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Melanophilin and myosin Va track the microtubule plus end on EB1.

Wu XS, Tsan GL, Hammer JA - J. Cell Biol. (2005)

Bottom Line: Moreover, myosin Va tracks the plus end in a Mlp-dependent manner.These results identify a novel +TIP and indicate that vertebrate cells possess a +TIP complex that is similar to the Myo2p-Kar9p-Bim1p complex in yeast.We suggest that the +TIP complex identified in this study may serve to focus the transfer of melanosomes from microtubules to actin at the microtubule plus end.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
In mouse melanocytes, myosin Va is recruited onto the surface of melanosomes by a receptor complex containing Rab27a that is present in the melanosome membrane and melanophilin (Mlp), which links myosin Va to Rab27a. In this study, we show that Mlp is also a microtubule plus end-tracking protein or +TIP. Moreover, myosin Va tracks the plus end in a Mlp-dependent manner. Data showing that overexpression and short inhibitory RNA knockdown of the +TIP EB1 have opposite effects on Mlp-microtubule interaction, that Mlp interacts directly with EB1, and that deletion from Mlp of a region similar to one in the adenomatous polyposis coli protein involved in EB1 binding blocks Mlp's ability to plus end track argue that Mlp tracks the plus end indirectly [corrected] by hitchhiking on EB1. These results identify a novel +TIP and indicate that vertebrate cells possess a +TIP complex that is similar to the Myo2p-Kar9p-Bim1p complex in yeast. We suggest that the +TIP complex identified in this study may serve to focus the transfer of melanosomes from microtubules to actin at the microtubule plus end.

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Myosin Va tracks the microtubule plus end. (A) A GFP-tagged version of the melanocyte-spliced isoform of myosin Va (MCMVa-GFP) plus end tracks together with Mlp-mRFP throughout the cell (Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). (B) In a fixed cell, the tail domain of this isoform (MCMVaTail-GFP) targets to microtubule plus ends together with FLAG-tagged Mlp (Mlp-FLAG; visualized by staining with α-FLAG antibody). The omission of Mlp-FLAG abrogated the interaction of MCMVaTail-GFP with microtubules (not depicted).
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fig4: Myosin Va tracks the microtubule plus end. (A) A GFP-tagged version of the melanocyte-spliced isoform of myosin Va (MCMVa-GFP) plus end tracks together with Mlp-mRFP throughout the cell (Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). (B) In a fixed cell, the tail domain of this isoform (MCMVaTail-GFP) targets to microtubule plus ends together with FLAG-tagged Mlp (Mlp-FLAG; visualized by staining with α-FLAG antibody). The omission of Mlp-FLAG abrogated the interaction of MCMVaTail-GFP with microtubules (not depicted).

Mentions: Although myosin Va and Rab27a are not required for Mlp to plus end track, one or both proteins might still track together with Mlp. To address this question, we used CV1 cells because they do not express Mlp, thereby allowing us to address the Mlp dependency of any possible plus end tracking behavior exhibited by myosin Va or Rab27a. When we cotransfected CV1 cells with Mlp-mRFP and a GFP-tagged version of the full-length melanocyte-spliced heavy chain isoform of myosin Va (MCMVa-GFP), which is fully capable of rescuing dilute melanocytes (Wu et al., 2002b), we observed a striking colocalization of the two proteins on comets whose dynamics were largely indistinguishable from those of Mlp alone (Fig. 4 A and Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). Moreover, both the myosin Va and Mlp components of these comets disappeared within 1 min after the addition of 100 nM nocodazole (not depicted). Importantly, CV1 cells that were transfected with MCMVa-GFP alone (Fig. S2 D) never exhibited GFP-labeled comets (n = 60 cells in five independent experiments). Together, these data demonstrate that myosin Va can track the plus end of growing microtubules and argue that this behavior is strictly Mlp dependent. In contrast, Rab27a-GFP, which is fully capable of rescuing ashen melanocytes (Wu et al., 2002a), did not exhibit plus end tracking behavior in CV1 cells when coexpressed with Mlp-mRFP (n = 45 cells in three independent experiments; unpublished data), indicating that Rab27a does not track together with Mlp.


