title: Rab5 isoforms orchestrate a "division of labor" in the endocytic network; Rab5C modulates Rac-mediated cell motility.
abstract: Rab5, the prototypical Rab GTPase and master regulator of the endocytic pathway, is encoded as three differentially expressed isoforms, Rab5A, Rab5B and Rab5C. Here, we examined the differential effects of Rab5 isoform silencing on cell motility and report that Rab5C, but neither Rab5A nor Rab5B, is selectively associated with the growth factor-activation of Rac1 and with enhanced cell motility. Initial observations revealed that silencing of Rab5C expression, but neither Rab5A nor Rab5C, led to spindle-shaped cells that displayed reduced formation of membrane ruffles. When subjected to a scratch wound assay, cells depleted of Rab5C, but not Rab5A or Rab5B, demonstrated reduced cell migration. U937 cells depleted of Rab5C also displayed reduced cell motility in a Transwell plate migration assay. To examine activation of Rac, HeLa cells stably expressing GFP-Rac1 were independently depleted of Rab5A, Rab5B or Rab5C and seeded onto coverslips imprinted with a crossbow pattern. 3-D GFP-Rac1 images of micro-patterned cells show that GFP-Rac1 was less localized to the cell periphery in the absence of Rab5C. To confirm the connection between Rab5C and Rac activation, HeLa cells depleted of Rab5 isoforms were starved and then stimulated with EGF. Rac1 pull-down assays revealed that EGF-stimulated Rac1 activity was significantly suppressed in Rab5C-suppressed cells. To determine whether events upstream of Rac activation were affected by Rab5C, we observed that EGF-stimulated Akt phosphorylation was suppressed in cells depleted of Rab5C. Finally, since spatio-temporal assembly/disassembly of adhesion complexes are essential components of cell migration, we examined the effect of Rab5 isoform depletion on the formation of focal adhesion complexes. Rab5C-depleted HeLa cells have significantly fewer focal adhesion foci, in accordance with the lack of persistent lamellipodial protrusions and reduced directional migration. We conclude that Rab5 isoforms selectively oversee the multiple signaling and trafficking events associated with the endocytic network.
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, United States of America.
authors.forename: Pin-I
authors.initials: PI
authors.lastname: Chen
authors.affiliation: Molecular Mechanisms of Intracellular Transport, Institut Curie, Paris, France.
authors.forename: Kristine
authors.initials: K
authors.lastname: Schauer
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, United States of America.
authors.forename: Chen
authors.initials: C
authors.lastname: Kong
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, United States of America.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Harding
authors.affiliation: Molecular Mechanisms of Intracellular Transport, Institut Curie, Paris, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Goud
authors.affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, United States of America.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Stahl
mesh_terms: D002448:Cell Adhesion; D002465:Cell Movement; D004705:Endocytosis / Q000235:genetics*; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D020696:rab5 GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D020033:Protein Isoforms; C107953:RAC1 protein, human; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; C427755:RAB5C protein, human; D020696:rab5 GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein