Identification of a third myosin-5a-melanophilin interaction that mediates the association of myosin-5a with melanosomes
- Group of Cell Motility and Muscle Contraction, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, China
- University of Chinese Academy of Sciences, China
Figures
Figure 1
The actin-binding domain of melanophilin (Mlph-ABD) interacts with the middle tail domain (MTD) of Myo5a.
(A) Diagram of the melanocyte-spliced isoform of Myo5a. Myo5a-MTD, Myo5a coiled-coils (residues 1106–1467). IQ, the CaM binding site; GTD, the C-terminal globular tail domain. In blue box is the sequence of exon-F and -G, annotated with the predicted coiled-coil heptad repeats. (B) Diagram of melanophilin (Mlph). RBD, Rab27a-binding domain; GTBM, globular tail domain-binding motif; EFBD, exon-F binding domain; ABD, actin-binding domain. (C, D) GST pulldown of GST-Mlph-ABD with the N-terminus truncated (C) or the C-terminus truncated (D) with Flag-Myo5a-MTD. GST-Mlph-ABD constructs were bound to GSH-Sepharose and then incubated with Flag-Myo5a-MTD. The GSH-Sepharose-bound proteins were eluted by GSH and analyzed by Western blot using anti-Flag antibody. The inputs were analyzed with SDS-PAGE and visualized by Coomassie brilliant blue (CBB) staining. GST was used as negative control. Note: The Sf9 cell-expressed Myo5a-MTD was used in the GST pulldown assays shown in this figure.
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Figure 1—source data 1
Original and uncropped gels and blots for Figure 1C.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig1-data1-v1.zip
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Figure 1—source data 2
Original and uncropped gels and blots for Figure 1D.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig1-data2-v1.zip
Figure 2 with 1 supplement
The exon-G of Myo5a interacts with Mlph-ABD.
(A) Summary of the interactions between the truncated Myo5a constructs and Mlph-ABD based on the GST pulldown assay shown in Figure 2—figure supplement 1A. +, strong interaction; +/-, weak interaction; -, no interaction. (B) Deletion of the C-terminal portion of exon-G abolishes the interaction between Myo5a-MTD and Mlph-ABD. (C) Deletion of the C-terminal portion of exon-G does not affect the interaction between Myo5a-MTD and Mlph-EFBD. GST pulldown assays were performed using GST-Mlph-ABD and Flag-Myo5a-MTD variants. The GSH-Sepharose-bound proteins were eluted by GSH and analyzed by Western blot using anti-FLAG antibody; the inputs were analyzed with SDS-PAGE and visualized by Coomassie brilliant blue (CBB) staining.
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Figure 2—source data 1
Original and uncropped gels and blots for Figure 2B.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig2-data1-v1.zip
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Figure 2—source data 2
Original and uncropped gels and blots for Figure 2C.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig2-data2-v1.zip
Figure 2—figure supplement 1
Identification of the Mlph-ABD-binding site in the middle tail of Myo5a.GST pulldown assays were performed using GST-Mlph-ABD and Flag-Myo5a-MTD variants.
The GSH-Sepharose-bound proteins were eluted by GSH and analyzed by Western blot using anti-FLAG antibody; the inputs were analyzed with SDS-PAGE and visualized by Coomassie brilliant blue (CBB) staining. (A) GST pulldown assays of GST-Mlph-ABD with Flag-Myo5a-MTD expressed in E. coli or Sf9 cells. (B and C) GST pulldown assay of GST-Mlph-ABD with Flag-tagged, truncated Myo5a tail.
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Figure 2—figure supplement 1—source data 1
Original and uncropped gels and blots for Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Original and uncropped gels and blots for Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig2-figsupp1-data2-v1.zip
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Figure 2—figure supplement 1—source data 3
Original and uncropped gels and blots for Figure 2—figure supplement 1C.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig2-figsupp1-data3-v1.zip
Figure 3
Identification of the key residues for the exon-G/ABD interaction.
(A) Dissociation constant (Kd) of Myo5a-MTD or Myo5a-MTDΔG binding to Mlph-ABD measured by MST. The solid curve was fit to the standard Kd-fit function. Results are presented as the mean ± SEM of three independent experiments. (B) Ionic strength dependence of the interaction between Myo5a-MTD and Mlph-ABD. GST pulldown was performed using GST-Mlph-ABD and Flag-Myo5a-MTD in the presence of different concentrations of NaCl. (C) Sequence alignments of the regions in Mlph-ABD essential for binding to Myo5a-MTD. Conserved charged residues are indicated. (D) Sequence alignments of the C-terminal portion of exon-G, an essential region for binding to Mlph-ABD. Two conserved basic residues (K1456 and K1460) are indicated. (E) Effects of alanine mutation of the conserved charged residues in Mlph-ABD on the interaction with Myo5a-MTD. E449A and E452A mutations in Mlph abolished the interaction between Mlph-ABD and Myo5a-MTD. (F) Effects of K1456A and K1460A mutations of Myo5a-MTD on the interaction with Mlph-ABD.
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Figure 3—source data 1
Original MST data for Figure 3A.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig3-data1-v1.zip
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Figure 3—source data 2
Original and uncropped gels and blots for Figure 3B.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig3-data2-v1.zip
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Figure 3—source data 3
Original and uncropped gels and blots for Figure 3E.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig3-data3-v1.zip
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Figure 3—source data 4
Original and uncropped gels and blots for Figure 3F.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig3-data4-v1.zip
Figure 4
The exon-F/EFBD and the exon-G/ABD interactions act synergistically.
(A) Myo5a-MTD bridges between Mlph-ABD and Mlph-EFBD. Upper, diagram shows Myo5a-MTD binds to both the EFBD and the ABD of Mlph. Lower, GST pulldown assays of GST-Mlph-ABD and Flag-Mlph-EFBD with or without Flag-Myo5a-MTD. (B) Both exon-F and exon-G of Myo5a-MTD are required for the strong interaction with Mlph-ΔRBD. GST pulldown of GST-Mlph-ΔRBD with Flag-Myo5a-MTD variants. (C) ABD is essential for the strong interaction between Mlph-ΔRBD and Myo5a-MTD. The input samples were analyzed by SDS-PAGE and visualized by CBB staining. The pulled down samples were analyzed by western blot using anti-Flag antibody (A and B) or by SDS-PAGE with CBB staining (C).
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Figure 4—source data 1
Original and uncropped gels and blots for Figure 4A.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig4-data1-v1.zip
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Figure 4—source data 2
Original and uncropped gels and blots for Figure 4B.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig4-data2-v1.zip
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Figure 4—source data 3
Original and uncropped gels for Figure 4C.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig4-data3-v1.zip
Figure 5
Myo5a-MTD antagonizes the interaction between Mlph-ABD and actin.
(A) GST-Mlph-ABD and/or Flag-Myo5a-MTD were incubated with actin and then subjected to ultracentrifugation. The supernatants (S) and the pellets (P) were analyzed by SDS-PAGE (10%) with CBB staining. (B) GST-Mlph-ABD was incubated with actin in the presence of different concentrations of Flag-Myo5a-MTD and then subjected to to ultracentrifugation. Upper panel, the supernatants (S) and the pellets (P) were analyzed by SDS-PAGE (10%) with CBB staining. Lower panel, the amounts of GST-Mlph-ABD co-sedimentated with actin in the presence of different concentration of Flag-Myo5a-MTD were quantified based on the density in the SDS-PAGE. Data are the mean ± SD of three independent experiments with one-way ANOVA with post-hoc Bonferroni test. **p<0.01, ****p<0.0001. Note: a Sf9 cell-expressed Myo5a-MTD was used in the actin cosedimentation assays shown in this figure.
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Figure 5—source data 1
Original and uncropped gels for Figure 5A.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig5-data1-v1.zip
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Figure 5—source data 2
Original and uncropped gels and statistical data for Figure 5B.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig5-data2-v1.zip
Figure 6 with 1 supplement
Both E449 and E452 of Mlph are essential for the perinuclear distribution of melanosomes induced by Mlph-ΔRBD overexpression.
Melan-a melanocytes were transfected to express EGFP-Mlph-ΔRBD (WT, E449A, or E452A) or EGFP. The distribution of melanosomes in the transfected melanocytes was imaged and the number of melanocytes with perinuclear distribution of melanosomes was counted. (A) Typical images of melanocytes expressing EGFP alone, EGFP-Mlph-ΔRBD WT, E449A, or E452A. Zooms areⅹ3. Cells are outlined with red dashed lines. Scale bars = 10 μm. The transfection efficiences were 11% for EGFP (n=275), 9.2% for EGFP-Mlph-ΔRBD WT (n=194), 11.2% for E449A (n=208), and 11.8% for E452A (n=267). n represents cell number. (B) The percentage of melanocytes exhibiting perinuclear melanosome aggregation among the transfected melanocytes. Results are presented as the mean ± SD of three independent experiments with one-way ANOVA with post-hoc Bonferroni test. Each point represents the percentage of perinuclear distribution of EGFP fusion proteins expressing cells in each fields. ****p<0.0001. (C) Western blot of the lysates of melan-a transfected with EGFP-Mlph-ΔRBD WT, E449A, or E452A. The expressed EGFP fusion proteins were probed with the antibody against EGFP. The relative band densities of EGFP-Mlph-ΔRBD WT, E449A, and E452A were 1, 1.29, and 1.25, respectively. Correction of the transfection efficiencies of those three constructs (A; Figure 6—figure supplement 1) gave rise to the relative protein expression levels of each transfected cells as 1, 1.05, and 0.97 for EGFP-Mlph-ΔRBD WT, E449A, and E452A, respectively.
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Figure 6—source data 1
Original and statistical data for Figure 6B.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig6-data1-v1.zip
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Figure 6—source data 2
Original and uncropped blots for Figure 6C.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig6-data2-v1.zip
Figure 6—figure supplement 1
Typical images of melanocytes expressing EGFP-Mlph-ΔRBD, its mutants, or EGFP.
Cells are outlined with a red dashed line. Scale bars = 10 μm. Zooms areⅹ0.75.
Figure 7 with 1 supplement
Exon-G region of Myo5a is essential for the perinuclear distribution of melanosomes induced by Myo5a-Tail overexpression.
(A) Diagram of Myo5a-Tail constructs used for transfecting melan-a melanocytes. (B) Melan-a melanocytes were transfected to express EGFP-Myo5a-Tail and the mutant. The distribution of melanosomes in the transfected melanocytes was imaged and the number of melanocytes with perinuclear distribution of melanosomes was counted. Typical images of melanocytes expressing EGFP-Myo5a-Tail, its mutants, or EGFP. Cells are outlined with a dashed line. Scale bars = 10 μm. Zooms areⅹ3. (C) The percentages of melanocytes exhibiting perinuclear melanosome aggregation. Data are the mean ± SD of three independent experiments with one-way ANOVA with post-hoc Bonferroni test. ****p<0.0001. (D) Western blots of whole cell extracts prepared from melan-a melanocytes transfected with each of the three Myo5a constructs EGFP fusion protein constructs probed with an antibody against EGFP. Each point represents the percentages of perinuclear distribution of EGFP fusion proteins expressing cells in each fields. Western blot of the lysates of melan-a transfected with EGFP-Myo5a-Tail or EGFP-Myo5a-TailΔG. The expressed EGFP fusion proteins were probed with the antibody against EGFP. The relative band densities of EGFP-Myo5a-Tail and EGFP-Myo5a-TailΔG were 1 and 0.9, respectively. Correction of the transfection efficiencies of those three constructs (B; Figure 7—figure supplement 1) gave rise to the relative protein expression levels of each transfected cells as 1, 0.93 for EGFP-Myo5a-Tail or EGFP-Myo5a-TailΔG, respectively.
