scholarly journals Keeping the Vimentin Network under Control: Cell–Matrix Adhesion–associated Plectin 1f Affects Cell Shape and Polarity of Fibroblasts

2010 ◽  
Vol 21 (19) ◽  
pp. 3362-3375 ◽  
Author(s):  
Gerald Burgstaller ◽  
Martin Gregor ◽  
Lilli Winter ◽  
Gerhard Wiche
Keyword(s):  
Cell Shape ◽  
Network Formation ◽  
De Novo ◽  
Control Cell ◽  
Focal Adhesions ◽  
Time Lapse ◽  
Cell Polarization ◽  
Dependent Manner ◽  
Cell Matrix ◽  
Contractile Stress

Focal adhesions (FAs) located at the ends of actin/myosin-containing contractile stress fibers form tight connections between fibroblasts and their underlying extracellular matrix. We show here that mature FAs and their derivative fibronectin fibril-aligned fibrillar adhesions (FbAs) serve as docking sites for vimentin intermediate filaments (IFs) in a plectin isoform 1f (P1f)-dependent manner. Time-lapse video microscopy revealed that FA-associated P1f captures mobile vimentin filament precursors, which then serve as seeds for de novo IF network formation via end-to-end fusion with other mobile precursors. As a consequence of IF association, the turnover of FAs is reduced. P1f-mediated IF network formation at FbAs creates a resilient cage-like core structure that encases and positions the nucleus while being stably connected to the exterior of the cell. We show that the formation of this structure affects cell shape with consequences for cell polarization.

scholarly journals Lipid rafts mediate internalization of β1-integrin in migrating intestinal epithelial cells

2008 ◽  
Vol 295 (5) ◽  
pp. G965-G976 ◽  
Author(s):  
Elena V. Vassilieva ◽  
Kirsten Gerner-Smidt ◽  
Andrei I. Ivanov ◽  
Asma Nusrat
Keyword(s):  
Epithelial Cells ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Intestinal Epithelial Cells ◽  
Focal Adhesions ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Caveolin 1 ◽  
Cell Matrix

Intestinal mucosal inflammation is associated with epithelial wounds that rapidly reseal by migration of intestinal epithelial cells (IECs). Cell migration involves cycles of cell-matrix adhesion/deadhesion that is mediated by dynamic turnover (assembly and disassembly) of integrin-based focal adhesions. Integrin endocytosis appears to be critical for deadhesion of motile cells. However, mechanisms of integrin internalization during remodeling of focal adhesions of migrating IECs are not understood. This study was designed to define the endocytic pathway that mediates internalization of β1-integrin in migrating model IECs. We observed that, in SK-CO15 and T84 colonic epithelial cells, β1-integrin is internalized in a dynamin-dependent manner. Pharmacological inhibition of clathrin-mediated endocytosis or macropinocytosis and small-interfering RNA (siRNA)-mediated knock down of clathrin did not prevent β1-integrin internalization. However, β1-integrin internalization was inhibited following cholesterol extraction and after overexpression of lipid raft protein, caveolin-1. Furthermore, internalized β1-integrin colocalized with the lipid rafts marker cholera toxin, and siRNA-mediated knockdown of caveolin-1 and flotillin-1/2 increased β1-integrin endocytosis. Our data suggest that, in migrating IEC, β1-integrin is internalized via a dynamin-dependent lipid raft-mediated pathway. Such endocytosis is likely to be important for disassembly of integrin-based cell-matrix adhesions and therefore in regulating IEC migration and wound closure.

scholarly journals Role of P120 Ras-Gap in Directed Cell Movement

2000 ◽  
Vol 149 (2) ◽  
pp. 457-470 ◽  
Author(s):  
Sarang V. Kulkarni ◽  
Gerald Gish ◽  
Peter van der Geer ◽  
Mark Henkemeyer ◽  
Tony Pawson
Keyword(s):  
Cell Polarity ◽  
Cell Movement ◽  
Focal Adhesions ◽  
Cell Polarization ◽  
Actin Stress Fiber ◽  
Dependent Manner ◽  
Directional Movement ◽  
Complete Cell ◽  
And Migration

