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J Cell Biol
1998 Dec 14;1436:1547-58. doi: 10.1083/jcb.143.6.1547.
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Heterotrimeric kinesin II is the microtubule motor protein responsible for pigment dispersion in Xenopus melanophores.
Tuma MC
,
Zill A
,
Le Bot N
,
Vernos I
,
Gelfand V
.
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Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720-3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles.
Figure 1. (A) All subunits of the heterotrimeric motor protein, kinesin II, are present on melanosomes. Western blots of cell extracts and purified melanosomes probed with antibodies against the motor subunits of kinesin II. Lanes C are cell extracts, and lanes M are purified melanosomes. The first two lanes were probed with K2.4 to detect the 85-kD motor subunit, the following two lanes were probed with a polyclonal anti-Xklp3 to detect the 95-kD motor subunit, and the last two lanes were probed with a polyclonal antiâsea urchin KAP 115 to detect the 115-kD accessory subunit. Notice that all the subunits are enriched in the melanosome fraction. (B) The overexpressed EGFP-headless Xklp3 protein forms a complex with the endogenous 85-kD motor subunit. Melanophore extracts were prepared from cells transfected with pEGFP-C1 or pEGFP-headless Xklp3, immunoprecipitated with an anti-GFP antibody, and probed with K2.4 to detect the 85-kD motor subunit. Shown are Western blots from both immunoprecipitates. The 85-kD subunit coimmunoprecipitates with EGFP-headless Xklp3 (lane 1), but not with EGFP alone (lane 2).
Figure 2. Quantitative analysis of pigment distribution in cells transfected with either control DNA (pEGFP-C1) or headless Xklp3 (pEGFP-headless Xklp3). Melanophores were transfected by electroporation and plated on coverslips, and after 72 h of expression, cells were incubated in serum-free medium containing MSH (A) or melatonin (C) for 1 h. In sequential treatments (B and D), cells were incubated for 1 h in melatonin followed by 1 h in MSH (B) or vice versa (D). The horizontal axis shows the percentage of cells that were scored as aggregated (white), partially dispersed (gray), or dispersed (black). For each treatment, 100 cells were scored. Data shown here are representative from one of four independent experiments.
Figure 4. Distribution of microtubules is not affected by expression of headless Xklp3. Immunofluorescence staining with an antitubulin primary antibody followed by a Texas red secondary in a melanophore overexpressing the headless Xklp3. Microtubules radiate from the cell center, forming a highly organized polar array. The inset is a fluorescence image taken at the green channel, showing that the cell in the large picture expresses the EGFP-headless Xklp3 fusion protein. Bar, 20 μm.
Figure 5. Headless Xklp3 affects the kinetics of pigment dispersion but not aggregation. Video-microscopic analysis of pigment movement was conducted on nontransfected melanophores, on melanophores 72 h after transfection, or immediately after treatment with nocodazole (see detailed description in Materials and Methods). Representative plots of changes in pigment distribution, determined by the ratio of pigment area to total cell area over time: (A) dispersion; (B) aggregation.
Figure 6. Distribution of lysosomes is not affected in melanophores overexpressing headless Xklp3. Cells exhibit normal lysosomal movement in response to changes in pH (dispersed in acidic conditions, clustered in alkaline conditions). Upon treatment with low pH, lysosomes redistribute through the entire cytoplasm (A). Normal perinuclear distribution is reestablished upon treatment with high pH (B). Transfected cells expressing the EGFP-headless Xklp3 fusion protein are shown in the insets, while in the large pictures the cell outlines are traced, and lysosomes stained with Texas red dextran are shown. Bar, 20 μm.
Figure 7. SUK4 inhibits dispersion of lysosomes but does not affect melanosomes. Melanophores with labeled lysosomes were injected with SUK4 and then exposed to acidic conditions in presence of MSH. Injected cells were identified by staining with a rhodamine antiâmouse antibody. (A) Overlay of bright field and fluorescence images, showing that melanosomes are normally dispersed in an injected cell. (B) Clustered distribution of lysosomes, labeled with FITC-dextran, in acidic conditions. Instead of normally dispersed through the cytoplasm, they remain clustered in the cell center. Bar, 20 μm.
Arhets,
Virulence and functions of myosin II are inhibited by overexpression of light meromyosin in Entamoeba histolytica.
1998, Pubmed
Arhets,
Virulence and functions of myosin II are inhibited by overexpression of light meromyosin in Entamoeba histolytica.
1998,
Pubmed
Bi,
Kinesin- and myosin-driven steps of vesicle recruitment for Ca2+-regulated exocytosis.
1997,
Pubmed
Blose,
10-nm filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin.
1984,
Pubmed
Burns,
Single-headed myosin II acts as a dominant negative mutation in Dictyostelium.
1995,
Pubmed
Cole,
Novel heterotrimeric kinesin-related protein purified from sea urchin eggs.
1993,
Pubmed
,
Xenbase
Cole,
Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons.
1998,
Pubmed
Cole,
Structural variations among the kinesins.
1995,
Pubmed
Cole,
Isolation of a sea urchin egg kinesin-related protein using peptide antibodies.
1992,
Pubmed
,
Xenbase
Daniolos,
Action of light on frog pigment cells in culture.
1990,
Pubmed
,
Xenbase
Diment,
Lysosomal hydrolases are present in melanosomes and are elevated in melanizing cells.
1995,
Pubmed
Gilbert,
Pathway of processive ATP hydrolysis by kinesin.
1995,
Pubmed
Graessmann,
"Early" simian-virus-40-specific RNA contains information for tumor antigen formation and chromatin replication.
