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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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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.
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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.

Mentions: To target the role of kinesin II in pigment transport, we generated a dominant-negative construct that encodes an EGFP-tagged headless form of Xklp3 (pEGFP-headless Xklp3). This headless form of the protein lacks the motor domain but contains the domain responsible for dimerization with the 85-kD subunit (Rashid et al., 1995). Consequently, it should complex with the 85-kD subunit but be unable to hydrolyze ATP and generate movement. The functional significance of dimerization of motor subunits, as proposed for conventional kinesin (Hackney, 1994; Gilbert et al., 1995; Vale et al., 1996), may be to allow the complex to move processively along microtubules in a “hand-over-hand” fashion, transporting its vesicular cargo (Cole and Scholey, 1995). As this mutant protein is overexpressed, it should compete with the endogenous Xklp3 pool for dimerization with the 85-kD subunit, thus behaving as a dominant-negative. We transfected melanophores with pEGFP-headless Xklp3 or the control plasmid pEGFP and used an anti-GFP antibody to immunoprecipitate the expressed proteins. The efficiency of transfection was typically ∼10% for the headless Xklp3 construct and ∼50% for pEGFP-C1. Immunoprecipitates were probed by Western blotting with K2.4, a mouse monoclonal antibody that recognizes the 85-kD subunit of kinesin II. Fig. 1 B shows that the anti-GFP antibody immunoprecipitated the 85-kD protein from headless transfected cells, but not from EGFP-transfected cells. Thus, the headless 95-kD subunit of kinesin II forms a complex with the 85-kD subunit, and therefore it can compete with the endogenous pool of 95-kD subunit for binding to the other components of the heterotrimeric kinesin II. Identical results were obtained in the accompanying paper with a similar headless Xklp3 construct (Le Bot et al., 1998). 72 h after transfection, cells were treated with MSH to induce pigment dispersion, or with melatonin to induce aggregation. Upon treatment with MSH, a smaller percentage of cells expressing headless Xklp3 had their pigment dispersed, compared with control cells expressing EGFP (Fig. 2 A). When transfected cells were induced to aggregate pigment by melatonin and then stimulated to redisperse in response to MSH, the number of headless-expressing cells with fully dispersed pigment was further reduced compared with EGFP- expressing cells (Fig. 2 B). Overexpression of wild-type Xklp3 did not have any effect on the ability of the cells to disperse their pigments (data not shown).


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132968&req=5

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.
Mentions: To target the role of kinesin II in pigment transport, we generated a dominant-negative construct that encodes an EGFP-tagged headless form of Xklp3 (pEGFP-headless Xklp3). This headless form of the protein lacks the motor domain but contains the domain responsible for dimerization with the 85-kD subunit (Rashid et al., 1995). Consequently, it should complex with the 85-kD subunit but be unable to hydrolyze ATP and generate movement. The functional significance of dimerization of motor subunits, as proposed for conventional kinesin (Hackney, 1994; Gilbert et al., 1995; Vale et al., 1996), may be to allow the complex to move processively along microtubules in a “hand-over-hand” fashion, transporting its vesicular cargo (Cole and Scholey, 1995). As this mutant protein is overexpressed, it should compete with the endogenous Xklp3 pool for dimerization with the 85-kD subunit, thus behaving as a dominant-negative. We transfected melanophores with pEGFP-headless Xklp3 or the control plasmid pEGFP and used an anti-GFP antibody to immunoprecipitate the expressed proteins. The efficiency of transfection was typically ∼10% for the headless Xklp3 construct and ∼50% for pEGFP-C1. Immunoprecipitates were probed by Western blotting with K2.4, a mouse monoclonal antibody that recognizes the 85-kD subunit of kinesin II. Fig. 1 B shows that the anti-GFP antibody immunoprecipitated the 85-kD protein from headless transfected cells, but not from EGFP-transfected cells. Thus, the headless 95-kD subunit of kinesin II forms a complex with the 85-kD subunit, and therefore it can compete with the endogenous pool of 95-kD subunit for binding to the other components of the heterotrimeric kinesin II. Identical results were obtained in the accompanying paper with a similar headless Xklp3 construct (Le Bot et al., 1998). 72 h after transfection, cells were treated with MSH to induce pigment dispersion, or with melatonin to induce aggregation. Upon treatment with MSH, a smaller percentage of cells expressing headless Xklp3 had their pigment dispersed, compared with control cells expressing EGFP (Fig. 2 A). When transfected cells were induced to aggregate pigment by melatonin and then stimulated to redisperse in response to MSH, the number of headless-expressing cells with fully dispersed pigment was further reduced compared with EGFP- expressing cells (Fig. 2 B). Overexpression of wild-type Xklp3 did not have any effect on the ability of the cells to disperse their pigments (data not shown).

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