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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 induces perinuclear clustering of endocytic recycling pathway membrane compartments. HeLa cells transfected with control siRNA or myosin Va siRNA for 72 h were fixed, permeabilized, and colabeled with antibodies to the TfR and TGN46 (A), EEA1 and GM130 (B), or LBPA and TGN46 (C). (D) Quantification of the fluorescence intensity of TfR, EEA1, or LBPA in the pericentrosomal region relative to the total cellular fluorescence intensity and expressed as a “compaction factor.” Data were normalized to the control (mean ± SEM; n = 30–50 cells). *p <10−4, Student's t test.
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Figure 6: Myosin Va depletion induces perinuclear clustering of endocytic recycling pathway membrane compartments. HeLa cells transfected with control siRNA or myosin Va siRNA for 72 h were fixed, permeabilized, and colabeled with antibodies to the TfR and TGN46 (A), EEA1 and GM130 (B), or LBPA and TGN46 (C). (D) Quantification of the fluorescence intensity of TfR, EEA1, or LBPA in the pericentrosomal region relative to the total cellular fluorescence intensity and expressed as a “compaction factor.” Data were normalized to the control (mean ± SEM; n = 30–50 cells). *p <10−4, Student's t test.

Mentions: We next investigated the effects of myosin Va knockdown on organelle distribution. RNAi-mediated knockdown of myosin Va resulted in the accumulation of TfR-positive membranes in the perinuclear region of the cell (Figure 6A). Quantification revealed an almost 60% increase in TfR fluorescence near the Golgi in comparison with the control cells (Figure 6D). Myosin Va depletion also caused a similar accumulation of EEA1-labeled early endosomes in the perinuclear region (Figure 6, B and D), whereas the distribution of late endosomes/lysosomes was unaffected (Figure 6, C and D). This juxtanuclear vesicle accumulation is reminiscent of the post-Golgi vesicle clustering observed in yeast cells when Myo2 binding to Sro7 is disrupted (Rossi and Brennwald, 2011).


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 induces perinuclear clustering of endocytic recycling pathway membrane compartments. HeLa cells transfected with control siRNA or myosin Va siRNA for 72 h were fixed, permeabilized, and colabeled with antibodies to the TfR and TGN46 (A), EEA1 and GM130 (B), or LBPA and TGN46 (C). (D) Quantification of the fluorescence intensity of TfR, EEA1, or LBPA in the pericentrosomal region relative to the total cellular fluorescence intensity and expressed as a “compaction factor.” Data were normalized to the control (mean ± SEM; n = 30–50 cells). *p <10−4, Student's t test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Myosin Va depletion induces perinuclear clustering of endocytic recycling pathway membrane compartments. HeLa cells transfected with control siRNA or myosin Va siRNA for 72 h were fixed, permeabilized, and colabeled with antibodies to the TfR and TGN46 (A), EEA1 and GM130 (B), or LBPA and TGN46 (C). (D) Quantification of the fluorescence intensity of TfR, EEA1, or LBPA in the pericentrosomal region relative to the total cellular fluorescence intensity and expressed as a “compaction factor.” Data were normalized to the control (mean ± SEM; n = 30–50 cells). *p <10−4, Student's t test.
Mentions: We next investigated the effects of myosin Va knockdown on organelle distribution. RNAi-mediated knockdown of myosin Va resulted in the accumulation of TfR-positive membranes in the perinuclear region of the cell (Figure 6A). Quantification revealed an almost 60% increase in TfR fluorescence near the Golgi in comparison with the control cells (Figure 6D). Myosin Va depletion also caused a similar accumulation of EEA1-labeled early endosomes in the perinuclear region (Figure 6, B and D), whereas the distribution of late endosomes/lysosomes was unaffected (Figure 6, C and D). This juxtanuclear vesicle accumulation is reminiscent of the post-Golgi vesicle clustering observed in yeast cells when Myo2 binding to Sro7 is disrupted (Rossi and Brennwald, 2011).

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