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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 plus end tracks in melanocytes. (A) In a typical transfected melanocyte, Mlp-GFP targets to both melanosomes (inset) and microtubule plus ends (arrowheads; Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). (B and C) Examples in which Mlp-GFP targets almost exclusively to microtubule plus ends/actin (B) or to melanosomes (C; Video 7). (D) A subset of endogenous EB1 comets stain for endogenous Mlp (arrowheads), whereas endogenous Mlp is recruited along the length of microtubules in melanocytes overexpressing EB1-GFP (E).
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fig3: Mlp plus end tracks in melanocytes. (A) In a typical transfected melanocyte, Mlp-GFP targets to both melanosomes (inset) and microtubule plus ends (arrowheads; Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). (B and C) Examples in which Mlp-GFP targets almost exclusively to microtubule plus ends/actin (B) or to melanosomes (C; Video 7). (D) A subset of endogenous EB1 comets stain for endogenous Mlp (arrowheads), whereas endogenous Mlp is recruited along the length of microtubules in melanocytes overexpressing EB1-GFP (E).

Mentions: We found that Mlp-GFP exhibited robust plus end tracking behavior in other cell types, including normal rat kidney fibroblasts, COS, CV1, and HeLa (unpublished data). That said, none of these cell types possess detectable levels of endogenous Mlp despite the fact that Mlp mRNA is present in a wide range of mouse tissues (Matesic et al., 2001). In contrast, Mlp is highly expressed in melanocytes. Given this and the fact that Mlp's role as an adaptor protein for organelle–myosin Va interaction was established in melanocytes, we sought to characterize the dynamic behavior of Mlp in these cells. Fig. 3 A and Video 5 (available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1) show that Mlp-GFP exhibits clear plus end tracking behavior in primary wild-type melanocytes in addition to targeting to melanosomes. Mlp-GFP also tracked the plus end in a variety of melanocyte cell lines, including melan-c melanocytes that make unpigmented melanosomes (see below). Although transfected melanocytes overexpressed Mlp-GFP an average of ∼12-fold based on Western blotting (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1), correlative time-lapse microscopy coupled with quantitative immunofluorescence staining using Mlp antibody to detect both endogenous Mlp and overexpressed Mlp-GFP showed that individual transfected melanocytes can show prominent Mlp plus end tracking behavior with less than twofold overexpression (i.e., without huge overexpression; Fig. S2 A and Video 6). Although 67% (n = 300) of transfected melanocytes showed targeting of Mlp-GFP to both melanosomes and the plus end, the remaining cells showed almost exclusive targeting to either the plus end/actin or to melanosomes (Fig. 3, B and C; and Video 7). We do not know the basis for this differential targeting. Finally, melanosomes do not plus end track. Whether visualized with transmitted light or as Mlp-GFP–tagged structures (Fig. 3, A and C; Wu et. al. 1998, 2002a), the properties of their movement (intermittent, bidirectional, and ∼1–1.5 μm/s) are quite distinct from those of +TIPs (persistent, unidirectional, and ∼0.25 μm/s).


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

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

Mlp plus end tracks in melanocytes. (A) In a typical transfected melanocyte, Mlp-GFP targets to both melanosomes (inset) and microtubule plus ends (arrowheads; Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). (B and C) Examples in which Mlp-GFP targets almost exclusively to microtubule plus ends/actin (B) or to melanosomes (C; Video 7). (D) A subset of endogenous EB1 comets stain for endogenous Mlp (arrowheads), whereas endogenous Mlp is recruited along the length of microtubules in melanocytes overexpressing EB1-GFP (E).
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Related In: Results  -  Collection

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fig3: Mlp plus end tracks in melanocytes. (A) In a typical transfected melanocyte, Mlp-GFP targets to both melanosomes (inset) and microtubule plus ends (arrowheads; Video 5, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1). (B and C) Examples in which Mlp-GFP targets almost exclusively to microtubule plus ends/actin (B) or to melanosomes (C; Video 7). (D) A subset of endogenous EB1 comets stain for endogenous Mlp (arrowheads), whereas endogenous Mlp is recruited along the length of microtubules in melanocytes overexpressing EB1-GFP (E).
Mentions: We found that Mlp-GFP exhibited robust plus end tracking behavior in other cell types, including normal rat kidney fibroblasts, COS, CV1, and HeLa (unpublished data). That said, none of these cell types possess detectable levels of endogenous Mlp despite the fact that Mlp mRNA is present in a wide range of mouse tissues (Matesic et al., 2001). In contrast, Mlp is highly expressed in melanocytes. Given this and the fact that Mlp's role as an adaptor protein for organelle–myosin Va interaction was established in melanocytes, we sought to characterize the dynamic behavior of Mlp in these cells. Fig. 3 A and Video 5 (available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1) show that Mlp-GFP exhibits clear plus end tracking behavior in primary wild-type melanocytes in addition to targeting to melanosomes. Mlp-GFP also tracked the plus end in a variety of melanocyte cell lines, including melan-c melanocytes that make unpigmented melanosomes (see below). Although transfected melanocytes overexpressed Mlp-GFP an average of ∼12-fold based on Western blotting (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200503028/DC1), correlative time-lapse microscopy coupled with quantitative immunofluorescence staining using Mlp antibody to detect both endogenous Mlp and overexpressed Mlp-GFP showed that individual transfected melanocytes can show prominent Mlp plus end tracking behavior with less than twofold overexpression (i.e., without huge overexpression; Fig. S2 A and Video 6). Although 67% (n = 300) of transfected melanocytes showed targeting of Mlp-GFP to both melanosomes and the plus end, the remaining cells showed almost exclusive targeting to either the plus end/actin or to melanosomes (Fig. 3, B and C; and Video 7). We do not know the basis for this differential targeting. Finally, melanosomes do not plus end track. Whether visualized with transmitted light or as Mlp-GFP–tagged structures (Fig. 3, A and C; Wu et. al. 1998, 2002a), the properties of their movement (intermittent, bidirectional, and ∼1–1.5 μm/s) are quite distinct from those of +TIPs (persistent, unidirectional, and ∼0.25 μm/s).

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