1976,
Pubmed
Graminski,
Pigment dispersion in frog melanophores can be induced by a phorbol ester or stimulation of a recombinant receptor that activates phospholipase C.
1993,
Pubmed
,
Xenbase
Hackney,
Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis.
1994,
Pubmed
Henson,
Immunolocalization of the heterotrimeric kinesin-related protein KRP(85/95) in the mitotic apparatus of sea urchin embryos.
1995,
Pubmed
Heuser,
Changes in lysosome shape and distribution correlated with changes in cytoplasmic pH.
1989,
Pubmed
Hollenbeck,
Radial extension of macrophage tubular lysosomes supported by kinesin.
1990,
Pubmed
Ingold,
Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains.
1988,
Pubmed
,
Xenbase
Johnson,
Localization of a kinesin-related protein to the central pair apparatus of the Chlamydomonas reinhardtii flagellum.
1994,
Pubmed
Kondo,
KIF3A is a new microtubule-based anterograde motor in the nerve axon.
1994,
Pubmed
Laemmli,
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
1970,
Pubmed
Le Bot,
Role of xklp3, a subunit of the Xenopus kinesin II heterotrimeric complex, in membrane transport between the endoplasmic reticulum and the Golgi apparatus.
1998,
Pubmed
,
Xenbase
Lombillo,
Antibodies to the kinesin motor domain and CENP-E inhibit microtubule depolymerization-dependent motion of chromosomes in vitro.
1995,
Pubmed
Luo,
Cotransfection of genes encoding human tyrosinase and tyrosinase-related protein-1 prevents melanocyte death and enhances melanin pigmentation and gene expression of Lamp-1.
1994,
Pubmed
McNiven,
Microtubule polarity and the direction of pigment transport reverse simultaneously in surgically severed melanophore arms.
1984,
Pubmed
Morris,
Heterotrimeric kinesin-II is required for the assembly of motile 9+2 ciliary axonemes on sea urchin embryos.
1997,
Pubmed
Muresan,
KIF3C and KIF3A form a novel neuronal heteromeric kinesin that associates with membrane vesicles.
1998,
Pubmed
Nakata,
Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport.
1995,
Pubmed
Obika,
The effects of lumicolchicine, colchicine and vinblastine on pigment migration in fish chromatophores.
1978,
Pubmed
Orlow,
Melanosomes are specialized members of the lysosomal lineage of organelles.
1995,
Pubmed
Pesavento,
Characterization of the KLP68D kinesin-like protein in Drosophila: possible roles in axonal transport.
1994,
Pubmed
Rashid,
Heterodimerization of the two motor subunits of the heterotrimeric kinesin, KRP85/95.
1995,
Pubmed
Reilein,
Regulation of organelle movement in melanophores by protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2A (PP2A).
1998,
Pubmed
,
Xenbase
Rodionov,
Kinesin is responsible for centrifugal movement of pigment granules in melanophores.
1991,
Pubmed
Rodionov,
Functional coordination of microtubule-based and actin-based motility in melanophores.
1998,
Pubmed
Rogers,
In vitro motility assay for melanophore pigment organelles.
1998,
Pubmed
,
Xenbase
Rogers,
Myosin cooperates with microtubule motors during organelle transport in melanophores.
1998,
Pubmed
,
Xenbase
Rogers,
Regulated bidirectional motility of melanophore pigment granules along microtubules in vitro.
1997,
Pubmed
,
Xenbase
Schliwa,
Pigment movements in fish melanophores: morphological and physiolgical studies. IV. The effect of cyclic adenosine monophosphate on normal and vinblastine treated melanophores.
1974,
Pubmed
Schliwa,
Chromatophores: their use in understanding microtubule-dependent intracellular transport.
1982,
Pubmed
Scholey,
Kinesin-II, a membrane traffic motor in axons, axonemes, and spindles.
1996,
Pubmed
Schraermeyer,
Transport of endocytosed material into melanin granules in cultured choroidal melanocytes of cattle--new insights into the relationship of melanosomes with lysosomes.
1995,
Pubmed
Stewart,
Identification and partial characterization of six members of the kinesin superfamily in Drosophila.
1991,
Pubmed
Tanaka,
Targeted disruption of mouse conventional kinesin heavy chain, kif5B, results in abnormal perinuclear clustering of mitochondria.
1998,
Pubmed
Towbin,
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
1979,
Pubmed
Vale,
Direct observation of single kinesin molecules moving along microtubules.
1996,
Pubmed
Vashishtha,
The kinesin-homologous protein encoded by the Chlamydomonas FLA10 gene is associated with basal bodies and centrioles.
1996,
Pubmed
,
Xenbase
Vernos,
Multiple kinesin-like transcripts in Xenopus oocytes.
1993,
Pubmed
,
Xenbase
Walther,
The Chlamydomonas FLA10 gene encodes a novel kinesin-homologous protein.
1994,
Pubmed
Wedaman,
Sequence and submolecular localization of the 115-kD accessory subunit of the heterotrimeric kinesin-II (KRP85/95) complex.
1996,
Pubmed
Welte,
Developmental regulation of vesicle transport in Drosophila embryos: forces and kinetics.
1998,
Pubmed
Wright,
Roles of kinesin and kinesin-like proteins in sea urchin embryonic cell division: evaluation using antibody microinjection.
1993,
Pubmed
Yamazaki,
KIF3A/B: a heterodimeric kinesin superfamily protein that works as a microtubule plus end-directed motor for membrane organelle transport.
1995,
Pubmed
Yang,
Characterization of the KIF3C neural kinesin-like motor from mouse.
1998,
Pubmed
