brain as well as other tissues such as melanocytes in vertebrates (2,(19)(20)(21). Defective mutations of MyoVa lead to "dilute-lethal" and neurological impairments in rodents (2,(22)(23)(24) and a severe hereditary disease known as Griscelli syndrome (GS) in humans (25).…”

mentioning

confidence: 99%

Structural basis of cargo recognitions for class V myosins

Wei

Liu

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Class V myosins (MyoV), the most studied unconventional myosins, recognize numerous cargos mainly via the motor's globular tail domain (GTD). Little is known regarding how MyoV-GTD recognizes such a diverse array of cargos specifically. Here, we solved the crystal structures of MyoVa-GTD in its apo-form and in complex with two distinct cargos, melanophilin and Rab interacting lysosomal protein-like 2. The apo-MyoVa-GTD structure indicates that most mutations found in patients with Griscelli syndrome, microvillus inclusion disease, or cancers or in "dilute" rodents likely impair the folding of GTD. The MyoVa-GTD/cargo complex structure reveals two distinct cargo-binding surfaces, one primarily via charge-charge interaction and the other mainly via hydrophobic interactions. Structural and biochemical analysis reveal the specific cargo-binding specificities of various isoforms of mammalian MyoV as well as very different cargo recognition mechanisms of MyoV between yeast and higher eukaryotes. The MyoVa-GTD structures resolved here provide a framework for future functional studies of vertebrate class V myosins.

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Subcellular Localization of Myosin-V in the B16 Melanoma Cells, a Wild-type Cell Line for thediluteGene

Cited by 91 publications

References 48 publications

Intracellular actin-based transport: How far you go depends on how often you switch

Intracellular actin-based transport: How far you go depends on how often you switch

Inhibition of dendrite formation in mouse melanocytes transiently transfected with antisense DNA to myosin Va

Structural basis of cargo recognitions for class V myosins

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