scholarly journals Coupling of Fibrin Reorganization and Fibronectin Patterning by Corneal Fibroblasts in Response to PDGF BB and TGFβ1

2020 ◽  
Vol 7 (3) ◽  
pp. 89
Author(s):  
Miguel Miron-Mendoza ◽  
Dalia Vazquez ◽  
Nerea García-Rámila ◽  
Hikaru R. Ikebe ◽  
W. Matthew Petroll
Keyword(s):  
Leading Edge ◽  
Cell Spreading ◽  
Interconnected Network ◽  
Corneal Injury ◽  
Corneal Fibroblasts ◽  
Track Formation ◽  
Tractional Forces ◽  
Highly Correlated ◽  
And Migration ◽  
Matrix Contraction

We previously reported that corneal fibroblasts within 3D fibrin matrices secrete, bind, and organize fibronectin into tracks that facilitate cell spreading and migration. Other cells use these fibronectin tracks as conduits, which leads to the development of an interconnected cell/fibronectin network. In this study, we investigate how cell-induced reorganization of fibrin correlates with fibronectin track formation in response to two growth factors present during wound healing: PDGF BB, which stimulates cell spreading and migration; and TGFβ1, which stimulates cellular contraction and myofibroblast transformation. Both PDGF BB and TGFβ1 stimulated global fibrin matrix contraction (p < 0.005); however, the cell and matrix patterning were different. We found that, during PDGF BB-induced cell spreading, fibronectin was organized simultaneously with the generation of tractional forces at the leading edge of pseudopodia. Over time this led to the formation of an interconnected network consisting of cells, fibronectin and compacted fibrin tracks. Following culture in TGFβ1, cells were less motile, produced significant local fibrin reorganization, and formed fewer cellular connections as compared to PDGF BB (p < 0.005). Although bands of compacted fibrin tracks developed in between neighboring cells, fibronectin labeling was not generally present along these tracks, and the correlation between fibrin and fibronectin labeling was significantly less than that observed in PDGF BB (p < 0.001). Taken together, our results show that cell-induced extracellular matrix (ECM) reorganization can occur independently from fibronectin patterning. Nonetheless, both events seem to be coordinated, as corneal fibroblasts in PDGF BB secrete and organize fibronectin as they preferentially spread along compacted fibrin tracks between cells, producing an interconnected network in which cells, fibronectin and compacted fibrin tracks are highly correlated. This mechanism of patterning could contribute to the formation of organized cellular networks that have been observed following corneal injury and refractive surgery.

scholarly journals Coupling of Fibrin Reorganization and Fibronectin Patterning by Corneal Fibroblasts in Response to PDGF BB and TGFβ1

2020 ◽  
Author(s):  
Miguel Miron-Mendoza ◽  
Dalia Vazquez ◽  
Nerea García-Rámila ◽  
W. Matthew Petroll ◽  
Hikaru R. Ikebe
Keyword(s):  
Leading Edge ◽  
Cell Spreading ◽  
Interconnected Network ◽  
Corneal Injury ◽  
Corneal Fibroblasts ◽  
Track Formation ◽  
Tractional Forces ◽  
Highly Correlated ◽  
And Migration ◽  
Matrix Contraction

We previously reported that corneal fibroblasts within 3D fibrin matrices secrete, bind, and organize fibronectin into tracks that facilitate cell spreading and migration. Other cells use these fibronectin tracks as conduits, which leads to the development of an interconnected cell/fibronectin network. In this study, we investigate how cell induced reorganization of fibrin correlates with fibronectin track formation in response to two growth factors present during wound healing: PDGF BB, which stimulates cell spreading and migration; and TGF&beta;1, which stimulates cellular contraction and myofibroblast transformation. Both PDGF BB and TGFb1 stimulated global fibrin matrix contraction (P &lt; 0.005), however cell and matrix patterning were different. We found that during PDGF BB induced cell spreading, fibronectin was organized simultaneously with the generation of tractional forces at the leading edge of pseudopodia. Over time this led to the formation of an interconnected network consisting of cells, fibronectin and compacted fibrin tracks. Following culture in TGF&beta;1, cells were less motile, produced significant local fibrin reorganization, and formed fewer cellular connections as compared to PDGF BB (P &lt; 0.005). Although bands of compacted fibrin tracks developed in between neighboring cells, fibronectin labeling was not generally present along these tracks, and the correlation between fibrin and fibronectin labeling was significantly less than that observed in PDGF BB (P &lt; 0.001). Taken together, our results show that cell-induced ECM reorganization can occur independently from fibronectin patterning. Nonetheless, both events seem to be coordinated, as corneal fibroblasts in PDGF BB secrete and organize fibronectin as they preferentially spread along compacted fibrin tracks between cells, producing an interconnected network in which cells, fibronectin and compacted fibrin tracks are highly correlated. This mechanism of patterning could contribute to the formation of organized cellular networks that have been observed following corneal injury and refractive surgery.

mRNA encoding WAVE–Arp2/3-associated proteins is co-localized with foci of active protein synthesis at the leading edge of MRC5 fibroblasts during cell migration

2013 ◽  
Vol 452 (1) ◽  
pp. 45-55 ◽  
Author(s):  
Mark Willett ◽  
Michele Brocard ◽  
Hilary J. Pollard ◽  
Simon J. Morley
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Structural Characteristics ◽  
Leading Edge ◽  
Cell Spreading ◽  
Active Protein ◽  
Associated Proteins ◽  
Steady State Levels ◽  
And Migration ◽  
Syndrome Protein

During cell spreading, mammalian cells migrate using lamellipodia formed from a large dense branched actin network which produces the protrusive force required for leading edge advancement. The formation of lamellipodia is a dynamic process and is dependent on a variety of protein cofactors that mediate their local regulation, structural characteristics and dynamics. In the present study, we show that mRNAs encoding some structural and regulatory components of the WAVE [WASP (Wiskott–Aldrich syndrome protein) verprolin homologous] complex are localized to the leading edge of the cell and associated with sites of active translation. Furthermore, we demonstrate that steady-state levels of ArpC2 and Rac1 proteins increase at the leading edge during cell spreading, suggesting that localized protein synthesis has a pivotal role in controlling cell spreading and migration.

scholarly journals The endocytic protein GRAF1 is directed to cell-matrix adhesion sites and regulates cell spreading

2011 ◽  
Vol 22 (22) ◽  
pp. 4380-4389 ◽  
Author(s):  
Gary J. Doherty ◽  
Monika K. Åhlund ◽  
Mark T. Howes ◽  
Björn Morén ◽  
Robert G. Parton ◽  
...  
Keyword(s):  
Rho Gtpase ◽  
Morphological Changes ◽  
Leading Edge ◽  
Cell Spreading ◽  
Endocytic Pathway ◽  
Membrane Microdomains ◽  
Membrane Turnover ◽  
Cell Matrix ◽  
Adhesion Sites ◽  
And Migration

The rho GTPase-activating protein GTPase regulator associated with focal adhesion kinase-1 (GRAF1) remodels membranes into tubulovesicular clathrin-independent carriers (CLICs) mediating lipid-anchored receptor endocytosis. However, the cell biological functions of this highly prevalent endocytic pathway are unclear. In this article, we present biochemical and cell biological evidence that GRAF1 interacted with a network of endocytic and adhesion proteins and was found enriched at podosome-like adhesions and src-induced podosomes. We further demonstrate that these sites comprise microdomains of highly ordered lipid enriched in GRAF1 endocytic cargo. GRAF1 activity was upregulated in spreading cells and uptake via CLICs was concentrated at the leading edge of migrating cells. Depletion of GRAF1, which inhibits CLIC generation, resulted in profound defects in cell spreading and migration. We propose that GRAF1 remodels membrane microdomains at adhesion sites into endocytic carriers, facilitating membrane turnover during cell morphological changes.

scholarly journals Tiam1 interaction with the PAR complex promotes talin-mediated Rac1 activation during polarized cell migration

2012 ◽  
Vol 199 (2) ◽  
pp. 331-345 ◽  
Author(s):  
Shujie Wang ◽  
Takashi Watanabe ◽  
Kenji Matsuzawa ◽  
Akira Katsumi ◽  
Mai Kakeno ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Cell Migration ◽  
Protein Kinase ◽  
Leading Edge ◽  
Cell Spreading ◽  
Kinase C ◽  
Rac1 Activity ◽  
Directional Movement ◽  
And Migration ◽  
Migrating Cells

Migrating cells acquire front-rear polarity with a leading edge and a trailing tail for directional movement. The Rac exchange factor Tiam1 participates in polarized cell migration with the PAR complex of PAR3, PAR6, and atypical protein kinase C. However, it remains largely unknown how Tiam1 is regulated and contributes to the establishment of polarity in migrating cells. We show here that Tiam1 interacts directly with talin, which binds and activates integrins to mediate their signaling. Tiam1 accumulated at adhesions in a manner dependent on talin and the PAR complex. The interactions of talin with Tiam1 and the PAR complex were required for adhesion-induced Rac1 activation, cell spreading, and migration toward integrin substrates. Furthermore, Tiam1 acted with talin to regulate adhesion turnover. Thus, we propose that Tiam1, with the PAR complex, binds to integrins through talin and, together with the PAR complex, thereby regulates Rac1 activity and adhesion turnover for polarized migration.

