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Identification and characterization of multiple novel Rab-myosin Va interactions.

Lindsay AJ, Jollivet F, Horgan CP, Khan AR, Raposo G, McCaffrey MW, Goud B - Mol. Biol. Cell (2013)

Bottom Line: Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems.Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes.We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology Laboratory, School of Biochemistry and Cell Biology, Biosciences Institute, University College Cork, Cork, Ireland Centre de Recherche, Molecular Mechanisms of Intracellular Transport, Institut Curie, CNRS UMR144, F-75248 Paris, France School of Biochemistry and Immunology, Trinity College, Dublin 2, Ireland Structure and Membrane Compartments, Institut Curie, CNRS UMR144, F-75248 Paris, France Cell and Tissue Imaging Facility (PICT-IBiSA), Institut Curie, CNRS UMR144, F-75248 Paris, France.

ABSTRACT
Myosin Va is a widely expressed actin-based motor protein that binds members of the Rab GTPase family (3A, 8A, 10, 11A, 27A) and is implicated in many intracellular trafficking processes. To our knowledge, myosin Va has not been tested in a systematic screen for interactions with the entire Rab GTPase family. To that end, we report a yeast two-hybrid screen of all human Rabs for myosin Va-binding ability and reveal 10 novel interactions (3B, 3C, 3D, 6A, 6A', 6B, 11B, 14, 25, 39B), which include interactions with three new Rab subfamilies (Rab6, Rab14, Rab39B). Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems. We demonstrate that myosin Va has three distinct Rab-binding domains on disparate regions of the motor (central stalk, an alternatively spliced exon, and the globular tail). Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes. We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes.

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Myosin Va depletion affects Rab motility and organelle distribution. (A) A HeLa cell line stably expressing GFP-Rab6A′ was transfected with control or myosin Va siRNA for 72 h. Two-minute time-lapse movies were recorded on a spinning disk confocal microscope. Representative frames from Supplemental Movie S1. Black arrows indicate tubules emerging from the Golgi complex. (B) Quantification of the number of Rab6A′-positive tubules connected to the Golgi for each condition (mean ± SEM; n = 11–15 cells). Two independent siRNAs targeting myosin Va were used. **p < 10−3. (C) HeLa cells were transfected with control or myosin Va siRNA for 72 h and plasmid DNA encoding GFP-Rab11A for the final 18 h. Two-minute time-lapse movies were recorded on a spinning disk confocal microscope. Representative frames from Supplemental Movie S2. Insets depict zoomed images of the boxed region. Indicated are examples of immobile vesicles (orange arrowheads) and rapidly moving vesicles (blue arrowheads). Closed arrowheads indicate the position of the vesicle in the first frame (recorded at 32 s), and the open arrowheads indicate the position of the same vesicle in the given frame. (D) HeLa cells transfected with control or myosin Va siRNA for 72 h and plasmid DNA encoding GFP-Rab14 for the final 18 h. Two-minute time-lapse movies were recorded on a spinning disk confocal microscope. Representative frames from Supplemental Movie S3.
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Figure 7: Myosin Va depletion affects Rab motility and organelle distribution. (A) A HeLa cell line stably expressing GFP-Rab6A′ was transfected with control or myosin Va siRNA for 72 h. Two-minute time-lapse movies were recorded on a spinning disk confocal microscope. Representative frames from Supplemental Movie S1. Black arrows indicate tubules emerging from the Golgi complex. (B) Quantification of the number of Rab6A′-positive tubules connected to the Golgi for each condition (mean ± SEM; n = 11–15 cells). Two independent siRNAs targeting myosin Va were used. **p < 10−3. (C) HeLa cells were transfected with control or myosin Va siRNA for 72 h and plasmid DNA encoding GFP-Rab11A for the final 18 h. Two-minute time-lapse movies were recorded on a spinning disk confocal microscope. Representative frames from Supplemental Movie S2. Insets depict zoomed images of the boxed region. Indicated are examples of immobile vesicles (orange arrowheads) and rapidly moving vesicles (blue arrowheads). Closed arrowheads indicate the position of the vesicle in the first frame (recorded at 32 s), and the open arrowheads indicate the position of the same vesicle in the given frame. (D) HeLa cells transfected with control or myosin Va siRNA for 72 h and plasmid DNA encoding GFP-Rab14 for the final 18 h. Two-minute time-lapse movies were recorded on a spinning disk confocal microscope. Representative frames from Supplemental Movie S3.

Mentions: To investigate the functional relationship(s) between myosin Va and its partner Rabs, we performed live-cell spinning-disk confocal microscopy in control or myosin Va-depleted HeLa cells expressing GFP fusions of each Rab. Myosin Va knockdown had no observable effect on the distribution/motility of GFP-Rab3D, GFP-Rab8A, or GFP-Rab10 (unpublished data); however, effects were observed in the distribution of Rab6-, Rab11A-, and Rab14-positive structures (Figure 7 and Supplemental Movies S1–S3).


Identification and characterization of multiple novel Rab-myosin Va interactions.

Lindsay AJ, Jollivet F, Horgan CP, Khan AR, Raposo G, McCaffrey MW, Goud B - Mol. Biol. Cell (2013)

Myosin Va depletion affects Rab motility and organelle distribution. (A) A HeLa cell line stably expressing GFP-Rab6A′ was transfected with control or myosin Va siRNA for 72 h. Two-minute time-lapse movies were recorded on a spinning disk confocal microscope. Representative frames from Supplemental Movie S1. Black arrows indicate tubules emerging from the Golgi complex. (B) Quantification of the number of Rab6A′-positive tubules connected to the Golgi for each condition (mean ± SEM; n = 11–15 cells). Two independent siRNAs targeting myosin Va were used. **p < 10−3. (C) HeLa cells were transfected with control or myosin Va siRNA for 72 h and plasmid DNA encoding GFP-Rab11A for the final 18 h. Two-minute time-lapse movies were recorded on a spinning disk confocal microscope. Representative frames from Supplemental Movie S2. Insets depict zoomed images of the boxed region. Indicated are examples of immobile vesicles (orange arrowheads) and rapidly moving vesicles (blue arrowheads). Closed arrowheads indicate the position of the vesicle in the first frame (recorded at 32 s), and the open arrowheads indicate the position of the same vesicle in the given frame. (D) HeLa cells transfected with control or myosin Va siRNA for 72 h and plasmid DNA encoding GFP-Rab14 for the final 18 h. Two-minute time-lapse movies were recorded on a spinning disk confocal microscope. Representative frames from Supplemental Movie S3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814135&req=5

Figure 7: Myosin Va depletion affects Rab motility and organelle distribution. (A) A HeLa cell line stably expressing GFP-Rab6A′ was transfected with control or myosin Va siRNA for 72 h. Two-minute time-lapse movies were recorded on a spinning disk confocal microscope. Representative frames from Supplemental Movie S1. Black arrows indicate tubules emerging from the Golgi complex. (B) Quantification of the number of Rab6A′-positive tubules connected to the Golgi for each condition (mean ± SEM; n = 11–15 cells). Two independent siRNAs targeting myosin Va were used. **p < 10−3. (C) HeLa cells were transfected with control or myosin Va siRNA for 72 h and plasmid DNA encoding GFP-Rab11A for the final 18 h. Two-minute time-lapse movies were recorded on a spinning disk confocal microscope. Representative frames from Supplemental Movie S2. Insets depict zoomed images of the boxed region. Indicated are examples of immobile vesicles (orange arrowheads) and rapidly moving vesicles (blue arrowheads). Closed arrowheads indicate the position of the vesicle in the first frame (recorded at 32 s), and the open arrowheads indicate the position of the same vesicle in the given frame. (D) HeLa cells transfected with control or myosin Va siRNA for 72 h and plasmid DNA encoding GFP-Rab14 for the final 18 h. Two-minute time-lapse movies were recorded on a spinning disk confocal microscope. Representative frames from Supplemental Movie S3.
Mentions: To investigate the functional relationship(s) between myosin Va and its partner Rabs, we performed live-cell spinning-disk confocal microscopy in control or myosin Va-depleted HeLa cells expressing GFP fusions of each Rab. Myosin Va knockdown had no observable effect on the distribution/motility of GFP-Rab3D, GFP-Rab8A, or GFP-Rab10 (unpublished data); however, effects were observed in the distribution of Rab6-, Rab11A-, and Rab14-positive structures (Figure 7 and Supplemental Movies S1–S3).

Bottom Line: Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems.Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes.We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology Laboratory, School of Biochemistry and Cell Biology, Biosciences Institute, University College Cork, Cork, Ireland Centre de Recherche, Molecular Mechanisms of Intracellular Transport, Institut Curie, CNRS UMR144, F-75248 Paris, France School of Biochemistry and Immunology, Trinity College, Dublin 2, Ireland Structure and Membrane Compartments, Institut Curie, CNRS UMR144, F-75248 Paris, France Cell and Tissue Imaging Facility (PICT-IBiSA), Institut Curie, CNRS UMR144, F-75248 Paris, France.

ABSTRACT
Myosin Va is a widely expressed actin-based motor protein that binds members of the Rab GTPase family (3A, 8A, 10, 11A, 27A) and is implicated in many intracellular trafficking processes. To our knowledge, myosin Va has not been tested in a systematic screen for interactions with the entire Rab GTPase family. To that end, we report a yeast two-hybrid screen of all human Rabs for myosin Va-binding ability and reveal 10 novel interactions (3B, 3C, 3D, 6A, 6A', 6B, 11B, 14, 25, 39B), which include interactions with three new Rab subfamilies (Rab6, Rab14, Rab39B). Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems. We demonstrate that myosin Va has three distinct Rab-binding domains on disparate regions of the motor (central stalk, an alternatively spliced exon, and the globular tail). Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes. We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes.

Show MeSH
Related in: MedlinePlus