Microtubule involvement in NIH 3T3 Golgi and MTOC polarity establishment

Abstract

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 WiskottAldrich 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.

A Novel Mechanism by Which Tissue Transglutaminase Activates Signaling Events That Promote Cell Survival

“…To determine the intrinsic capability of tTG to mediate actions relevant to cancer progression, we examined the effects of overexpressing this protein in a non-transformed cell type. The NIH3T3 mouse fibroblast cell line was chosen as our model system for this study, as it is commonly used to read-out various types of cellular outcomes (23)(24)(25). Fig.…”

Nanopattern-induced changes in morphology and motility of smooth muscle cells

“…Motility of the SMC on the patterned versus unpatterned surfaces was examined in terms of the polarization and reorientation of the MTOC after wounding [31]. When a confluent monolayer SMC on patterned PDMS and glass cover slip was observed, a wound was made parallel to the cell alignment by scraping the cell monolayer across the sample with the back of a surgical blade.…”

“…Previous studies show that microtubules (MT) are essential for the polarization of many cell types [31,33], and cell migration involves the MT and actin cytoskeletons. The MTOC would re-orientate towards the direction of migration.…”

“…The morphology of cells grown on such gratings was characterized with respect to their elongation and alignment. In addition to proliferation, the cell motility was studied using the in vitro wound healing model, where the polarization of the microtubule organizing centers (MTOC) of the SMC during the healing was examined [31].…”

Prenylated C17orf37 Induces Filopodia Formation to Promote Cell Migration and Metastasis

“…S4) in scratch wound assays. Scratch assays are commonly used to study the ability of cells to polarize and migrate into the wound with time (35,36). Cells expressing the C17orf37…”