Melanophilin and myosin Va track the microtubule plus end on EB1.

Wu XS, Tsan GL, Hammer JA - J. Cell Biol. (2005)

Myosin Va tracks the microtubule plus end. (A) A GFP-tagged version of the melanocyte-spliced isoform of myosin Va (MCMVa-GFP) plus end tracks together with Mlp-mRFP throughout the cell (Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). (B) In a fixed cell, the tail domain of this isoform (MCMVaTail-GFP) targets to microtubule plus ends together with FLAG-tagged Mlp (Mlp-FLAG; visualized by staining with α-FLAG antibody). The omission of Mlp-FLAG abrogated the interaction of MCMVaTail-GFP with microtubules (not depicted).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171176&req=5

fig4: Myosin Va tracks the microtubule plus end. (A) A GFP-tagged version of the melanocyte-spliced isoform of myosin Va (MCMVa-GFP) plus end tracks together with Mlp-mRFP throughout the cell (Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). (B) In a fixed cell, the tail domain of this isoform (MCMVaTail-GFP) targets to microtubule plus ends together with FLAG-tagged Mlp (Mlp-FLAG; visualized by staining with α-FLAG antibody). The omission of Mlp-FLAG abrogated the interaction of MCMVaTail-GFP with microtubules (not depicted).
Mentions: Although myosin Va and Rab27a are not required for Mlp to plus end track, one or both proteins might still track together with Mlp. To address this question, we used CV1 cells because they do not express Mlp, thereby allowing us to address the Mlp dependency of any possible plus end tracking behavior exhibited by myosin Va or Rab27a. When we cotransfected CV1 cells with Mlp-mRFP and a GFP-tagged version of the full-length melanocyte-spliced heavy chain isoform of myosin Va (MCMVa-GFP), which is fully capable of rescuing dilute melanocytes (Wu et al., 2002b), we observed a striking colocalization of the two proteins on comets whose dynamics were largely indistinguishable from those of Mlp alone (Fig. 4 A and Video 8, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). Moreover, both the myosin Va and Mlp components of these comets disappeared within 1 min after the addition of 100 nM nocodazole (not depicted). Importantly, CV1 cells that were transfected with MCMVa-GFP alone (Fig. S2 D) never exhibited GFP-labeled comets (n = 60 cells in five independent experiments). Together, these data demonstrate that myosin Va can track the plus end of growing microtubules and argue that this behavior is strictly Mlp dependent. In contrast, Rab27a-GFP, which is fully capable of rescuing ashen melanocytes (Wu et al., 2002a), did not exhibit plus end tracking behavior in CV1 cells when coexpressed with Mlp-mRFP (n = 45 cells in three independent experiments; unpublished data), indicating that Rab27a does not track together with Mlp.

Bottom Line: Moreover, myosin Va tracks the plus end in a Mlp-dependent manner.These results identify a novel +TIP and indicate that vertebrate cells possess a +TIP complex that is similar to the Myo2p-Kar9p-Bim1p complex in yeast.We suggest that the +TIP complex identified in this study may serve to focus the transfer of melanosomes from microtubules to actin at the microtubule plus end.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
In mouse melanocytes, myosin Va is recruited onto the surface of melanosomes by a receptor complex containing Rab27a that is present in the melanosome membrane and melanophilin (Mlp), which links myosin Va to Rab27a. In this study, we show that Mlp is also a microtubule plus end-tracking protein or +TIP. Moreover, myosin Va tracks the plus end in a Mlp-dependent manner. Data showing that overexpression and short inhibitory RNA knockdown of the +TIP EB1 have opposite effects on Mlp-microtubule interaction, that Mlp interacts directly with EB1, and that deletion from Mlp of a region similar to one in the adenomatous polyposis coli protein involved in EB1 binding blocks Mlp's ability to plus end track argue that Mlp tracks the plus end indirectly [corrected] by hitchhiking on EB1. These results identify a novel +TIP and indicate that vertebrate cells possess a +TIP complex that is similar to the Myo2p-Kar9p-Bim1p complex in yeast. We suggest that the +TIP complex identified in this study may serve to focus the transfer of melanosomes from microtubules to actin at the microtubule plus end.

Show MeSH
Related in: MedlinePlus