Rab1A regulates anterograde melanosome transport by recruiting kinesin-1 to melanosomes through interaction with SKIP - PubMed (original) (raw)
Rab1A regulates anterograde melanosome transport by recruiting kinesin-1 to melanosomes through interaction with SKIP
Morié Ishida et al. Sci Rep. 2015.
Abstract
Melanosomes are lysosome-related organelles in melanocytes that are transported from the perinucleus to the cell periphery by coordination between bidirectional (anterograde and retrograde) microtubule-dependent transport and unidirectional actin-dependent transport. Although the molecular machineries that mediate retrograde transport and actin-dependent transport have already been identified, little is known about the anterograde transport complex on microtubules in mammalian cells. Here we discovered that small GTPase Rab1A on melanosomes recruits SKIP/PLEKHM2 as a Rab1A-specific effector and that Rab1A, SKIP, and a kinesin-1/(Kif5b+KLC2) motor form a transport complex that mediates anterograde melanosome transport in melanocytes. Interestingly, Arl8, Arf-like small GTPase that also interacts with SKIP, is specifically localized at lysosomes and regulates their anterograde transport in melanocytes. Our findings suggest that the anterograde microtubule-dependent transport of melanosomes and lysosomes are differently regulated by independent cargo receptors, i.e., Rab1A and Arl8, respectively, but that a SKIP-kinesin-1 mechanism is responsible for the transport of both.
Figures
Figure 1. Functional involvement of SKIP in melanosome distribution in melanocytes.
(A) Rab binding specificity of the RUN domain of SKIP as revealed by yeast two-hybrid panels. Yeast cells containing pGBD-C1 plasmid expressing each of the 60 constitutively active/constitutively negative (CA/CN) Rabs (positions indicated in the left panels) and pAct2-SKIP-RUN were streaked on SC-AHLW and incubated at 30°C for 1 week. Note the specific, GTP-dependent interaction between SKIP-RUN and Rab1A. (B) Knockdown of endogenous SKIP protein in melan-a cells with the specific SKIP shRNA. The positions of the molecular mass markers are shown on the left. (C) Characterization of an shRNA-resistant (SR) SKIPSR mutant. (D) Knockdown of SKIP in melanocytes resulted in perinuclear melanosome aggregation. Melan-a cells were cotransfected with pSilencer-mStr-SKIP and either pEGFP-C1 (top row) or pEGFP-C1-SKIPSR (bottom row). Cells exhibiting perinuclear melanosome aggregation are outlined with broken lines. Note the clear perinuclear melanosome aggregation in the SKIP-deficient cells (top left panel), whereas re-expression of SKIPSR completely restored peripheral melanosome distribution (bottom left panel). (E) Quantification of the results shown in (D). The results are expressed as the percentages of transfected cells that exhibited perinuclear melanosome aggregation, and the bars represent the means and S.E. of the data from three independent experiments (n >50). **, p <0.01 in comparison with the control cells (Dunnett's test). Scale bars, 20 μm.
Figure 2. Effect of KLC2 knockdown in melanocytes on melanosome distribution.
(A) Knockdown of endogenous KLC2 protein in melan-a cells with specific KLC2 siRNAs. The positions of the molecular mass markers are shown on the left. (B) Knockdown of KLC2 in melanocytes resulted in perinuclear melanosome aggregation. Melan-a cells were treated with a control siRNA (left panel) or KLC2 siRNAs (right two panels). Cells exhibiting perinuclear melanosome aggregation are outlined with broken lines. Note the clear perinuclear melanosome aggregation in the KLC2-deficient cells (right two panels). Scale bars, 20 μm. (C) Quantification of the results shown in (B). The results are expressed as the percentages of cells that exhibited perinuclear melanosome aggregation, and the bars represent the means and S.E. of the data from three independent experiments (n >100). **, p <0.01 in comparison with the control cells (Dunnett's test).
Figure 3. Effect of Kif5b knockdown in melanocytes on melanosome distribution.
(A) Expression of Kif5b mRNA and not of Kif5a or Kif5c mRNA in melan-a cells as revealed by an RT-PCR analysis. The results are shown in the form of white/black-reversal images. GAPDH mRNA expression (bottom panel) is shown as a loading control to ensure that equivalent amounts of first-strand cDNA were used for the RT-PCR analysis. The size of the molecular mass markers (bp, base pairs) is shown on the left side of the panel. (B) Knockdown of endogenous Kif5b protein in melan-a cells with the specific Kif5b siRNAs. The positions of the molecular mass markers are shown on the left. (C) Knockdown of Kif5b in melanocytes resulted in perinuclear melanosome aggregation. Melan-a cells were treated with a control siRNA (top panel) or Kif5b siRNAs (bottom two panels). Cells exhibiting perinuclear melanosome aggregation are outlined with a broken line. Note the clear perinuclear melanosome aggregation in the Kif5b-deficient cells (bottom two panels). Scale bars, 20 μm. (D) Quantification of the results shown in (C). The results are expressed as the percentages of cells that exhibited perinuclear melanosome aggregation, and the bars represent the means and S.E. of the data from four independent experiments (n >100). **, p <0.01 in comparison with the control cells (Dunnett's test).
