Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Oct 1;135(1):153–167. doi: 10.1083/jcb.135.1.153

Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts

PMCID: PMC2121014  PMID: 8858170

Abstract

Rab8 is a small Ras-like GTPase that regulates polarized membrane transport to the basolateral membrane in epithelial cells and to the dendrites in neurons. It has recently been demonstrated that fibroblasts sort newly synthesized proteins into two different pathways for delivery to the cell surface that are equivalent to the apical and the basolateral post-Golgi routes in epithelial cells (Yoshimori, T., P. Keller, M.G. Roth, and K. Simons. 1996. J. Cell Biol. 133:247-256). To determine the role of Rab8 in fibroblasts, we used both transient expression systems and stable cell lines expressing mutant or wild-type (wt) Rab8. A dramatic change in cell morphology occurred in BHK cells expressing both the wt Rab8 and the activated form of the GTPase, the Rab8Q67L mutant. These cells formed processes as a result of a reorganization of both their actin filaments and microtubules. Newly synthesized vesicular stomatitis virus G glycoprotein, a basolateral marker protein in MDCK cells, was preferentially delivered into these cell outgrowths. Based on these findings, we propose that Rab8 provides a link between the machinery responsible for the formation of cell protrusions and polarized biosynthetic membrane traffic.

Full Text

The Full Text of this article is available as a PDF (4.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bender A., Pringle J. R. Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9976–9980. doi: 10.1073/pnas.86.24.9976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bergmann J. E., Kupfer A., Singer S. J. Membrane insertion at the leading edge of motile fibroblasts. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1367–1371. doi: 10.1073/pnas.80.5.1367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bershadsky A. D., Futerman A. H. Disruption of the Golgi apparatus by brefeldin A blocks cell polarization and inhibits directed cell migration. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5686–5689. doi: 10.1073/pnas.91.12.5686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  5. Bretscher A., Drees B., Harsay E., Schott D., Wang T. What are the basic functions of microfilaments? Insights from studies in budding yeast. J Cell Biol. 1994 Aug;126(4):821–825. doi: 10.1083/jcb.126.4.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brändli A. W., Parton R. G., Simons K. Transcytosis in MDCK cells: identification of glycoproteins transported bidirectionally between both plasma membrane domains. J Cell Biol. 1990 Dec;111(6 Pt 2):2909–2921. doi: 10.1083/jcb.111.6.2909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bucci C., Parton R. G., Mather I. H., Stunnenberg H., Simons K., Hoflack B., Zerial M. The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell. 1992 Sep 4;70(5):715–728. doi: 10.1016/0092-8674(92)90306-w. [DOI] [PubMed] [Google Scholar]
  8. Burstein E. S., Brondyk W. H., Macara I. G. Amino acid residues in the Ras-like GTPase Rab3A that specify sensitivity to factors that regulate the GTP/GDP cycling of Rab3A. J Biol Chem. 1992 Nov 15;267(32):22715–22718. [PubMed] [Google Scholar]
  9. Chant J., Herskowitz I. Genetic control of bud site selection in yeast by a set of gene products that constitute a morphogenetic pathway. Cell. 1991 Jun 28;65(7):1203–1212. doi: 10.1016/0092-8674(91)90015-q. [DOI] [PubMed] [Google Scholar]
  10. Chavrier P., Vingron M., Sander C., Simons K., Zerial M. Molecular cloning of YPT1/SEC4-related cDNAs from an epithelial cell line. Mol Cell Biol. 1990 Dec;10(12):6578–6585. doi: 10.1128/mcb.10.12.6578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chen Y. T., Holcomb C., Moore H. P. Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6508–6512. doi: 10.1073/pnas.90.14.6508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cid-Arregui A., Parton R. G., Simons K., Dotti C. G. Nocodazole-dependent transport, and brefeldin A--sensitive processing and sorting, of newly synthesized membrane proteins in cultured neurons. J Neurosci. 1995 Jun;15(6):4259–4269. doi: 10.1523/JNEUROSCI.15-06-04259.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Craighead M. W., Bowden S., Watson R., Armstrong J. Function of the ypt2 gene in the exocytic pathway of Schizosaccharomyces pombe. Mol Biol Cell. 1993 Oct;4(10):1069–1076. doi: 10.1091/mbc.4.10.1069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Der C. J., Finkel T., Cooper G. M. Biological and biochemical properties of human rasH genes mutated at codon 61. Cell. 1986 Jan 17;44(1):167–176. doi: 10.1016/0092-8674(86)90495-2. [DOI] [PubMed] [Google Scholar]
  15. Deretic D., Huber L. A., Ransom N., Mancini M., Simons K., Papermaster D. S. rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis. J Cell Sci. 1995 Jan;108(Pt 1):215–224. doi: 10.1242/jcs.108.1.215. [DOI] [PubMed] [Google Scholar]
  16. Dotti C. G., Simons K. Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture. Cell. 1990 Jul 13;62(1):63–72. doi: 10.1016/0092-8674(90)90240-f. [DOI] [PubMed] [Google Scholar]
  17. Feig L. A., Cooper G. M. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol. 1988 Aug;8(8):3235–3243. doi: 10.1128/mcb.8.8.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ferro-Novick S., Novick P. The role of GTP-binding proteins in transport along the exocytic pathway. Annu Rev Cell Biol. 1993;9:575–599. doi: 10.1146/annurev.cb.09.110193.003043. [DOI] [PubMed] [Google Scholar]
  19. Fiedler K., Lafont F., Parton R. G., Simons K. Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane. J Cell Biol. 1995 Mar;128(6):1043–1053. doi: 10.1083/jcb.128.6.1043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Glotzer M., Hyman A. A. Cell polarity. The importance of being polar. Curr Biol. 1995 Oct 1;5(10):1102–1105. doi: 10.1016/s0960-9822(95)00221-1. [DOI] [PubMed] [Google Scholar]
  22. Gottlieb T. A., Ivanov I. E., Adesnik M., Sabatini D. D. Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells. J Cell Biol. 1993 Feb;120(3):695–710. doi: 10.1083/jcb.120.3.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Govindan B., Bowser R., Novick P. The role of Myo2, a yeast class V myosin, in vesicular transport. J Cell Biol. 1995 Mar;128(6):1055–1068. doi: 10.1083/jcb.128.6.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gundersen G. G., Bulinski J. C. Selective stabilization of microtubules oriented toward the direction of cell migration. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5946–5950. doi: 10.1073/pnas.85.16.5946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hasson T., Mooseker M. S. Molecular motors, membrane movements and physiology: emerging roles for myosins. Curr Opin Cell Biol. 1995 Aug;7(4):587–594. doi: 10.1016/0955-0674(95)80017-4. [DOI] [PubMed] [Google Scholar]
  26. Haubruck H., Engelke U., Mertins P., Gallwitz D. Structural and functional analysis of ypt2, an essential ras-related gene in the fission yeast Schizosaccharomyces pombe encoding a Sec4 protein homologue. EMBO J. 1990 Jun;9(6):1957–1962. doi: 10.1002/j.1460-2075.1990.tb08323.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hiti A. L., Davis A. R., Nayak D. P. Complete sequence analysis shows that the hemagglutinins of the H0 and H2 subtypes of human influenza virus are closely related. Virology. 1981 May;111(1):113–124. doi: 10.1016/0042-6822(81)90658-9. [DOI] [PubMed] [Google Scholar]
  28. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  29. Huber L. A., Dupree P., Dotti C. G. A deficiency of the small GTPase rab8 inhibits membrane traffic in developing neurons. Mol Cell Biol. 1995 Feb;15(2):918–924. doi: 10.1128/mcb.15.2.918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Huber L. A., Pimplikar S., Parton R. G., Virta H., Zerial M., Simons K. Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane. J Cell Biol. 1993 Oct;123(1):35–45. doi: 10.1083/jcb.123.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Huber L. A., de Hoop M. J., Dupree P., Zerial M., Simons K., Dotti C. Protein transport to the dendritic plasma membrane of cultured neurons is regulated by rab8p. J Cell Biol. 1993 Oct;123(1):47–55. doi: 10.1083/jcb.123.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ikonen E., Tagaya M., Ullrich O., Montecucco C., Simons K. Different requirements for NSF, SNAP, and Rab proteins in apical and basolateral transport in MDCK cells. Cell. 1995 May 19;81(4):571–580. doi: 10.1016/0092-8674(95)90078-0. [DOI] [PubMed] [Google Scholar]
