Microtubule involvement in NIH 3T3 Golgi and MTOC polarity establishment.
Scratch-wound assays are commonly used to study the ability of cells to polarize and migrate. In a previous study we showed that Golgi reorientation in response to a scratch wound is actin-dependent in NIH 3T3 cells but not in astrocytes. In this investigation, to study cell polarity and motility further, we used the polarization of the Golgi and microtubule organizing center (MTOC), as well as the ability of NIH 3T3 cells to migrate, in a scratch-wound assay. Unlike Golgi polarization, MTOC polarization was not dependent on actin, the Arp2/3 complex or Wiskott-Aldrich syndrome protein (WASP)-family proteins. By contrast, disruption of microtubules inhibited MTOC polarity, but not Golgi polarity. Migration was found to be dependent both on actin and microtubules. Expression of the formin-homology 2 (FH2) region of mDia1 inhibited Golgi polarization and migration but not MTOC polarization. Similarly, ST638, a Src inhibitor, inhibited Golgi polarization and migration but not MTOC polarization, whereas expression of the actin regulator IRSp53 only inhibited cell migration. Interestingly, the inhibition of cell migration by the mDia1 FH2 domain could be overcome by addition of Y27632, an inhibitor of ROCK (Rho-associated kinase). In fact, in the presence of ROCK inhibitor, cell migration was accelerated but polarization of both the Golgi and MTOC were inhibited. These data show that, in NIH 3T3 cells, different aspects of cell polarization and migration occur by different mechanisms, and both actin and microtubule networks are required. In addition, this study indicates that MTOC and Golgi polarization events are separately controlled.
Magdalena
Juana
J
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Millard
Thomas H
TH
Machesky
Laura M
LM
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology*; D016764:Cell Polarity / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005057:Eukaryotic Cells / Q000378:metabolism* / Q000648:ultrastructure; D000080865:Formins; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D022101:Microtubule-Organizing Center / Q000378:metabolism* / Q000648:ultrastructure; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D009419:Nerve Tissue Proteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D014945:Wound Healing / Q000502:physiology; D054460:rho-Associated Kinases
D000199:Actins; C119539:BAIAP2 protein, human; D002352:Carrier Proteins; C491464:Diap1 protein, mouse; D004791:Enzyme Inhibitors; D000080865:Formins; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases