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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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Endosomes are tethered to the actin cytoskeleton by Rab14 and myosin Va. (A) Model depicting the tethering of endosomes to the actin cytoskeleton by myosin Va, which is associated with the endosomal membrane via a direct interaction with Rab14. (B) HeLa cells expressing GFP-Rab5Q79L for ∼24 h were fixed and labeled with coumarin–phalloidin and an antibody to myosin Va. Inset contains a zoomed image of the indicated enlarged endosome. (C) Quantification of the number of endogenous myosin Va puncta on GFP-Rab5AQ79L enlarged endosomes in cells transfected with the indicated siRNA for 72 h (mean ± SEM; n ≥ 60 endosomes per condition). **p <10−3. (D) A431 cells treated with solvent alone, 0.5 μM latrunculin A, or 20 μM nocodazole for 1 h at 37°C before fixation and labeling with anti-EEA1 (green) and DAPI (blue). (E) The fluorescence intensity in the perinuclear region of the cell was expressed as a ratio of the total cellular fluorescence using the Radial Profile plug-in in ImageJ (mean ± SEM; n ≥ 100 cells per condition). **p <10−3.
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Figure 8: Endosomes are tethered to the actin cytoskeleton by Rab14 and myosin Va. (A) Model depicting the tethering of endosomes to the actin cytoskeleton by myosin Va, which is associated with the endosomal membrane via a direct interaction with Rab14. (B) HeLa cells expressing GFP-Rab5Q79L for ∼24 h were fixed and labeled with coumarin–phalloidin and an antibody to myosin Va. Inset contains a zoomed image of the indicated enlarged endosome. (C) Quantification of the number of endogenous myosin Va puncta on GFP-Rab5AQ79L enlarged endosomes in cells transfected with the indicated siRNA for 72 h (mean ± SEM; n ≥ 60 endosomes per condition). **p <10−3. (D) A431 cells treated with solvent alone, 0.5 μM latrunculin A, or 20 μM nocodazole for 1 h at 37°C before fixation and labeling with anti-EEA1 (green) and DAPI (blue). (E) The fluorescence intensity in the perinuclear region of the cell was expressed as a ratio of the total cellular fluorescence using the Radial Profile plug-in in ImageJ (mean ± SEM; n ≥ 100 cells per condition). **p <10−3.

Mentions: The foregoing findings led us to speculate that myosin Va may tether Rab14-positive endosomes to peripheral actin patches in order to maintain their correct intracellular distribution. To investigate this hypothesis further, we examined the localization of endogenous myosin Va to endosomal structures in cells in which Rab14 had been depleted (Figure 8, B and C). Because the size of these endosomal organelles is at the resolution limit of optical microscopy, we induced their enlargement by expressing a DA mutant of Rab5 (GFP-Rab5AQ79L), which promotes endosomal fusion (Stenmark et al., 1994). Cells expressing GFP-Rab5AQ79L were labeled with an antibody that specifically recognizes endogenous myosin Va (Supplemental Figure S2, B and C) and with coumarin-labeled phalloidin. These enlarged endosomal structures possessed an average of three myosin Va puncta, which were present on the endosomal membrane, where they frequently overlapped with actin patches (Figure 8, B and C). This is reminiscent of the findings of Morel et al. (2009), which demonstrate the presence of actin patches on endosomal membranes that are important for endosome biogenesis and remodeling. These puncta are indeed myosin Va rather than nonspecific antibody labeling, as depletion of myosin Va with two independent siRNAs resulted in ∼50% reduction in the number of puncta per endosome (Figure 8C). In addition, the puncta remaining after siRNA treatment had much lower fluorescence intensity. Depletion of Rab14 also significantly reduced the average number of myosin Va puncta per endosome (Figure 8C), raising the possibility that Rab14 is involved in the targeting of myosin Va to these enlarged endosomes.


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)

Endosomes are tethered to the actin cytoskeleton by Rab14 and myosin Va. (A) Model depicting the tethering of endosomes to the actin cytoskeleton by myosin Va, which is associated with the endosomal membrane via a direct interaction with Rab14. (B) HeLa cells expressing GFP-Rab5Q79L for ∼24 h were fixed and labeled with coumarin–phalloidin and an antibody to myosin Va. Inset contains a zoomed image of the indicated enlarged endosome. (C) Quantification of the number of endogenous myosin Va puncta on GFP-Rab5AQ79L enlarged endosomes in cells transfected with the indicated siRNA for 72 h (mean ± SEM; n ≥ 60 endosomes per condition). **p <10−3. (D) A431 cells treated with solvent alone, 0.5 μM latrunculin A, or 20 μM nocodazole for 1 h at 37°C before fixation and labeling with anti-EEA1 (green) and DAPI (blue). (E) The fluorescence intensity in the perinuclear region of the cell was expressed as a ratio of the total cellular fluorescence using the Radial Profile plug-in in ImageJ (mean ± SEM; n ≥ 100 cells per condition). **p <10−3.
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Related In: Results  -  Collection

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Figure 8: Endosomes are tethered to the actin cytoskeleton by Rab14 and myosin Va. (A) Model depicting the tethering of endosomes to the actin cytoskeleton by myosin Va, which is associated with the endosomal membrane via a direct interaction with Rab14. (B) HeLa cells expressing GFP-Rab5Q79L for ∼24 h were fixed and labeled with coumarin–phalloidin and an antibody to myosin Va. Inset contains a zoomed image of the indicated enlarged endosome. (C) Quantification of the number of endogenous myosin Va puncta on GFP-Rab5AQ79L enlarged endosomes in cells transfected with the indicated siRNA for 72 h (mean ± SEM; n ≥ 60 endosomes per condition). **p <10−3. (D) A431 cells treated with solvent alone, 0.5 μM latrunculin A, or 20 μM nocodazole for 1 h at 37°C before fixation and labeling with anti-EEA1 (green) and DAPI (blue). (E) The fluorescence intensity in the perinuclear region of the cell was expressed as a ratio of the total cellular fluorescence using the Radial Profile plug-in in ImageJ (mean ± SEM; n ≥ 100 cells per condition). **p <10−3.
Mentions: The foregoing findings led us to speculate that myosin Va may tether Rab14-positive endosomes to peripheral actin patches in order to maintain their correct intracellular distribution. To investigate this hypothesis further, we examined the localization of endogenous myosin Va to endosomal structures in cells in which Rab14 had been depleted (Figure 8, B and C). Because the size of these endosomal organelles is at the resolution limit of optical microscopy, we induced their enlargement by expressing a DA mutant of Rab5 (GFP-Rab5AQ79L), which promotes endosomal fusion (Stenmark et al., 1994). Cells expressing GFP-Rab5AQ79L were labeled with an antibody that specifically recognizes endogenous myosin Va (Supplemental Figure S2, B and C) and with coumarin-labeled phalloidin. These enlarged endosomal structures possessed an average of three myosin Va puncta, which were present on the endosomal membrane, where they frequently overlapped with actin patches (Figure 8, B and C). This is reminiscent of the findings of Morel et al. (2009), which demonstrate the presence of actin patches on endosomal membranes that are important for endosome biogenesis and remodeling. These puncta are indeed myosin Va rather than nonspecific antibody labeling, as depletion of myosin Va with two independent siRNAs resulted in ∼50% reduction in the number of puncta per endosome (Figure 8C). In addition, the puncta remaining after siRNA treatment had much lower fluorescence intensity. Depletion of Rab14 also significantly reduced the average number of myosin Va puncta per endosome (Figure 8C), raising the possibility that Rab14 is involved in the targeting of myosin Va to these enlarged endosomes.

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