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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 is recruited to membranes by Rab10 and Rab11. HeLa cells expressing GFP-MyoVaTail(F)WT (A) or GFP-MyoVaTail(D)WT (B) or their mutants were fixed and imaged by fluorescence deconvolution microscopy. HeLa cells were transfected for 72 h with control siRNA or siRNA targeting the indicated Rab GTPase, and GFP-MyoVaTail(F)WT (C), GFP-MyoVaTail(D)WT (D), or GFP-MyoVaTail(D)Q1753R (E) was transfected into these cells for the final 18 h. The cells were fixed and the nuclei labeled with DAPI and imaged by fluorescence deconvolution microscopy. (See Figure S5D for Western blots analyzing the knockdown efficiency of the siRNA duplexes.)
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Figure 3: Myosin Va is recruited to membranes by Rab10 and Rab11. HeLa cells expressing GFP-MyoVaTail(F)WT (A) or GFP-MyoVaTail(D)WT (B) or their mutants were fixed and imaged by fluorescence deconvolution microscopy. HeLa cells were transfected for 72 h with control siRNA or siRNA targeting the indicated Rab GTPase, and GFP-MyoVaTail(F)WT (C), GFP-MyoVaTail(D)WT (D), or GFP-MyoVaTail(D)Q1753R (E) was transfected into these cells for the final 18 h. The cells were fixed and the nuclei labeled with DAPI and imaged by fluorescence deconvolution microscopy. (See Figure S5D for Western blots analyzing the knockdown efficiency of the siRNA duplexes.)

Mentions: Although it is believed that Rab GTPases recruit class V myosins to membranes, to our knowledge this has not yet been proven in mammalian cells. To address this, we expressed the tail regions of the D isoform (residues 1100–1855; henceforth referred to as the D isoform tail or MyoVaT(D)) and the F isoform (residues 1100–1852; henceforth referred to as the F isoform tail or MyoVaT(F)) of myosin Va bearing the Y1203A (abolishes Rab6 and Rab14 binding) or Q1753R (abolishes Rab3, Rab11, and Rab39B binding) mutations as GFP fusions in HeLa cells. At low levels of expression the F isoform displayed a punctate pattern reminiscent of the endogenous protein (Figure 3A). The Y1203A mutation does not affect this localization pattern, but introduction of the Q1753R mutation renders the F isoform mostly cytosolic (Figure 3A). This suggests that one or all of the Rabs that bind at this site are involved in the recruitment of the F isoform to membranes. To confirm this and determine which Rabs are important for myosin Va recruitment, we expressed MyoVaT(F)WT in HeLa cells in which many of its interacting Rabs had been individually depleted by RNA interference (RNAi). MyoVaT(F)WT was found to be cytosolic in cells in which Rab11A and Rab11B had been depleted (Figure 3C), whereas depletion of the other Rabs, including Rab3D, which is expressed in our HeLa cells (Supplemental Figure S2F), had no such effect (Figure 3C). To rule out the possibility that the level of overexpression of GFP-MyoVaT(F)WT was so high as to obscure any punctate staining, we recorded confocal images of siRab11A/B–transfected cells expressing low, medium, or high levels of the fusion protein, and in each case the myosin Va fusion was found to be predominantly cytosolic (Supplemental Figure S5A). A second small interfering RNA (siRNA) targeting each Rab was used in an independent series of experiments, and the same results were observed (Supplemental Figure S5, B, C, and E). To further verify these results, we cotransfected DN mutants of each GFP-Rab with mCherry-MyoVaT(F)WT (Supplemental Figure S6A). In agreement with the RNAi data, only Rab11A DN resulted in the redistribution of myosin Va into the cytosol (Supplemental Figure S6A).


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 is recruited to membranes by Rab10 and Rab11. HeLa cells expressing GFP-MyoVaTail(F)WT (A) or GFP-MyoVaTail(D)WT (B) or their mutants were fixed and imaged by fluorescence deconvolution microscopy. HeLa cells were transfected for 72 h with control siRNA or siRNA targeting the indicated Rab GTPase, and GFP-MyoVaTail(F)WT (C), GFP-MyoVaTail(D)WT (D), or GFP-MyoVaTail(D)Q1753R (E) was transfected into these cells for the final 18 h. The cells were fixed and the nuclei labeled with DAPI and imaged by fluorescence deconvolution microscopy. (See Figure S5D for Western blots analyzing the knockdown efficiency of the siRNA duplexes.)
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Figure 3: Myosin Va is recruited to membranes by Rab10 and Rab11. HeLa cells expressing GFP-MyoVaTail(F)WT (A) or GFP-MyoVaTail(D)WT (B) or their mutants were fixed and imaged by fluorescence deconvolution microscopy. HeLa cells were transfected for 72 h with control siRNA or siRNA targeting the indicated Rab GTPase, and GFP-MyoVaTail(F)WT (C), GFP-MyoVaTail(D)WT (D), or GFP-MyoVaTail(D)Q1753R (E) was transfected into these cells for the final 18 h. The cells were fixed and the nuclei labeled with DAPI and imaged by fluorescence deconvolution microscopy. (See Figure S5D for Western blots analyzing the knockdown efficiency of the siRNA duplexes.)
Mentions: Although it is believed that Rab GTPases recruit class V myosins to membranes, to our knowledge this has not yet been proven in mammalian cells. To address this, we expressed the tail regions of the D isoform (residues 1100–1855; henceforth referred to as the D isoform tail or MyoVaT(D)) and the F isoform (residues 1100–1852; henceforth referred to as the F isoform tail or MyoVaT(F)) of myosin Va bearing the Y1203A (abolishes Rab6 and Rab14 binding) or Q1753R (abolishes Rab3, Rab11, and Rab39B binding) mutations as GFP fusions in HeLa cells. At low levels of expression the F isoform displayed a punctate pattern reminiscent of the endogenous protein (Figure 3A). The Y1203A mutation does not affect this localization pattern, but introduction of the Q1753R mutation renders the F isoform mostly cytosolic (Figure 3A). This suggests that one or all of the Rabs that bind at this site are involved in the recruitment of the F isoform to membranes. To confirm this and determine which Rabs are important for myosin Va recruitment, we expressed MyoVaT(F)WT in HeLa cells in which many of its interacting Rabs had been individually depleted by RNA interference (RNAi). MyoVaT(F)WT was found to be cytosolic in cells in which Rab11A and Rab11B had been depleted (Figure 3C), whereas depletion of the other Rabs, including Rab3D, which is expressed in our HeLa cells (Supplemental Figure S2F), had no such effect (Figure 3C). To rule out the possibility that the level of overexpression of GFP-MyoVaT(F)WT was so high as to obscure any punctate staining, we recorded confocal images of siRab11A/B–transfected cells expressing low, medium, or high levels of the fusion protein, and in each case the myosin Va fusion was found to be predominantly cytosolic (Supplemental Figure S5A). A second small interfering RNA (siRNA) targeting each Rab was used in an independent series of experiments, and the same results were observed (Supplemental Figure S5, B, C, and E). To further verify these results, we cotransfected DN mutants of each GFP-Rab with mCherry-MyoVaT(F)WT (Supplemental Figure S6A). In agreement with the RNAi data, only Rab11A DN resulted in the redistribution of myosin Va into the cytosol (Supplemental Figure S6A).

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