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Figure 7—source data 1
Original and statistical data for Figure 7C.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig7-data1-v1.zip
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Figure 7—source data 2
Original and uncropped blots for Figure 7D.
- https://cdn.elifesciences.org/articles/93662/elife-93662-fig7-data2-v1.zip
Figure 7—figure supplement 1
Typical images of melanocytes expressing EGFP-Myo5a-Tail, its mutants, or EGFP.
Cells are outlined with a red dashed line. Scale bars = 10 μm. Zooms areⅹ0.75.
Figure 8
Effects of deletion of exon-F or exon-G on the localization of Myo5a-MTD in melan-a cells.
Melan-a melanocytes were transfected to express EGFP-Myo5a-MTD (A), EGFP-Myo5a-MTDΔF (B), or EGFP-Myo5a-MTDΔG (C) and stained for endogenous Mlph. Insets represent higher magnification photomicrographs of a cell within the region outlined by frames. Scale bars = 10 μm.
Figure 9
A model for the Mlph-mediated Myo5a transportation of melanosome.
The Mlph-mediated Myo5a transportation of melanosome is comprised of four stages. At stage 1, Mlph associates with melanosome via its interaction with Rab27a, which directly binds to the membrane of melanosome; the unattached Myo5a is in a folded conformation, in which the GTD binds to and inhibits the motor domain. At stage 2, Mlph interacts with the folded Myo5a via the interactions of EFBD/exon-F and ABD/exon-G; the attached Myo5a is still in folded conformation, because the GTBM-binding surface in the GTD is buried at the GTD-GTD interface. At stage 3, the buried GTBM-binding surface between the GTD-GTD interface is exposed and thus facilitate the binding of GTBM, causing the dissociation of the GTD from the motor domain and inducing the extended conformation of Myo5a (This step is probably regulated by the binding of Rab36/RilpL2 to the GTD). At stage 4, Mlph-ABD dissociates from exon-G and then binds to actin filament, thus enhancing the processive movement of Myo5a (This step might be regulated by the phosphorylation of Mlph-ABD).
Tables
Author response table 1
Percentage of the EGFP-expressing cells with perinuclear aggregation of melanosomes.
| Transfected construct | High expression | Low expression |
|---|---|---|
| EGFP-Mlph-ΔRBD WT | 59.2%(n=94) | 23.8%(n=74) |
| EGFP-Mlph-ΔRBD E449A | 22.5%(n=103) | 4.9%(n=71) |
| EGFP-Mlph-ΔRBD E452A | 20.8%(n=120) | 3.7%(n=77) |
| EGFP-Myo5a-Tail | 69.3%(n=96) | 11.9%(n=63) |
| EGFP-Myo5a-TailΔG | 16.7%(n=110) | 2.1%(n=72) |
Additional files
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Supplementary file 1
The primer sequences used in PCR for Myo5a constructs in pET30HFa.
- https://cdn.elifesciences.org/articles/93662/elife-93662-supp1-v1.xlsx
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Supplementary file 2
The primer sequences used in PCR for Mlph constructs in pGEX4T1.
- https://cdn.elifesciences.org/articles/93662/elife-93662-supp2-v1.xlsx
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Supplementary file 3
The primer sequences used in PCR for Myo5a or Mlph constructs in pEGFP-C1.
- https://cdn.elifesciences.org/articles/93662/elife-93662-supp3-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/93662/elife-93662-mdarchecklist1-v1.docx
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Identification of a third myosin-5a-melanophilin interaction that mediates the association of myosin-5a with melanosomes
eLife 13:RP93662.
https://doi.org/10.7554/eLife.93662.3
