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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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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 anti–mouse 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.
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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 anti–mouse 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.

Mentions: Melanophores were injected with SUK4, allowed to recover for 4 h, and then were incubated in melatonin to induce pigment aggregation, followed by MSH, to induce dispersion. Dispersion of melanosomes was not affected by the antibody injections (Fig. 7 A). Although SUK4 recognizes conventional kinesin from Xenopus melanophores on Western blots (Rogers et al., 1997), we did not know if this antibody would inhibit its motor activity, as all the inhibition data were obtained with either sea urchin or mammalian kinesins (Hollenbeck and Swanson, 1990; Wright et al., 1993; Bi et al., 1997). We therefore wanted to determine if SUK4 inhibits organelle movement powered by Xenopus kinesin. To do so, we analyzed lysosomal movement in cells injected with SUK4. Lysosomes were labeled with Texas red dextran, and then cells were injected with SUK4. The pH shift assay performed 4 h after injection. Lysosome dispersion in response to low pH was strongly inhibited by SUK4 (Fig. 7 B), but aggregation of these organelles to the cell center in response to high pH was not affected, as reflected by the quantitative measurements of lysosomal distribution (Table III). We conclude that conventional kinesin does not play a role in the dispersion of melanosomes.


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)

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 anti–mouse 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.
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Related In: Results  -  Collection

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

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 anti–mouse 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.
Mentions: Melanophores were injected with SUK4, allowed to recover for 4 h, and then were incubated in melatonin to induce pigment aggregation, followed by MSH, to induce dispersion. Dispersion of melanosomes was not affected by the antibody injections (Fig. 7 A). Although SUK4 recognizes conventional kinesin from Xenopus melanophores on Western blots (Rogers et al., 1997), we did not know if this antibody would inhibit its motor activity, as all the inhibition data were obtained with either sea urchin or mammalian kinesins (Hollenbeck and Swanson, 1990; Wright et al., 1993; Bi et al., 1997). We therefore wanted to determine if SUK4 inhibits organelle movement powered by Xenopus kinesin. To do so, we analyzed lysosomal movement in cells injected with SUK4. Lysosomes were labeled with Texas red dextran, and then cells were injected with SUK4. The pH shift assay performed 4 h after injection. Lysosome dispersion in response to low pH was strongly inhibited by SUK4 (Fig. 7 B), but aggregation of these organelles to the cell center in response to high pH was not affected, as reflected by the quantitative measurements of lysosomal distribution (Table III). We conclude that conventional kinesin does not play a role in the dispersion of melanosomes.

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