RhoA and Rac1 are both required for efficient wound closure of airway epithelial cells

191

176

Programs that direct cellular differentiation are dependent on the strict temporal expression of regulatory factors that can be provided by Rho GTPases. Ciliogenesis is a complex sequence of events involving the generation and docking of basal bodies at the apical membrane, followed by ciliary axoneme generation. Although a cilia proteome has been assembled, programs that direct ciliated cell differentiation are not well established, particularly in mammalian systems. Using mouse primary culture airway epithelial cells, we identified a critical stage of ciliogenesis requiring the temporal establishment of an apical web-like structure of actin for basal body docking and subsequent axoneme growth. Apical web formation and basal body docking were prevented by interruption of actin remodeling and were dependent on RhoA activation. Additional evidence for this program was provided by analysis of Foxj1-null mice that failed to dock basal bodies and lacked apical actin. Foxj1 expression coincided with actin web formation, activated RhoA and RhoB, and persisted despite RhoA inhibition, suggesting that Foxj1 promoted RhoA during ciliogenesis. Apical ezrin localization was also dependent on Foxj1, actin remodeling, and RhoA, but was not critical for ciliogenesis. Thus, temporal Foxj1 and RhoA activity are essential regulatory events for cytoskeletal remodeling during mammalian ciliogenesis.

Section: Journal Of Cell Science 120 (11)supporting

confidence: 93%

RhoA-mediated apical actin enrichment is required for ciliogenesis and promoted by Foxj1

Pan

You

Huang

et al. 2007

191

176

“…Cells that are protruding at any given time display less marginal F-actin staining, and formation and contraction of marginal actomyosin bundles and stress fibers in MDCK cells may be antagonistic to membrane protrusion and spreading (Figs 5,6). It has also been shown that inhibition of the Rho pathway results in greater protrusive activity at the margin of wounded rat liver epithelial monolayers (Omelchenko et al, 2003) and slightly accelerated wound closure in bronchial epithelial cells (Desai et al, 2004).…”

Section: Wound Closure In Cultured Epithelial Cells Provides a Tractablementioning

confidence: 99%

Multiple rows of cells behind an epithelial wound edge extend cryptic lamellipodia to collectively drive cell-sheet movement

Farooqui

Fenteany

2005

391

373

The mechanism by which epithelial, endothelial and other strongly cell-cell adhesive cells migrate collectively as continuous sheets is not clear, even though this process is crucial for embryonic development and tissue repair in virtually all multicellular animals. Wound closure in Madin-Darby canine kidney (MDCK) epithelial cell monolayers involves Rac GTPase-dependent migration of cells both at and behind the wound edge. We report here for the first time that cells behind the margin of wounded MDCK cell monolayers, even hundreds of microns from the edge, extend `cryptic' lamellipodia against the substratum beneath cells in front of them, toward the wound, as determined by confocal, two-photon and transmission electron microscopy. These so-called submarginal cells nevertheless strictly maintain their more apical cell-cell contacts when they migrate as part of a coherent cell sheet, hiding their basal protrusions from conventional microscopy. The submarginal protrusions display the hallmarks of traditional lamellipodia based on morphology and dynamics. Cells behind the margin therefore actively crawl, instead of just moving passively when cells at the margin pull on them. The rate of migration is inversely proportional to the distance from the margin, and cells move co-ordinately, yet still in part autonomously, toward the wound area. We also clarify the ancillary role played by nonprotrusive contractile actin bundles that assemble in a Rho GTPase-dependent manner at the margin after wounding. In addition, some cell proliferation occurs at a delay after wounding but does not contribute to closure. Instead, it apparently serves to replace damaged cells so that intact spread cells can revert to their normal cuboidal morphology and the original cell density of the unbroken sheet can be restored.

“…Adenoviruses expressing DN-RhoA (T19N-RhoA) and DN-Rac1 (T17N-Rac1) were a gift from Christopher M. Waters (University of Tennessee, Memphis, TN) (Desai et al, 2004).…”

Section: Plasmids and Adenovirusesmentioning

confidence: 99%

Actin coating and compression of fused secretory vesicles are essential for surfactant secretion: a role for Rho, formins and myosin II

Miklavc¹,

Hecht²,

Hobi³

et al. 2012

130

SummarySecretion of vesicular contents by exocytosis is a fundamental cellular process. Increasing evidence suggests that post-fusion events play an important role in determining the composition and quantity of the secretory output. In particular, regulation of fusion pore dilation and closure is considered a key regulator of the post-fusion phase. However, depending on the nature of the cargo, additional mechanisms might be essential to facilitate effective release. We have recently described that in alveolar type II (ATII) cells, lamellar bodies (LBs), which are secretory vesicles that store lung surfactant, are coated with actin following fusion with the plasma membrane. Surfactant, a lipoprotein complex, does not readily diffuse out of fused LBs following opening and dilation of the fusion pore. Using fluorescence microscopy, atomic force microscopy and biochemical assays, we present evidence that actin coating and subsequent contraction of the actin coat is essential to facilitate surfactant secretion. Latrunculin B prevents actin coating of fused LBs and inhibits surfactant secretion almost completely. Simultaneous imaging of the vesicle membrane and the actin coat revealed that contraction of the actin coat compresses the vesicle following fusion. This leads to active extrusion of vesicle contents. Initial actin coating of fused vesicles is dependent on activation of Rho and formin-dependent actin nucleation. Actin coat contraction is facilitated by myosin II. In summary, our data suggest that fusion pore opening and dilation itself is not sufficient for release of bulky vesicle cargos and that active extrusion mechanisms are required.