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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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(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 anti–sea 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).
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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 anti–sea 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).

Mentions: To investigate the role of kinesin II in dispersion of melanosomes, the first step was to show that this protein is actually present on isolated organelles. Previous Western blotting analysis of isolated Xenopus melanosomes with the monoclonal antibody K2.4, which specifically recognizes the 85-kD motor subunit of kinesin II, showed the presence of this motor in the organelle fraction (Rogers et al., 1997). The presence of the 95-kD motor subunit was only verified with a pan-kinesin antibody, HIPYR, which recognizes motor domains of many kinesin-like proteins (Rogers et al., 1997). However, the 85-kD subunit of mouse kinesin II (KIF3A) can form complexes with two different motor subunits of ∼95 kD: KIF3B and KIF3C (Muresan et al., 1998; Yang and Goldstein, 1998). Therefore, we needed to know which of these two 95-kD subunits associates with the 85-kD subunit in the complex copurified with melanosomes. To answer this question, we probed Western blots of purified melanosomes with antibodies directed against the two motor subunits known to form a complex with the 85-kD subunit: an antibody against Xklp3, the Xenopus homologue of KIF3B, and an antibody against mouse KIF3C. Western blots showed that Xklp3 is present not only in the cell extract, but also on purified melanosomes (Fig. 1). The homologue of KIF3C in Xenopus is present in the extract, but unlike Xklp3, it does not copurify with melanosomes (data not shown). Thus, Xklp3, and not the Xenopus form of KIF3C, is the 95-kD motor subunit that, together with the 85-kD subunit, is present on melanosomes. Xklp3 has been fully characterized and shown to share a high degree of homology with sea urchin KRP95 and with mouse KIF3B. The motor domain has 90% amino acid similarity with KIF3B, suggesting that Xklp3 is the Xenopus counterpart of mouse KIF3B (Le Bot et al., 1998). Also, like the mouse and the sea urchin proteins, Xklp3 forms a complex with another motor subunit of 80 kD, and with a 100-kD accessory protein that cross-reacts with an antibody against the sea urchin KAP115 (Le Bot et al., 1998). To test whether the accessory subunit was present on melanosomes, we used a polyclonal antibody against the sea urchin KAP115 and verified that the third subunit of the kinesin II complex also copurifies with the pigment organelles. Thus, the presence of all the three subunits of kinesin II on melanosomes represents a strong indication that kinesin II might play a role in transporting these organelles.


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)

(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 anti–sea 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).
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Related In: Results  -  Collection

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

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 anti–sea 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).
Mentions: To investigate the role of kinesin II in dispersion of melanosomes, the first step was to show that this protein is actually present on isolated organelles. Previous Western blotting analysis of isolated Xenopus melanosomes with the monoclonal antibody K2.4, which specifically recognizes the 85-kD motor subunit of kinesin II, showed the presence of this motor in the organelle fraction (Rogers et al., 1997). The presence of the 95-kD motor subunit was only verified with a pan-kinesin antibody, HIPYR, which recognizes motor domains of many kinesin-like proteins (Rogers et al., 1997). However, the 85-kD subunit of mouse kinesin II (KIF3A) can form complexes with two different motor subunits of ∼95 kD: KIF3B and KIF3C (Muresan et al., 1998; Yang and Goldstein, 1998). Therefore, we needed to know which of these two 95-kD subunits associates with the 85-kD subunit in the complex copurified with melanosomes. To answer this question, we probed Western blots of purified melanosomes with antibodies directed against the two motor subunits known to form a complex with the 85-kD subunit: an antibody against Xklp3, the Xenopus homologue of KIF3B, and an antibody against mouse KIF3C. Western blots showed that Xklp3 is present not only in the cell extract, but also on purified melanosomes (Fig. 1). The homologue of KIF3C in Xenopus is present in the extract, but unlike Xklp3, it does not copurify with melanosomes (data not shown). Thus, Xklp3, and not the Xenopus form of KIF3C, is the 95-kD motor subunit that, together with the 85-kD subunit, is present on melanosomes. Xklp3 has been fully characterized and shown to share a high degree of homology with sea urchin KRP95 and with mouse KIF3B. The motor domain has 90% amino acid similarity with KIF3B, suggesting that Xklp3 is the Xenopus counterpart of mouse KIF3B (Le Bot et al., 1998). Also, like the mouse and the sea urchin proteins, Xklp3 forms a complex with another motor subunit of 80 kD, and with a 100-kD accessory protein that cross-reacts with an antibody against the sea urchin KAP115 (Le Bot et al., 1998). To test whether the accessory subunit was present on melanosomes, we used a polyclonal antibody against the sea urchin KAP115 and verified that the third subunit of the kinesin II complex also copurifies with the pigment organelles. Thus, the presence of all the three subunits of kinesin II on melanosomes represents a strong indication that kinesin II might play a role in transporting these organelles.

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