scholarly journals Lulu2 regulates the circumferential actomyosin tensile system in epithelial cells through p114RhoGEF

2011 ◽  
Vol 195 (2) ◽  
pp. 245-261 ◽  
Author(s):  
Hiroyuki Nakajima ◽  
Takuji Tanoue
Keyword(s):  
Epithelial Cells ◽  
Cell Shape ◽  
Molecular Mechanisms ◽  
Molecular System ◽  
Apical Cell ◽  
Cell Junctions ◽  
Cellular Processes ◽  
Kinase C ◽  
Important Regulator ◽  
Cell Cell

Myosin II–driven mechanical forces control epithelial cell shape and morphogenesis. In particular, the circumferential actomyosin belt, which is located along apical cell–cell junctions, regulates many cellular processes. Despite its importance, the molecular mechanisms regulating the belt are not fully understood. In this paper, we characterize Lulu2, a FERM (4.1 protein, ezrin, radixin, moesin) domain–containing molecule homologous to Drosophila melanogaster Yurt, as an important regulator. In epithelial cells, Lulu2 is localized along apical cell–cell boundaries, and Lulu2 depletion by ribonucleic acid interference results in disorganization of the circumferential actomyosin belt. In its regulation of the belt, Lulu2 interacts with and activates p114RhoGEF, a Rho-specific guanine nucleotide exchanging factor (GEF), at apical cell–cell junctions. This interaction is negatively regulated via phosphorylation events in the FERM-adjacent domain of Lulu2 catalyzed by atypical protein kinase C. We further found that Patj, an apical cell polarity regulator, recruits p114RhoGEF to apical cell–cell boundaries via PDZ (PSD-95/Dlg/ZO-1) domain–mediated interaction. These findings therefore reveal a novel molecular system regulating the circumferential actomyosin belt in epithelial cells.

scholarly journals Comprehensive analysis of formin localization in Xenopus epithelial cells

2019 ◽  
Vol 30 (1) ◽  
pp. 82-95 ◽  
Author(s):  
Tomohito Higashi ◽  
Rachel E. Stephenson ◽  
Ann L. Miller
Keyword(s):  
Epithelial Cells ◽  
Comprehensive Analysis ◽  
Cell Junctions ◽  
Actin Dynamics ◽  
Cell Junction ◽  
Contractile Ring ◽  
Dimerization Domain ◽  
Cellular Processes ◽  
And Function ◽  
Cell Cell

Reorganization of the actin cytoskeleton is crucial for cellular processes, including cytokinesis and cell–cell junction remodeling. Formins are conserved processive actin-polymerizing machines that regulate actin dynamics by nucleating, elongating, and bundling linear actin filaments. Because the formin family is large, with at least 15 members in vertebrates, there have not been any comprehensive studies examining formin localization and function within a common cell type. Here, we characterized the localization of all 15 formins in epithelial cells of Xenopus laevis gastrula-stage embryos. Dia1 and Dia2 localized to tight junctions, while Fhod1 and Fhod3 localized to adherens junctions. Only Dia3 strongly localized at the cytokinetic contractile ring. The Diaphanous inhibitory domain–dimerization domain (DID-DD) region of Dia1 was sufficient for Dia1 localization, and overexpression of a Dia1 DID-DD fragment competitively removed Dia1 and Dia2 from cell–cell junctions. In Dia1 DID-DD–overexpressing cells, Dia1 and Dia2 were mislocalized to the contractile ring, and cells exhibited increased cytokinesis failure. This work provides a comprehensive analysis of the localization of all 15 vertebrate formins in epithelial cells and suggests that misregulated formin localization results in epithelial cytokinesis failure.

scholarly journals A role for a micron-scale supramolecular myosin array in adherens junction cytoskeletal assembly

2021 ◽  
Author(s):  
Hui-Chia Yu-Kemp ◽  
Rachel A. Szymanski ◽  
Nicole C. Gadda ◽  
Madeline L. Lillich ◽  
Mark Peifer
Keyword(s):  
Epithelial Cells ◽  
Cell Shape ◽  
Shape Change ◽  
Cell Junctions ◽  
Actin Assembly ◽  
Cell Shape Change ◽  
Micrometer Scale ◽  
Assembly Pathways ◽  
E Cadherin ◽  
Cell Cell

AbstractEpithelial cells assemble specialized actomyosin structures at E-Cadherin-based cell-cell junctions, and the force exerted drives cell shape change during morphogenesis. The mechanisms used to build this supramolecular actomyosin structure remain unclear. We used ZO-knockdown MDCK cells, which assemble a robust, polarized and highly organized actomyosin cytoskeleton at the zonula adherens, and combined genetic and pharmacological approaches with super-resolution microscopy to define molecular machines required. To our surprise, inhibiting individual actin assembly pathways (Arp2/3, formins or Ena/VASP) did not prevent or delay assembly of this polarized actomyosin structure. Instead, as junctions matured, micrometer-scale supramolecular myosin arrays assembled, with aligned stacks of myosin filaments adjacent to the apical membrane, while associated actin filaments remained disorganized. This suggested these myosin arrays might bundle actin at mature junctions. Consistent with this, inhibiting ROCK or myosin ATPase disrupted myosin localization/organization, and prevented actin bundling and polarization. These results suggest a novel mechanism by which myosin self-assembly helps drive actin organization to facilitate cell shape change.SummaryWe explored mechanisms epithelial cells use to assemble supramolecular actomyosin structures at E-Cadherin-based cell-cell junctions. Our data suggest individual actin assembly pathways are not essential. Instead, microscopy and pharmacological inhibition suggest micrometer-scale supramolecular myosin arrays help bundle actin at mature junctions.

scholarly journals Heat Shock Protein 27 Associates with Basolateral Cell Boundaries in Heat-Shocked and ATP-Depleted Epithelial Cells

2002 ◽  
Vol 13 (2) ◽  
pp. 332-341
Author(s):  
Eric A. Shelden ◽  
Michael J. Borrelli ◽  
Fiona M. Pollock ◽  
Rita Bonham
Keyword(s):  
Heat Stress ◽  
Heat Shock ◽  
Epithelial Cells ◽  
Apical Cell ◽  
Atp Depletion ◽  
Cell Junctions ◽  
Cell Contact ◽  
Double Labeling ◽  
Cell Substrate ◽  
Cell Cell

ABSTRACT. Heat stress alters epithelial barrier function, and heat stress preconditioning protects epithelial function from injury. Hsp27 is a small stress protein that has previously been shown to modulate actin assembly. Thus, by regulating actin filaments associated with cell junctions, hsp27 could alter epithelial function. To begin to address this hypothesis, the regulation and distribution of a human hsp27-green fluorescence fusion protein (EGFPhHsp27) that is expressed in cultured renal epithelial cells was assessed. EGFPhHsp27, like the endogenous hsp27, associated with the cytoskeleton in heat-stressed and chemically ATP-depleted cells, and both proteins were regulated similarly. Confocal microscopy of intact and detergent-lysed cells revealed novel distribution patterns in which EGFPhHsp27 associated with basolateral, but not apical, cell borders in injured cells. Double labeling studies revealed EGFPhHsp27 and actin filament colocalization in ATP-depleted cells. However, during heat shock, granules of EGFPhHsp27 were found at sites of cell-cell contact and in the cell body, but colocalization with actin was not apparent. Thus, heat stress and ATP depletion induce distinct patterns of hsp27 redistribution in epithelial cells, and sites of cell-cell and cell-substrate attachment are unique in their ability to recruit hsp27 during injury. The association of EGFPhHsp27 with basolateral cell boundaries supports a potential role for hsp27 in protection or regulation of epithelial cell-cell and cell-substrate attachments.

scholarly journals Cdc42 Regulates Apical Junction Formation in Human Bronchial Epithelial Cells through PAK4 and Par6B

2010 ◽  
Vol 21 (17) ◽  
pp. 2996-3006 ◽  
Author(s):  
Sean W. Wallace ◽  
Joanne Durgan ◽  
Dan Jin ◽  
Alan Hall
Keyword(s):  
Epithelial Cells ◽  
Apical Cell ◽  
Lung Epithelial Cells ◽  
Cell Junctions ◽  
Epithelial Cell Line ◽  
Dependent Manner ◽  
Bronchial Epithelial ◽  
Junction Formation ◽  
Apical Junctions ◽  
Cell Cell

