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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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Mlp is a +TIP. (A) Mlp remains at the end of growing microtubules (Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). A time series inside the boxed region is shown to the right (arrows mark the growing microtubule end). (B) Mlp and EB1 plus end track together (Video 3). A time series of the boxed regions is shown to the right. (C) Mlp comets disappear within 1 min after the addition of 100 nM nocodazole (Nz; Video 4).
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fig2: Mlp is a +TIP. (A) Mlp remains at the end of growing microtubules (Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). A time series inside the boxed region is shown to the right (arrows mark the growing microtubule end). (B) Mlp and EB1 plus end track together (Video 3). A time series of the boxed regions is shown to the right. (C) Mlp comets disappear within 1 min after the addition of 100 nM nocodazole (Nz; Video 4).

Mentions: We used four approaches to prove that Mlp is a +TIP. First, we showed that in fibroblasts cotransfected with mRFP-tagged Mlp (Mlp-mRFP) and GFP-tagged α-tubulin, Mlp comets localized at the distal end of microtubules and remained there as the microtubules grew (Fig. 2 A and Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). Second, we showed that in fibroblasts cotransfected with Mlp-mRFP and EB1-GFP, which is a well-characterized +TIP, the two proteins tracked together throughout the cell (Fig. 2 B and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). As reported previously (Mimori-Kiyosue et al., 2000), we observed that EB1-GFP comets disappear when growing microtubules reach the edge of the cell because EB1 does not remain at the end of pausing or shrinking microtubules. In most cases (Fig. 1 B and Video 1), we could not be certain that Mlp-GFP also disappeared at the periphery because the abundant signal from Mlp-GFP that is associated with cortical actin usually obscured the protein's microtubule plus end signal near the cell margin. Where this was not a problem (e.g., the cell in Video 3), Mlp and EB1 blinked out together, suggesting that Mlp, like EB1, does not usually associate with the plus ends of pausing or shrinking microtubules. Third, we found that Mlp-GFP comets moved at a uniform rate of 0.23 ± 0.1 μm/s (n = 100 from five cells), which is very similar to the rate of microtubule growth reported previously using the +TIPs CLIP-170 (Komarova et al., 2002) and EB1 (Mimori-Kiyosue et al., 2000) as reporters. Fourth, we showed that a low dose of nocodazole (100 nM), which leaves the interphase microtubule array largely intact but blocks growth at the plus end and dissociates +TIPs, caused Mlp-GFP comets to vanish within 1 min (Fig. 2 C and Video 4).


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

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

Mlp is a +TIP. (A) Mlp remains at the end of growing microtubules (Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). A time series inside the boxed region is shown to the right (arrows mark the growing microtubule end). (B) Mlp and EB1 plus end track together (Video 3). A time series of the boxed regions is shown to the right. (C) Mlp comets disappear within 1 min after the addition of 100 nM nocodazole (Nz; Video 4).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171176&req=5

fig2: Mlp is a +TIP. (A) Mlp remains at the end of growing microtubules (Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). A time series inside the boxed region is shown to the right (arrows mark the growing microtubule end). (B) Mlp and EB1 plus end track together (Video 3). A time series of the boxed regions is shown to the right. (C) Mlp comets disappear within 1 min after the addition of 100 nM nocodazole (Nz; Video 4).
Mentions: We used four approaches to prove that Mlp is a +TIP. First, we showed that in fibroblasts cotransfected with mRFP-tagged Mlp (Mlp-mRFP) and GFP-tagged α-tubulin, Mlp comets localized at the distal end of microtubules and remained there as the microtubules grew (Fig. 2 A and Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). Second, we showed that in fibroblasts cotransfected with Mlp-mRFP and EB1-GFP, which is a well-characterized +TIP, the two proteins tracked together throughout the cell (Fig. 2 B and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). As reported previously (Mimori-Kiyosue et al., 2000), we observed that EB1-GFP comets disappear when growing microtubules reach the edge of the cell because EB1 does not remain at the end of pausing or shrinking microtubules. In most cases (Fig. 1 B and Video 1), we could not be certain that Mlp-GFP also disappeared at the periphery because the abundant signal from Mlp-GFP that is associated with cortical actin usually obscured the protein's microtubule plus end signal near the cell margin. Where this was not a problem (e.g., the cell in Video 3), Mlp and EB1 blinked out together, suggesting that Mlp, like EB1, does not usually associate with the plus ends of pausing or shrinking microtubules. Third, we found that Mlp-GFP comets moved at a uniform rate of 0.23 ± 0.1 μm/s (n = 100 from five cells), which is very similar to the rate of microtubule growth reported previously using the +TIPs CLIP-170 (Komarova et al., 2002) and EB1 (Mimori-Kiyosue et al., 2000) as reporters. Fourth, we showed that a low dose of nocodazole (100 nM), which leaves the interphase microtubule array largely intact but blocks growth at the plus end and dissociates +TIPs, caused Mlp-GFP comets to vanish within 1 min (Fig. 2 C and Video 4).

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