  33. Johnson D. I., Pringle J. R. Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity. J Cell Biol. 1990 Jul;111(1):143–152. doi: 10.1083/jcb.111.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kelly R. B. Microtubules, membrane traffic, and cell organization. Cell. 1990 Apr 6;61(1):5–7. doi: 10.1016/0092-8674(90)90206-t. [DOI] [PubMed] [Google Scholar]
  35. Kreis T. E. Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface. EMBO J. 1986 May;5(5):931–941. doi: 10.1002/j.1460-2075.1986.tb04306.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lafont F., Burkhardt J. K., Simons K. Involvement of microtubule motors in basolateral and apical transport in kidney cells. Nature. 1994 Dec 22;372(6508):801–803. doi: 10.1038/372801a0. [DOI] [PubMed] [Google Scholar]
  37. Langford G. M. Actin- and microtubule-dependent organelle motors: interrelationships between the two motility systems. Curr Opin Cell Biol. 1995 Feb;7(1):82–88. doi: 10.1016/0955-0674(95)80048-4. [DOI] [PubMed] [Google Scholar]
  38. Lauffenburger D. A., Horwitz A. F. Cell migration: a physically integrated molecular process. Cell. 1996 Feb 9;84(3):359–369. doi: 10.1016/s0092-8674(00)81280-5. [DOI] [PubMed] [Google Scholar]
  39. Lian J. P., Stone S., Jiang Y., Lyons P., Ferro-Novick S. Ypt1p implicated in v-SNARE activation. Nature. 1994 Dec 15;372(6507):698–701. doi: 10.1038/372698a0. [DOI] [PubMed] [Google Scholar]
  40. Liljeström P., Garoff H. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (N Y) 1991 Dec;9(12):1356–1361. doi: 10.1038/nbt1291-1356. [DOI] [PubMed] [Google Scholar]
  41. Luo L., Liao Y. J., Jan L. Y., Jan Y. N. Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes Dev. 1994 Aug 1;8(15):1787–1802. doi: 10.1101/gad.8.15.1787. [DOI] [PubMed] [Google Scholar]
  42. Martenson C., Stone K., Reedy M., Sheetz M. Fast axonal transport is required for growth cone advance. Nature. 1993 Nov 4;366(6450):66–69. doi: 10.1038/366066a0. [DOI] [PubMed] [Google Scholar]
  43. Mitchison T. J., Cramer L. P. Actin-based cell motility and cell locomotion. Cell. 1996 Feb 9;84(3):371–379. doi: 10.1016/s0092-8674(00)81281-7. [DOI] [PubMed] [Google Scholar]
  44. Muallem S., Kwiatkowska K., Xu X., Yin H. L. Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells. J Cell Biol. 1995 Feb;128(4):589–598. doi: 10.1083/jcb.128.4.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Nelson W. J. Regulation of cell surface polarity from bacteria to mammals. Science. 1992 Nov 6;258(5084):948–955. doi: 10.1126/science.1439806. [DOI] [PubMed] [Google Scholar]
  46. Nobes C. D., Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell. 1995 Apr 7;81(1):53–62. doi: 10.1016/0092-8674(95)90370-4. [DOI] [PubMed] [Google Scholar]
  47. Novick P., Brennwald P. Friends and family: the role of the Rab GTPases in vesicular traffic. Cell. 1993 Nov 19;75(4):597–601. doi: 10.1016/0092-8674(93)90478-9. [DOI] [PubMed] [Google Scholar]
  48. Rindler M. J., Ivanov I. E., Sabatini D. D. Microtubule-acting drugs lead to the nonpolarized delivery of the influenza hemagglutinin to the cell surface of polarized Madin-Darby canine kidney cells. J Cell Biol. 1987 Feb;104(2):231–241. doi: 10.1083/jcb.104.2.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rodriguez-Boulan E., Powell S. K. Polarity of epithelial and neuronal cells. Annu Rev Cell Biol. 1992;8:395–427. doi: 10.1146/annurev.cb.08.110192.002143. [DOI] [PubMed] [Google Scholar]