We have used cell lines deficient in p120 Ras GTPase activating protein (Ras-GAP) to investigate the roles of Ras-GAP and the associated p190 Rho-GAP (p190) in cell polarity and cell migration. Cell wounding assays showed that Ras-GAP–deficient cells were incapable of establishing complete cell polarity and migration into the wound. Stimulation of mutant cells with growth factor rescued defects in cell spreading, Golgi apparatus fragmentation, and polarized vesicular transport and partially rescued migration in a Ras-dependent manner. However, for directional movement, the turnover of stress fibers and focal adhesions to produce an elongate morphology was dependent on the constitutive association between Ras-GAP and p190, independent of Ras regulation. Disruption of the phosphotyrosine-mediated Ras-GAP/p190 complex by microinjecting synthetic peptides derived from p190 sequences in wild-type cells caused a suppression of actin filament reorientation and migration. From these observations we suggest that although Ras-GAP is not directly required for motility per se, it is important for cell polarization by regulating actin stress fiber and focal adhesion reorientation when complexed with 190. This observation suggests a specific function for Ras-GAP separate from Ras regulation in cell motility.

scholarly journals FGFR/Heartless and Smog interact synergistically to negatively regulate Fog mediated GPCR signaling

2019 ◽  
Author(s):  
Kumari Shweta ◽  
Anagha Basargekar ◽  
Anuradha Ratnaparkhi
Keyword(s):  
G Protein ◽  
Cell Shape ◽  
Shape Change ◽  
Control Cell ◽  
Negative Regulator ◽  
Fine Tuning ◽  
Dependent Manner ◽  
Gpcr Signaling ◽  
Cell Shape Change ◽  
Embryonic Cns

AbstractG-protein coupled receptor (GPCR) signaling triggered by Folded gastrulation (Fog) is one of the pathways known to regulate glial organization and morphogenesis in the embryonic CNS in Drosophila. Fog is best known for its role in epithelial morphogenesis during gastrulation. Here, the signaling pathway includes GPCRs Mist and Smog and the G-Protein Concertina (Cta) which activate downstream effectors to bring about cytoskeletal changes essential for cell shape changeIn this study, we identify molecular players that mediate and serve as important regulators of Fog signaling in the embryonic CNS. We find that while Cta is essential for Fog signaling neither receptors, Mist nor Smog mediates signaling in the CNS. On the contrary, we find that Smog functions as a negative regulator of the pathway. Surprisingly, Heartless which encodes a fibroblast growth factor receptor, also functions as a negative regulator of Fog signaling. Further, we find that both heartless and smog interact in a synergistic manner to regulate Fog signaling.This study thus identifies novel regulators of Fog signaling that may play an important role in fine-tuning the pathway to control cell morphogenesis. It also suggests the likelihood of there being multiple receptors for Fog that mediate and regulate signaling in a context specific manner.Author SummaryIn Drosophila, Folded gastrulation (Fog) functions as ligand that signals via GPCRs to regulate cell shape during gastrulation -one of the earliest events in embryogenesis. Here, Fog signals via receptors Mist and Smog to activate the G-protein Concertina to elicit change in cell shape. In the embryonic central nervous system (CNS) this pathway regulates shape and organization of glia important for functions such as insulation of neurons and synapses.The mechanism of Fog signal transduction in the CNS and its regulation is not well understood. We have sought to address these questions in our study. We find that Concertina is an essential factor for Fog signaling in the CNS but interestingly Mist is not. In contrast, Smog functions as a negative regulator such that loss of Smog enhances Fog signaling. A similar role is played by the receptor tyrosine kinase-Heartless. Interestingly, we find that Smog and Heartless interact as part of a common genetic network to regulate Fog signaling. Our results thus provide novel insights into the regulation of Fog signaling and shed light on how signaling can be fine-tuned in a context dependent manner to control cell shape change which plays a critical role during development and organ formation.

scholarly journals Cdc42 regulates junctional actin but not cell polarization in the Caenorhabditis elegans epidermis

2017 ◽  
Vol 216 (11) ◽  
pp. 3729-3744 ◽  
Author(s):  
Yuliya Zilberman ◽  
Joshua Abrams ◽  
Dorian C. Anderson ◽  
Jeremy Nance
Keyword(s):  
Caenorhabditis Elegans ◽  
Epidermal Cell ◽  
Cell Shape ◽  
Time Lapse ◽  
Cell Polarization ◽  
Epithelial Morphogenesis ◽  
Actin Organization ◽  
Protein Levels ◽  
Guanosine Triphosphatase ◽  
Shape Changes

During morphogenesis, adherens junctions (AJs) remodel to allow changes in cell shape and position while preserving adhesion. Here, we examine the function of Rho guanosine triphosphatase CDC-42 in AJ formation and regulation during Caenorhabditis elegans embryo elongation, a process driven by asymmetric epidermal cell shape changes. cdc-42 mutant embryos arrest during elongation with epidermal ruptures. Unexpectedly, we find using time-lapse fluorescence imaging that cdc-42 is not required for epidermal cell polarization or junction assembly, but rather is needed for proper junctional actin regulation during elongation. We show that the RhoGAP PAC-1/ARHGAP21 inhibits CDC-42 activity at AJs, and loss of PAC-1 or the interacting linker protein PICC-1/CCDC85A-C blocks elongation in embryos with compromised AJ function. pac-1 embryos exhibit dynamic accumulations of junctional F-actin and an increase in AJ protein levels. Our findings identify a previously unrecognized molecular mechanism for inhibiting junctional CDC-42 to control actin organization and AJ protein levels during epithelial morphogenesis.

Nitric oxide inhibits enterocyte migration through activation of RhoA-GTPase in a SHP-2-dependent manner

2007 ◽  
Vol 292 (5) ◽  
pp. G1347-G1358 ◽  
Author(s):  
Selma Cetin ◽  
Cynthia L. Leaphart ◽  
Jun Li ◽  
Irene Ischenko ◽  
Michael Hayman ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Intestinal Inflammation ◽  
Tyrosine Phosphatase ◽  
Focal Adhesions ◽  
Mucosal Healing ◽  
Dependent Manner ◽  
Ileal Mucosa ◽  
Cell Matrix ◽  
Rhoa Gtpase

Diseases of intestinal inflammation like necrotizing enterocolitis (NEC) are associated with impaired epithelial barrier integrity and the sustained release of intestinal nitric oxide (NO). NO modifies the cytoskeletal regulator RhoA-GTPase, suggesting that NO could affect barrier healing by inhibiting intestinal restitution. We now hypothesize that NO inhibits enterocyte migration through RhoA-GTPase and sought to determine the pathways involved. The induction of NEC was associated with increased enterocyte NO release and impaired migration of bromodeoxyuridine-labeled enterocytes from terminal ileal crypts to villus tips. In IEC-6 enterocytes, NO significantly inhibited enterocyte migration and activated RhoA-GTPase while increasing the formation of stress fibers. In parallel, exposure of IEC-6 cells to NO increased the phosphorylation of focal adhesion kinase (pFAK) and caused a striking increase in cell-matrix adhesiveness, suggesting a mechanism by which NO could impair enterocyte migration. NEC was associated with increased expression of pFAK in the terminal ileal mucosa of wild-type mice and a corresponding increase in disease severity compared with inducible NO synthase knockout mice, confirming the dependence of NO for FAK phosphorylation in vivo and its role in the pathogenesis of NEC. Strikingly, inhibition of the protein tyrosine phosphatase SHP-2 in IEC-6 cells prevented the activation of RhoA by NO, restored focal adhesions, and reversed the inhibitory effects of NO on enterocyte migration. These data indicate that NO impairs mucosal healing by inhibiting enterocyte migration through activation of RhoA in a SHP-2-dependent manner and support a possible role for SHP-2 as a therapeutic target in diseases of intestinal inflammation like NEC.

scholarly journals Affixin interacts with α-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell–substrate interaction

2004 ◽  
Vol 165 (4) ◽  
pp. 539-551 ◽  
Author(s):  
Satoshi Yamaji ◽  
Atsushi Suzuki ◽  
Heiwa Kanamori ◽  
Wataru Mishima ◽  
Ryusuke Yoshimi ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Critical Role ◽  
Focal Adhesions ◽  
Time Lapse ◽  
Integrin Signaling ◽  
Critical Event ◽  
Dependent Manner ◽  
Small Interference Rna ◽  
Substrate Adhesion ◽  
Integrin Linked Kinase

The linking of integrin to cytoskeleton is a critical event for an effective cell migration. Previously, we have reported that a novel integrin-linked kinase (ILK)–binding protein, affixin, is closely involved in the linkage between integrin and cytoskeleton in combination with ILK. In the present work, we demonstrated that the second calponin homology domain of affixin directly interacts with α-actinin in an ILK kinase activity–dependent manner, suggesting that integrin–ILK signaling evoked by substrate adhesion induces affixin–α-actinin interaction. The overexpression of a peptide corresponding to the α-actinin–binding site of affixin as well as the knockdown of endogenous affixin by small interference RNA resulted in the blockade of cell spreading. Time-lapse observation revealed that in both experiments cells were round with small peripheral blebs and failed to develop lamellipodia, suggesting that the ILK–affixin complex serves as an integrin-anchoring site for α-actinin and thereby mediates integrin signaling to α-actinin, which has been shown to play a critical role in actin polymerization at focal adhesions.

scholarly journals Hemidesmosome-Related Keratin Filament Bundling and Nucleation

2021 ◽  
Vol 22 (4) ◽  
pp. 2130
Author(s):  
Marcin Moch ◽  
Rudolf E. Leube
Keyword(s):  
Actin Filaments ◽  
Network Formation ◽  
Focal Adhesions ◽  
Time Lapse ◽  
Tubulin Polymerization ◽  
Latrunculin B ◽  
Keratin Filaments ◽  
Keratinocyte Cell Line Hacat ◽  
Filament Bundles ◽  
Keratin Filament

The epithelial cytoskeleton encompasses actin filaments, microtubules, and keratin intermediate filaments. They are interconnected and attached to the extracellular matrix via focal adhesions and hemidesmosomes. To study their interplay, we inhibited actin and tubulin polymerization in the human keratinocyte cell line HaCaT by latrunculin B and nocodazole, respectively. Using immunocytochemistry and time-lapse imaging of living cells, we found that inhibition of actin and tubulin polymerization alone or in combination induced keratin network re-organization albeit differently in each situation. Keratin filament network retraction towards the nucleus and formation of bundled and radial keratin filaments was most pronounced in latrunculin-B treated cells but less in doubly-treated cells and not detectable in the presence of nocodazole alone. Hemidesmosomal keratin filament anchorage was maintained in each instance, whereas focal adhesions were disassembled in the absence of actin filaments. Simultaneous inhibition of actin and tubulin polymerization, therefore, allowed us to dissect hemidesmosome-specific functions for keratin network properties. These included not only anchorage of keratin filament bundles but also nucleation of keratin filaments, which was also observed in migrating cells. The findings highlight the fundamental role of hemidesmosomal adhesion for keratin network formation and organization independent of other cytoskeletal filaments pointing to a unique mechanobiological function.

Squalamine, a novel cationic steroid, specifically inhibits the brush-border Na+/H+exchanger isoform NHE3

1999 ◽  
Vol 276 (1) ◽  
pp. C136-C144 ◽  
Author(s):  
S. Akhter ◽  
S. K. Nath ◽  
C. M. Tse ◽  
J. Williams ◽  
M. Zasloff ◽  
...  
Keyword(s):  
Brush Border ◽  
Intracellular Signaling ◽  
Cell Shape ◽  
Membrane Vesicle ◽  
Control Cell ◽  
Specific Inhibitor ◽  
Maximum Effect ◽  
Maximal Velocity ◽  
Dependent Manner ◽  
Delayed Effect

Squalamine, an endogenous molecule found in the liver and other tissues of Squalus acanthias, has antibiotic properties and causes changes in endothelial cell shape. The latter suggested that its potential targets might include transport proteins that control cell volume or cell shape. The effect of purified squalamine was examined on cloned Na+/H+exchanger isoforms NHE1, NHE2, and NHE3 stably transfected in PS120 fibroblasts. Squalamine (1-h pretreatment) decreased the maximal velocity of rabbit NHE3 in a concentration-dependent manner (13, 47, and 57% inhibition with 3, 5, and 7 μg/ml, respectively) and also increased K′[H+]i. Squalamine did not affect rabbit NHE1 or NHE2 function. The inhibitory effect of squalamine was 1) time dependent, with no effect of immediate addition and maximum effect with 1 h of exposure, and 2) fully reversible. Squalamine pretreatment of the ileum for 60 min inhibited brush-border membrane vesicle Na+/H+activity by 51%. Further investigation into the mechanism of squalamine’s effects showed that squalamine required the COOH-terminal 76 amino acids of NHE3. Squalamine had no cytotoxic effect at the concentrations studied, as indicated by monitoring lactate dehydrogenase release. These results indicate that squalamine 1) is a specific inhibitor of the brush-border NHE isoform NHE3 and not NHE1 or NHE2, 2) acts in a nontoxic and fully reversible manner, and 3) has a delayed effect, indicating that it may influence brush-border Na+/H+exchanger function indirectly, through an intracellular signaling pathway or by acting as an intracellular modulator.

Regulation of Adhesion Strength by Focal Adhesion Position and Cell Shape

2012 ◽  
Author(s):  
Nathan D. Gallant ◽  
Kranthi Kumar Elineni
Keyword(s):  
Cell Adhesion ◽  
Adhesion Strength ◽  
Cell Shape ◽  
Microcontact Printing ◽  
Control Cell ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Spreading Area ◽  
Hydrodynamic Shear ◽  
Cell Spreading Area

Cell adhesion to extracellular matrices is critical to numerous cellular functions and is primarily mediated by integrin receptors. Binding and aggregation of integrins leads to the formation of focal adhesions (FA) which connect the cytoskeleton to the extracellular matrix in order to reinforce adhesion and transmit signals [1]. Preliminary observations indicated preferential recruitment of FAs to the periphery of the cell spreading area on both uniformly coated and micropatterned fibronectin surfaces (Fig. 1). The current study investigates the biophysical regulation of cell adhesion strength based on the size and position of FA with the central hypothesis that peripheral FAs stabilize adhesion strength. The hypothesis was tested by delineating the cell spreading area from the total cell adhesive area by employing microcontact printing to pattern substrates with a series of circular and annular adhesive islands which control cell shape (Fig. 2). A well characterized hydrodynamic shear assay known as the spinning disk device was used to quantify the adhesion strength of cells adhered to the micropatterns [2].

scholarly journals YAP and TAZ limit cytoskeletal and focal adhesion maturation to enable persistent cell motility

2019 ◽  
Vol 218 (4) ◽  
pp. 1369-1389 ◽  
Author(s):  
Devon E. Mason ◽  
Joseph M. Collins ◽  
James H. Dawahare ◽  
Trung Dung Nguyen ◽  
Yang Lin ◽  
...  
Keyword(s):  
Cell Motility ◽  
Focal Adhesion ◽  
De Novo ◽  
Focal Adhesions ◽  
Cell Polarization ◽  
Cytoskeletal Remodeling ◽  
De Novo Gene ◽  
Cytoskeletal Tension ◽  
Persistent Cell ◽  
Transcriptional Feedback

Cell migration initiates by traction generation through reciprocal actomyosin tension and focal adhesion reinforcement, but continued motility requires adaptive cytoskeletal remodeling and adhesion release. Here, we asked whether de novo gene expression contributes to this cytoskeletal feedback. We found that global inhibition of transcription or translation does not impair initial cell polarization or migration initiation, but causes eventual migratory arrest through excessive cytoskeletal tension and over-maturation of focal adhesions, tethering cells to their matrix. The transcriptional coactivators YAP and TAZ mediate this feedback response, modulating cell mechanics by limiting cytoskeletal and focal adhesion maturation to enable persistent cell motility and 3D vasculogenesis. Motile arrest after YAP/TAZ ablation was partially rescued by depletion of the YAP/TAZ-dependent myosin phosphatase regulator, NUAK2, or by inhibition of Rho-ROCK-myosin II. Together, these data establish a transcriptional feedback axis necessary to maintain a responsive cytoskeletal equilibrium and persistent migration.

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