Cdc42 has been implicated in numerous biochemical pathways during epithelial morphogenesis, including the control of spindle orientation during mitosis, the establishment of apical-basal polarity, the formation of apical cell–cell junctions, and polarized secretion. To investigate the signaling pathways through which Cdc42 mediates these diverse effects, we have screened an siRNA library corresponding to the 36 known Cdc42 target proteins, in a human bronchial epithelial cell line. Two targets, PAK4 and Par6B, were identified as necessary for the formation of apical junctions. PAK4 is recruited to nascent cell–cell contacts in a Cdc42-dependent manner, where it is required for the maturation of primordial junctions into apical junctions. PAK4 kinase activity is essential for junction maturation, but overexpression of an activated PAK4 mutant disrupts this process. Par6B, together with its binding partner aPKC, is necessary both for junction maturation and for the retention of PAK4 at sites of cell–cell contact. This study demonstrates that controlled regulation of PAK4 is required for apical junction formation in lung epithelial cells and highlights potential cross-talk between two Cdc42 targets, PAK4 and Par6B.

scholarly journals Atypical protein kinase C induces cell transformation by disrupting Hippo/Yap signaling

2015 ◽  
Vol 26 (20) ◽  
pp. 3578-3595 ◽  
Author(s):  
Andrew Archibald ◽  
Maia Al-Masri ◽  
Alyson Liew-Spilger ◽  
Luke McCaffrey
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
3D Culture ◽  
Cell Junctions ◽  
Nuclear Accumulation ◽  
Cell Phenotype ◽  
Atypical Protein Kinase C ◽  
Atypical Protein Kinase ◽  
Kinase C

Epithelial cells are major sites of malignant transformation. Atypical protein kinase C (aPKC) isoforms are overexpressed and activated in many cancer types. Using normal, highly polarized epithelial cells (MDCK and NMuMG), we report that aPKC gain of function overcomes contact inhibited growth and is sufficient for a transformed epithelial phenotype. In 2D cultures, aPKC induced cells to grow as stratified epithelia, whereas cells grew as solid spheres of nonpolarized cells in 3D culture. aPKC associated with Mst1/2, which uncoupled Mst1/2 from Lats1/2 and promoted nuclear accumulation of Yap1. Of importance, Yap1 was necessary for aPKC-mediated overgrowth but did not restore cell polarity defects, indicating that the two are separable events. In MDCK cells, Yap1 was sequestered to cell–cell junctions by Amot, and aPKC overexpression resulted in loss of Amot expression and a spindle-like cell phenotype. Reexpression of Amot was sufficient to restore an epithelial cobblestone appearance, Yap1 localization, and growth control. In contrast, the effect of aPKC on Hippo/Yap signaling and overgrowth in NMuMG cells was independent of Amot. Finally, increased expression of aPKC in human cancers strongly correlated with increased nuclear accumulation of Yap1, indicating that the effect of aPKC on transformed growth by deregulating Hippo/Yap1 signaling may be clinically relevant.

scholarly journals Regulation of myosin activation during cell–cell contact formation by Par3-Lgl antagonism: entosis without matrix detachment

2012 ◽  
Vol 23 (11) ◽  
pp. 2076-2091 ◽  
Author(s):  
Qingwen Wan ◽  
Jing Liu ◽  
Zhen Zheng ◽  
Huabin Zhu ◽  
Xiaogang Chu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Molecular Mechanisms ◽  
Myosin Ii ◽  
Coiled Coil ◽  
Host Cells ◽  
Cell Contact ◽  
Contact Formation ◽  
Cell Internalization ◽  
Mammary Gland Epithelial Cells ◽  
Cell Cell

Cell–cell contact formation following cadherin engagement requires actomyosin contraction along the periphery of cell–cell contact. The molecular mechanisms that regulate myosin activation during this process are not clear. In this paper, we show that two polarity proteins, partitioning defective 3 homologue (Par3) and mammalian homologues of Drosophila Lethal (2) Giant Larvae (Lgl1/2), antagonize each other in modulating myosin II activation during cell–cell contact formation in Madin-Darby canine kidney cells. While overexpression of Lgl1/2 or depletion of endogenous Par3 leads to enhanced myosin II activation, knockdown of Lgl1/2 does the opposite. Intriguingly, altering the counteraction between Par3 and Lgl1/2 induces cell–cell internalization during early cell–cell contact formation, which involves active invasion of the lateral cell–cell contact underneath the apical-junctional complexes and requires activation of the Rho–Rho-associated, coiled-coil containing protein kinase (ROCK)–myosin pathway. This is followed by predominantly nonapoptotic cell-in-cell death of the internalized cells and frequent aneuploidy of the host cells. Such effects are reminiscent of entosis, a recently described process observed when mammary gland epithelial cells were cultured in suspension. We propose that entosis could occur without matrix detachment and that overactivation of myosin or unbalanced myosin activation between contacting cells may be the driving force for entosis in epithelial cells.

scholarly journals Direct laser manipulation reveals the mechanics of cell contacts in vivo

2015 ◽  
Vol 112 (5) ◽  
pp. 1416-1421 ◽  
Author(s):  
Kapil Bambardekar ◽  
Raphaël Clément ◽  
Olivier Blanc ◽  
Claire Chardès ◽  
Pierre-François Lenne
Keyword(s):  
Cell Shape ◽  
Constitutive Law ◽  
Cell Junctions ◽  
Scale Model ◽  
Tissue Morphogenesis ◽  
Cell Contacts ◽  
Cell Shape Changes ◽  
Shape Changes ◽  
Cell Cell

Cell-generated forces produce a variety of tissue movements and tissue shape changes. The cytoskeletal elements that underlie these dynamics act at cell–cell and cell–ECM contacts to apply local forces on adhesive structures. In epithelia, force imbalance at cell contacts induces cell shape changes, such as apical constriction or polarized junction remodeling, driving tissue morphogenesis. The dynamics of these processes are well-characterized; however, the mechanical basis of cell shape changes is largely unknown because of a lack of mechanical measurements in vivo. We have developed an approach combining optical tweezers with light-sheet microscopy to probe the mechanical properties of epithelial cell junctions in the early Drosophila embryo. We show that optical trapping can efficiently deform cell–cell interfaces and measure tension at cell junctions, which is on the order of 100 pN. We show that tension at cell junctions equilibrates over a few seconds, a short timescale compared with the contractile events that drive morphogenetic movements. We also show that tension increases along cell interfaces during early tissue morphogenesis and becomes anisotropic as cells intercalate during germ-band extension. By performing pull-and-release experiments, we identify time-dependent properties of junctional mechanics consistent with a simple viscoelastic model. Integrating this constitutive law into a tissue-scale model, we predict quantitatively how local deformations propagate throughout the tissue.

Assembly of adherens junctions is required for sphingosine 1-phosphate-induced matriptase accumulation and activation at mammary epithelial cell-cell contacts

2004 ◽  
Vol 286 (5) ◽  
pp. C1159-C1169 ◽  
Author(s):  
Ruei-Jiun Hung ◽  
Ia-Wen J. Hsu ◽  
Jennifer L. Dreiling ◽  
Mon-Juan Lee ◽  
Cicely A. Williams ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Adherens Junctions ◽  
Adherens Junction ◽  
Mammary Epithelial ◽  
Cell Junctions ◽  
Cell Contacts ◽  
Cytoskeletal Rearrangement ◽  
Sphingosine 1 Phosphate ◽  
Cell Cell

Sphingosine 1-phosphate (S1P), a bioactive phospholipid, simultaneously induces actin cytoskeletal rearrangements and activation of matriptase, a membrane-associated serine protease in human mammary epithelial cells. In this study, we used a monoclonal antibody selective for activated, two-chain matriptase to examine the functional relationship between these two S1P-induced events. Ten minutes after exposure of 184 A1N4 mammary epithelial cells to S1P, matriptase was observed to accumulate at cell-cell contacts. Activated matriptase first began to appear as small spots at cell-cell contacts, and then its deposits elongated along cell-cell contacts. Concomitantly, S1P induced assembly of adherens junctions and subcortical actin belts. Matriptase localization was observed to be coincident with markers of adherens junctions at cell-cell contacts but likely not to be incorporated into the tightly bound adhesion plaque. Disruption of subcortical actin belt formation and prevention of adherens junction assembly led to prevention of accumulation and activation of the protease at cell-cell contacts. These data suggest that S1P-induced accumulation and activation of matriptase depend on the S1P-induced adherens junction assembly. Although MAb M32, directed against one of the low-density lipoprotein receptor class A domains of matriptase, blocked S1P-induced activation of the enzyme, the antibody had no effect on S1P-induced actin cytoskeletal rearrangement. Together, these data indicate that actin cytoskeletal rearrangement is necessary but not sufficient for S1P-induced activation of matriptase at cell-cell contacts. The coupling of matriptase activation to adherens junction assembly and actin cytoskeletal rearrangement may serve to ensure tight control of matriptase activity, restricted to cell-cell junctions of mammary epithelial cells.

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