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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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Expression of the myosin Va tail induces the aggregation of Rab-positive vesicles. (A) HeLa cells expressing the indicated myosin Va tail fused to GFP were fixed and labeled with antibodies that detect endogenous Rab GTPases. Insets show zoomed images of the boxed areas. (B) Quantitative analysis of the colocalization coefficients of each of the GFP–myosin Va tail constructs in HeLa cells with the indicated Rab GTPase (mean ± SEM; n = 20–40 cells).
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Figure 4: Expression of the myosin Va tail induces the aggregation of Rab-positive vesicles. (A) HeLa cells expressing the indicated myosin Va tail fused to GFP were fixed and labeled with antibodies that detect endogenous Rab GTPases. Insets show zoomed images of the boxed areas. (B) Quantitative analysis of the colocalization coefficients of each of the GFP–myosin Va tail constructs in HeLa cells with the indicated Rab GTPase (mean ± SEM; n = 20–40 cells).

Mentions: Immunofluorescence microscopy revealed that the large D isoform tail aggregates are positive for Rab8A, Rab10, Rab11A, and Rab14, indicating that they are accumulations of Rab-positive endosomal membranes (Figure 4A). The smaller MyoVaT(D)Q1753R-induced structures also contain Rab8A, Rab10, and Rab14 but lack Rab11A (Figure 4, A and B), confirming that Rab11-positive vesicles disengaged due to their inability to associate with the mutant protein.


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)

Expression of the myosin Va tail induces the aggregation of Rab-positive vesicles. (A) HeLa cells expressing the indicated myosin Va tail fused to GFP were fixed and labeled with antibodies that detect endogenous Rab GTPases. Insets show zoomed images of the boxed areas. (B) Quantitative analysis of the colocalization coefficients of each of the GFP–myosin Va tail constructs in HeLa cells with the indicated Rab GTPase (mean ± SEM; n = 20–40 cells).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Expression of the myosin Va tail induces the aggregation of Rab-positive vesicles. (A) HeLa cells expressing the indicated myosin Va tail fused to GFP were fixed and labeled with antibodies that detect endogenous Rab GTPases. Insets show zoomed images of the boxed areas. (B) Quantitative analysis of the colocalization coefficients of each of the GFP–myosin Va tail constructs in HeLa cells with the indicated Rab GTPase (mean ± SEM; n = 20–40 cells).
Mentions: Immunofluorescence microscopy revealed that the large D isoform tail aggregates are positive for Rab8A, Rab10, Rab11A, and Rab14, indicating that they are accumulations of Rab-positive endosomal membranes (Figure 4A). The smaller MyoVaT(D)Q1753R-induced structures also contain Rab8A, Rab10, and Rab14 but lack Rab11A (Figure 4, A and B), confirming that Rab11-positive vesicles disengaged due to their inability to associate with the mutant protein.

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