Figure 4. Formation of the Rab1A–SKIP–kinesin-1 complex in melan-a cells.
Endogenous Rab1A molecules were immunoprecipitated from lysates of melan-a cells with anti-Rab1A specific IgG (lane 3) or control IgG (lane 2). Rab1A, SKIP, KLC2, kinesin heavy chain (KHC), and Arl8b were analyzed by immunoblotting with specific antibodies. The amount of IgG heavy chain used for immunoprecipitation was analyzed with an anti-rabbit IgG antibody (bottom panel). It should be noted that SKIP, KLC2, and KHC (Kif5b) were co-purified with Rab1A, but Arl8b was not. Input shows a 1/140 volume of the reaction mixture used for immunoprecipitation. The positions of the molecular mass markers are shown on the left.
Figure 5. Rab1A on melanosomes interacts with SKIP and regulates anterograde melanosome transport.
(A) Competition experiments revealed that Arl8b-binding and Rab1A-binding to SKIP are mutually exclusive. Glutathione Sepharose beads coupled with T7-GST-SKIP-RUN or beads alone were incubated with solutions containing FLAG-Rab1A(Q70L) and/or an increasing amount of FLAG-Arl8b(Q75L), and the proteins bound to the beads were analyzed by immunoblotting with the antibodies indicated. Input shows a 1/140 volume of the reaction mixture used for the GST pull-down assays. The positions of the molecular mass markers are shown on the left. Note that Rab1A binding to SKIP was reduced by an increasing amount of Arl8b (the signal intensity values of bound FLAG-Rab1A(Q70L) normalized to T7-GST-SKIP-RUN signals are 1.0 (lane 6), 0.95 (lane 7), and 0.36 (lane 8)). (B) Typical images of melan-a cells expressing EGFP-Rab1A(Q70L) together with mStr-SKIP are shown. The insets are magnified views of the boxed areas showing that Rab1A colocalized with SKIP at melanosomes (arrowheads). Note that cells expressing EGFP-Rab1A(Q70L) together with mStr-SKIP exhibited peripheral melanosome accumulation. (C) Live cell images of melan-a cells expressing EGFP-SKIP and mStr-Rab1A(Q70L), and montage images showing colocalization of both EGFP-SKIP and mStr-Rab1A(Q70L) at a melanosome and moving with it toward the cell periphery (arrowheads). Images of the cells in (B) and (C) exhibiting peripheral melanosome accumulation are outlined with a broken line. Scale bars, 20 μm. (D) Quantification of the results shown in (C). The results are expressed as the percentages of transfected cells that exhibited peripheral melanosome accumulation, and the bars represent the means and S.E. of the data from three independent experiments (n >50). **, p <0.01 in comparison with the mStr- and EGFP-SKIP-expressing cells (Student's unpaired t test).
Figure 6. The Rab1A–SKIP interaction is necessary for anterograde melanosome transport.
(A) Interaction between SKIP-RUN (wild-type or Δ177–186) and Arl8b or Rab1A as revealed by yeast two-hybrid assays. Yeast cells containing pGBD-C1-Arl8b(Q75L)ΔΝ or pGBD-C1-Rab1A(Q70L)ΔCys and pAct2-SKIP-RUN (wild-type or Δ177–186) were streaked on SC-AHLW and incubated at 30°C for 1 week. (B) Interaction between SKIP-RUN (wild-type or Δ177–186) and Arl8b or Rab1A as revealed by coimmunoprecipitation assays. Beads coupled with either FLAG-Arl8b(Q75L) (lanes 1–4) or FLAG-Rab1A(Q70L) (lanes 5–8) were incubated with COS-7 cell lysates containing T7-SKIP-RUN (lanes 1, 2, 5, and 6) or T7-SKIP(Δ177–186)-RUN (lanes 3, 4, 7, and 8), and the proteins bound to the beads were analyzed by immunoblotting with the antibodies indicated. (C) Typical images of melan-a cells expressing EGFP-SKIP(Δ177–186) together with Arl8b(Q75L)-mStr (top row), or mStr-Rab1A(Q70L) (bottom row) are shown (the lysosomal marker Lamp-1 images and their corresponding bright-field images). Cells exhibiting perinuclear melanosome aggregation are outlined with a broken line. The insets show magnified views of the boxed areas. The arrowheads point to the site of lysosome accumulation at the cell periphery. Note that melan-a cells expressing EGFP-SKIP(Δ177–186) together with mStr-Rab1A(Q70L) no longer exhibited peripheral melanosome accumulation (bottom row), whereas cells expressing Arl8b(Q75L)-mStr (top row) still exhibited peripheral lysosome accumulation. Scale bars, 20 μm. (D) The results are expressed as the percentages of transfected cells that exhibited peripheral melanosome accumulation, and the bars represent the means and S.E. of the data from three independent experiments (n >50). **, p <0.01 in comparison with the cells expressing both mStr-Rab1A(Q70L) and EGFP-SKIP (Student's unpaired t test).
References
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