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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 - J. Cell Biol. (1998)

Bottom Line: Natl.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

ABSTRACT
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.

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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.
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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.

Mentions: We characterized pigment movement in individual transfected cells by means of time-lapse video microscopy. This analysis demonstrated that the character of melanosome movement is dramatically altered in cells overexpressing headless Xklp3. Spreading of the pigment mass to the periphery of the cells in the presence of MSH was much slower in cells overexpressing headless Xklp3 than in cells expressing EGFP. To quantitate this difference, we measured the ratio of the area occupied by the pigment mass to the total cell area and plotted this ratio over time. Fig. 5 A shows typical plots for nontransfected cells and cells transfected with pEGFP-C1 and pEGFP-headless Xklp3. These curves demonstrate that cells expressing headless Xklp3 are indeed able to disperse their pigment, but at a much slower rate. From these curves, we calculated the time required for individual cells to reach 50% of maximal dispersion (midpoint between the initial ratio at the fully aggregated state and the final ratio of 1, equivalent to complete dispersion). The values obtained for cells transfected with pEGFP-headless Xklp3 (8–20 min) were substantially higher than the values for control cells (4–6 min) (Table I). The rates of pigment aggregation in headless-expressing cells were similar to those of control EGFP-expressing cells (Fig. 5 B, Table I). This confirms the indication obtained with the fixed time point assay that the effects of overexpression of headless Xklp3 are specific for dispersion.


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 - J. Cell Biol. (1998)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132968&req=5

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.
Mentions: We characterized pigment movement in individual transfected cells by means of time-lapse video microscopy. This analysis demonstrated that the character of melanosome movement is dramatically altered in cells overexpressing headless Xklp3. Spreading of the pigment mass to the periphery of the cells in the presence of MSH was much slower in cells overexpressing headless Xklp3 than in cells expressing EGFP. To quantitate this difference, we measured the ratio of the area occupied by the pigment mass to the total cell area and plotted this ratio over time. Fig. 5 A shows typical plots for nontransfected cells and cells transfected with pEGFP-C1 and pEGFP-headless Xklp3. These curves demonstrate that cells expressing headless Xklp3 are indeed able to disperse their pigment, but at a much slower rate. From these curves, we calculated the time required for individual cells to reach 50% of maximal dispersion (midpoint between the initial ratio at the fully aggregated state and the final ratio of 1, equivalent to complete dispersion). The values obtained for cells transfected with pEGFP-headless Xklp3 (8–20 min) were substantially higher than the values for control cells (4–6 min) (Table I). The rates of pigment aggregation in headless-expressing cells were similar to those of control EGFP-expressing cells (Fig. 5 B, Table I). This confirms the indication obtained with the fixed time point assay that the effects of overexpression of headless Xklp3 are specific for dispersion.

Bottom Line: Natl.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus