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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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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.
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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.

Mentions: In nontransfected melanophores, lysosomes are normally clustered near the nucleus. Incubation in acidic conditions leads to their dispersion throughout the cytoplasm, and this movement is reversed by alkaline conditions (data not shown). These responses to pH were not changed in melanophores expressing either EGFP or headless Xklp3 (Fig. 6). We quantitated the distribution of lysosomes by measuring the average distance between them and the cell center (see Materials and Methods). For all groups (nontransfected, transfected with pEGFP-C1, or transfected with pEGFP-headless Xklp3), the normalized average distance between the lysosomes and the cell center was substantially higher in acidic than in alkaline conditions (Table III). Moreover, there was no difference in the average normalized distance between the groups either in alkaline or in acidic conditions (Table III). Thus, neither dispersion nor aggregation of lysosomes was affected by overexpression of headless Xklp3, indicating that the effect observed on melanosomes is specific and that movements powered by conventional kinesin are not affected by the dominant-negative kinesin II.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: In nontransfected melanophores, lysosomes are normally clustered near the nucleus. Incubation in acidic conditions leads to their dispersion throughout the cytoplasm, and this movement is reversed by alkaline conditions (data not shown). These responses to pH were not changed in melanophores expressing either EGFP or headless Xklp3 (Fig. 6). We quantitated the distribution of lysosomes by measuring the average distance between them and the cell center (see Materials and Methods). For all groups (nontransfected, transfected with pEGFP-C1, or transfected with pEGFP-headless Xklp3), the normalized average distance between the lysosomes and the cell center was substantially higher in acidic than in alkaline conditions (Table III). Moreover, there was no difference in the average normalized distance between the groups either in alkaline or in acidic conditions (Table III). Thus, neither dispersion nor aggregation of lysosomes was affected by overexpression of headless Xklp3, indicating that the effect observed on melanosomes is specific and that movements powered by conventional kinesin are not affected by the dominant-negative kinesin II.

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