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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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Cytoskeletal targeting of Mlp. (A) Mlp-GFP targets to actin stress fibers, cortical actin, and the MTOC (arrows) in fixed fibroblasts. (B) B1–B6 are designed to show (using still images) that Mlp-GFP exhibits a relatively stationary signal that is associated with cortical actin and a dynamic signal that is associated with microtubule plus ends (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). A fibroblast expressing Mlp-GFP was imaged at 1 frame/s for 30 s. B1 and B2 are the first and last frames (B2 is pseudocolored red), whereas B3 is the merge of these two images. The prominent yellow shows where Mlp has not changed position over 30 s. B4 shows a projection of all 30 frames, whereas B5 (pseudocolored red) shows the signal remaining in the projected image after subtraction of the first frame (B1). B6 shows the merge of B1 and B5. The dynamic microtubule-associated signal for Mlp-GFP appears in red, whereas the stationary actin-associated signal appears in green.
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fig1: Cytoskeletal targeting of Mlp. (A) Mlp-GFP targets to actin stress fibers, cortical actin, and the MTOC (arrows) in fixed fibroblasts. (B) B1–B6 are designed to show (using still images) that Mlp-GFP exhibits a relatively stationary signal that is associated with cortical actin and a dynamic signal that is associated with microtubule plus ends (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). A fibroblast expressing Mlp-GFP was imaged at 1 frame/s for 30 s. B1 and B2 are the first and last frames (B2 is pseudocolored red), whereas B3 is the merge of these two images. The prominent yellow shows where Mlp has not changed position over 30 s. B4 shows a projection of all 30 frames, whereas B5 (pseudocolored red) shows the signal remaining in the projected image after subtraction of the first frame (B1). B6 shows the merge of B1 and B5. The dynamic microtubule-associated signal for Mlp-GFP appears in red, whereas the stationary actin-associated signal appears in green.

Mentions: While using full-length GFP-tagged Mlp (Mlp-GFP) to determine the protein's distribution in primary melanocytes, we noticed that, in addition to melanosomes, Mlp showed variable targeting to three apparent cytoskeletal structures: actin stress fibers, cortical actin, and a single perinuclear spot presumably corresponding to the MTOC (unpublished data). These cytoskeletal-like distributions were even more pronounced in primary fibroblasts that contaminated the melanocyte cultures. Staining of transfected fibroblasts with phalloidin and an antibody to α-tubulin confirmed that Mlp-GFP concentrates on actin stress fibers, cortical actin, and at the MTOC (Fig. 1 A). To extend these observations, we examined the dynamic behavior of Mlp-GFP in fibroblasts (Fig. 1 B and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). Time-lapse images contained two distinct types of fluorescent signal: nearly stationary fluorescence that appeared to correspond to the actin-rich structures (Fig. 1, B1–B3) and, to our surprise, highly dynamic cometlike fluorescent signals radiating from the centrosome (Fig. 1, B4–B6; and Video 1). These latter structures emanated continuously from the bright spot at the MTOC and moved in a persistent, roughly linear path to the cell periphery in a fashion similar to that described previously for +TIPs (Carvalho et al., 2003).


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

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

Cytoskeletal targeting of Mlp. (A) Mlp-GFP targets to actin stress fibers, cortical actin, and the MTOC (arrows) in fixed fibroblasts. (B) B1–B6 are designed to show (using still images) that Mlp-GFP exhibits a relatively stationary signal that is associated with cortical actin and a dynamic signal that is associated with microtubule plus ends (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). A fibroblast expressing Mlp-GFP was imaged at 1 frame/s for 30 s. B1 and B2 are the first and last frames (B2 is pseudocolored red), whereas B3 is the merge of these two images. The prominent yellow shows where Mlp has not changed position over 30 s. B4 shows a projection of all 30 frames, whereas B5 (pseudocolored red) shows the signal remaining in the projected image after subtraction of the first frame (B1). B6 shows the merge of B1 and B5. The dynamic microtubule-associated signal for Mlp-GFP appears in red, whereas the stationary actin-associated signal appears in green.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171176&req=5

fig1: Cytoskeletal targeting of Mlp. (A) Mlp-GFP targets to actin stress fibers, cortical actin, and the MTOC (arrows) in fixed fibroblasts. (B) B1–B6 are designed to show (using still images) that Mlp-GFP exhibits a relatively stationary signal that is associated with cortical actin and a dynamic signal that is associated with microtubule plus ends (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). A fibroblast expressing Mlp-GFP was imaged at 1 frame/s for 30 s. B1 and B2 are the first and last frames (B2 is pseudocolored red), whereas B3 is the merge of these two images. The prominent yellow shows where Mlp has not changed position over 30 s. B4 shows a projection of all 30 frames, whereas B5 (pseudocolored red) shows the signal remaining in the projected image after subtraction of the first frame (B1). B6 shows the merge of B1 and B5. The dynamic microtubule-associated signal for Mlp-GFP appears in red, whereas the stationary actin-associated signal appears in green.
Mentions: While using full-length GFP-tagged Mlp (Mlp-GFP) to determine the protein's distribution in primary melanocytes, we noticed that, in addition to melanosomes, Mlp showed variable targeting to three apparent cytoskeletal structures: actin stress fibers, cortical actin, and a single perinuclear spot presumably corresponding to the MTOC (unpublished data). These cytoskeletal-like distributions were even more pronounced in primary fibroblasts that contaminated the melanocyte cultures. Staining of transfected fibroblasts with phalloidin and an antibody to α-tubulin confirmed that Mlp-GFP concentrates on actin stress fibers, cortical actin, and at the MTOC (Fig. 1 A). To extend these observations, we examined the dynamic behavior of Mlp-GFP in fibroblasts (Fig. 1 B and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). Time-lapse images contained two distinct types of fluorescent signal: nearly stationary fluorescence that appeared to correspond to the actin-rich structures (Fig. 1, B1–B3) and, to our surprise, highly dynamic cometlike fluorescent signals radiating from the centrosome (Fig. 1, B4–B6; and Video 1). These latter structures emanated continuously from the bright spot at the MTOC and moved in a persistent, roughly linear path to the cell periphery in a fashion similar to that described previously for +TIPs (Carvalho et al., 2003).

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