Nanopatterned Adhesive, Stretchable Hydrogel to Control Ligand Spacing and Regulate Cell Spreading and Migration

ACS Nano ◽  
2017 ◽  
Vol 11 (8) ◽  
pp. 8282-8291 ◽  
Author(s):  
Jie Deng ◽  
Changsheng Zhao ◽  
Joachim P. Spatz ◽  
Qiang Wei
Keyword(s):  
Cell Spreading ◽  
And Migration

Phospholipase C-γ1 potentiates integrin-dependent cell spreading and migration through Pyk2/paxillin activation

2007 ◽  
Vol 19 (8) ◽  
pp. 1784-1796 ◽  
Author(s):  
J CHOI ◽  
Y YANG ◽  
S LEE ◽  
I KIM ◽  
S HA ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Cell Spreading ◽  
Dependent Cell ◽  
And Migration

Distinct signals via Rho GTPases and Src drive shape changes by thrombin and sphingosine-1-phosphate in endothelial cells

2002 ◽  
Vol 115 (12) ◽  
pp. 2475-2484 ◽  
Author(s):  
Valérie Vouret-Craviari ◽  
Christine Bourcier ◽  
Etienne Boulter ◽  
Ellen Van Obberghen-Schilling
Keyword(s):  
Endothelial Cells ◽  
Rho Gtpases ◽  
Actin Polymerization ◽  
Morphological Changes ◽  
Umbilical Vein ◽  
Cell Spreading ◽  
Actin Binding ◽  
Sphingosine 1 Phosphate ◽  
And Migration ◽  
Umbilical Vein Endothelial Cells

Soluble mediators such as thrombin and sphingosine-1-phosphate regulate morphological changes in endothelial cells that affect vascular permeability and new blood vessel formation. Although these ligands activate a similar set of heterotrimeric G proteins, thrombin causes cell contraction and rounding whereas sphingosine-1-phosphate induces cell spreading and migration. A functional requirement for Rho family GTPases in the cytoskeletal responses to both ligands has been established, yet the dynamics of their regulation and additional signaling mechanisms that lead to such opposite effects remain poorly understood. Using a pull-down assay to monitor the activity of Rho GTPases in human umbilical vein endothelial cells, we find significant temporal and quantitative differences in RhoA and Rac1 activation. High levels of active RhoA rapidly accumulate in cells in response to thrombin whereas Rac1 is inhibited. In contrast, sphingosine-1-phosphate addition leads to comparatively weak and delayed activation of RhoA and it activates Rac1. In addition, we show here that sphingosine-1-phosphate treatment activates a Src family kinase and triggers recruitment of the F-actin-binding protein cortactin to sites of actin polymerization at the rim of membrane ruffles. Both Src and Rac pathways are essential for lamellipodia targeting of cortactin. Further, Src plays a determinant role in sphingosine-1-phosphate-induced cell spreading and migration. Taken together these data demonstrate that the thrombin-induced contractile and immobile phenotype in endothelial cells reflects both robust RhoA activation and Rac inhibition, whereas Src- and Rac-dependent events couple sphingosine-1-phosphate receptors to the actin polymerizing machinery that drives the extension of lamellipodia and cell migration.

scholarly journals CaM kinase IIδ2-dependent regulation of vascular smooth muscle cell polarization and migration

2008 ◽  
Vol 294 (6) ◽  
pp. C1465-C1475 ◽  
Author(s):  
Melissa Z. Mercure ◽  
Roman Ginnan ◽  
Harold A. Singer
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Vascular Smooth Muscle ◽  
Cell Monolayer ◽  
Leading Edge ◽  
Cell Polarization ◽  
Loss Of Function ◽  
Downstream Signaling ◽  
Transient Activation ◽  
And Migration

Previous studies indicate involvement of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) in vascular smooth muscle (VSM) cell migration. In the present study, molecular loss-of-function studies were used specifically to assess the role of the predominant CaMKIIδ2 isoform on VSM cell migration using a scratch wound healing assay. Targeted CaMKIIδ2 knockdown using siRNA or inhibition of activity by overexpressing a kinase-negative mutant resulted in attenuation of VSM cell migration. Temporal and spatial assessments of kinase autophosphorylation indicated rapid and transient activation in response to wounding, in addition to a sustained activation in the leading edge of migrating and spreading cells. Furthermore, siRNA-mediated suppression of CaMKIIδ2 resulted in the inhibition of wound-induced Rac activation and Golgi reorganization, and disruption of leading edge morphology, indicating an important function for CaMKIIδ2 in regulating VSM cell polarization. Numerous previous reports link activation of CaMKII to ERK1/2 signaling in VSM. Wound-induced ERK1/2 activation was also found to be dependent on CaMKII; however, ERK activity did not account for effects of CaMKII in regulating Golgi polarization, indicating alternative mechanisms by which CaMKII affects the complex events involved in cell migration. Wounding a VSM cell monolayer results in CaMKIIδ2 activation, which positively regulates VSM cell polarization and downstream signaling, including Rac and ERK1/2 activation, leading to cell migration.

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