  50. Rogalski A. A., Bergmann J. E., Singer S. J. Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane. J Cell Biol. 1984 Sep;99(3):1101–1109. doi: 10.1083/jcb.99.3.1101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Rose J. K., Bergmann J. E. Altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein. Cell. 1983 Sep;34(2):513–524. doi: 10.1016/0092-8674(83)90384-7. [DOI] [PubMed] [Google Scholar]
  52. Rothman J. E. Mechanisms of intracellular protein transport. Nature. 1994 Nov 3;372(6501):55–63. doi: 10.1038/372055a0. [DOI] [PubMed] [Google Scholar]
  53. Sauvé D. M., Ho D. T., Roberge M. Concentration of dilute protein for gel electrophoresis. Anal Biochem. 1995 Apr 10;226(2):382–383. doi: 10.1006/abio.1995.1242. [DOI] [PubMed] [Google Scholar]
  54. Simons K., Dupree P., Fiedler K., Huber L. A., Kobayashi T., Kurzchalia T., Olkkonen V., Pimplikar S., Parton R., Dotti C. Biogenesis of cell-surface polarity in epithelial cells and neurons. Cold Spring Harb Symp Quant Biol. 1992;57:611–619. doi: 10.1101/sqb.1992.057.01.067. [DOI] [PubMed] [Google Scholar]
  55. Simons K., Wandinger-Ness A. Polarized sorting in epithelia. Cell. 1990 Jul 27;62(2):207–210. doi: 10.1016/0092-8674(90)90357-k. [DOI] [PubMed] [Google Scholar]
  56. Singer S. J., Kupfer A. The directed migration of eukaryotic cells. Annu Rev Cell Biol. 1986;2:337–365. doi: 10.1146/annurev.cb.02.110186.002005. [DOI] [PubMed] [Google Scholar]
  57. Stenmark H., Parton R. G., Steele-Mortimer O., Lütcke A., Gruenberg J., Zerial M. Inhibition of rab5 GTPase activity stimulates membrane fusion in endocytosis. EMBO J. 1994 Mar 15;13(6):1287–1296. doi: 10.1002/j.1460-2075.1994.tb06381.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Stenmark H., Vitale G., Ullrich O., Zerial M. Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion. Cell. 1995 Nov 3;83(3):423–432. doi: 10.1016/0092-8674(95)90120-5. [DOI] [PubMed] [Google Scholar]
  59. Stowers L., Yelon D., Berg L. J., Chant J. Regulation of the polarization of T cells toward antigen-presenting cells by Ras-related GTPase CDC42. Proc Natl Acad Sci U S A. 1995 May 23;92(11):5027–5031. doi: 10.1073/pnas.92.11.5027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Söllner T., Whiteheart S. W., Brunner M., Erdjument-Bromage H., Geromanos S., Tempst P., Rothman J. E. SNAP receptors implicated in vesicle targeting and fusion. Nature. 1993 Mar 25;362(6418):318–324. doi: 10.1038/362318a0. [DOI] [PubMed] [Google Scholar]
  61. Søgaard M., Tani K., Ye R. R., Geromanos S., Tempst P., Kirchhausen T., Rothman J. E., Söllner T. A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles. Cell. 1994 Sep 23;78(6):937–948. doi: 10.1016/0092-8674(94)90270-4. [DOI] [PubMed] [Google Scholar]
  62. TerBush D. R., Novick P. Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae. J Cell Biol. 1995 Jul;130(2):299–312. doi: 10.1083/jcb.130.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Ting A. E., Hazuka C. D., Hsu S. C., Kirk M. D., Bean A. J., Scheller R. H. rSec6 and rSec8, mammalian homologs of yeast proteins essential for secretion. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9613–9617. doi: 10.1073/pnas.92.21.9613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Vitale M. L., Seward E. P., Trifaró J. M. Chromaffin cell cortical actin network dynamics control the size of the release-ready vesicle pool and the initial rate of exocytosis. Neuron. 1995 Feb;14(2):353–363. doi: 10.1016/0896-6273(95)90291-0. [DOI] [PubMed] [Google Scholar]
  65. Wandinger-Ness A., Bennett M. K., Antony C., Simons K. Distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells. J Cell Biol. 1990 Sep;111(3):987–1000. doi: 10.1083/jcb.111.3.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Yoshimori T., Keller P., Roth M. G., Simons K. Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells. J Cell Biol. 1996 Apr;133(2):247–256. doi: 10.1083/jcb.133.2.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Zerial M., Stenmark H. Rab GTPases in vesicular transport. Curr Opin Cell Biol. 1993 Aug;5(4):613–620. doi: 10.1016/0955-0674(93)90130-i. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES