scholarly journals Integrin-mediated Signals Regulated by Members of the Rho Family of GTPases

1998 ◽  
Vol 142 (2) ◽  
pp. 573-586 ◽  
Author(s):  
Edwin A. Clark ◽  
Warren G. King ◽  
Joan S. Brugge ◽  
Marc Symons ◽  
Richard O. Hynes
Keyword(s):  
Signaling Pathways ◽  
Matrix Protein ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Extracellular Matrix Protein ◽  
Cell Periphery ◽  
Dependent Manner ◽  
Membrane Ruffling ◽  
Initial Adhesion ◽  
Rho Family

The organization of the actin cytoskeleton can be regulated by soluble factors that trigger signal transduction events involving the Rho family of GTPases. Since adhesive interactions are also capable of organizing the actin-based cytoskeleton, we examined the role of Cdc42-, Rac-, and Rho-dependent signaling pathways in regulating the cytoskeleton during integrin-mediated adhesion and cell spreading using dominant-inhibitory mutants of these GTPases. When Rat1 cells initially adhere to the extracellular matrix protein fibronectin, punctate focal complexes form at the cell periphery. Concomitant with focal complex formation, we observed some phosphorylation of the focal adhesion kinase (FAK) and Src, which occurred independently of Rho family GTPases. However, subsequent phosphorylation of FAK and paxillin occurs in a Rho-dependent manner. Moreover, we found Rho dependence of the assembly of large focal adhesions from which actin stress fibers radiate. Initial adhesion to fibronectin also stimulates membrane ruffling; we show that this ruffling is independent of Rho but is dependent on both Cdc42 and Rac. Furthermore, we observed that Cdc42 controls the integrin-dependent activation of extracellular signal–regulated kinase 2 and of Akt, a kinase whose activity has been demonstrated to be dependent on phosphatidylinositol (PI) 3-kinase. Since Rac-dependent membrane ruffling can be stimulated by PI 3-kinase, it appears that Cdc42, PI 3-kinase, and Rac lie on a distinct pathway that regulates adhesion-induced membrane ruffling. In contrast to the differential regulation of integrin-mediated signaling by Cdc42, Rac, and Rho, we observed that all three GTPases regulate cell spreading, an event that may indirectly control cellular architecture. Therefore, several separable signaling pathways regulated by different members of the Rho family of GTPases converge to control adhesion-dependent changes in the organization of the cytoskeleton, changes that regulate cell morphology and behavior.

scholarly journals Signaling Mechanisms in the Regulation of Renal Matrix Metabolism in Diabetes

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Meenalakshmi M. Mariappan
Keyword(s):  
Extracellular Matrix ◽  
Signaling Pathways ◽  
Matrix Protein ◽  
Structural Changes ◽  
Mammalian Target Of Rapamycin ◽  
Adverse Outcomes ◽  
Matrix Proteins ◽  
Extracellular Matrix Protein ◽  
Renal Hypertrophy ◽  
Matrix Metabolism

Renal hypertrophy and accumulation of extracellular matrix proteins are among cardinal manifestations of diabetic nephropathy. TGF beta system has been implicated in the pathogenesis of these manifestations. Among signaling pathways activated in the kidney in diabetes, mTOR- (mammalian target of rapamycin-)regulated pathways are pivotal in orchestrating high glucose-induced production of ECM proteins leading to functional and structural changes in the kidney culminating in adverse outcomes. Understanding signaling pathways that influence individual matrix protein expression could lead to the development of new interventional strategies. This paper will highlight some of the diverse components of the signaling network stimulated by hyperglycemia with an emphasis on extracellular matrix protein metabolism in the kidney in diabetes.

Domains of tenascin involved in glioma migration

1998 ◽  
Vol 111 (8) ◽  
pp. 1095-1104
Author(s):  
G.R. Phillips ◽  
L.A. Krushel ◽  
K.L. Crossin
Keyword(s):  
Cell Migration ◽  
Matrix Protein ◽  
Glioma Cells ◽  
C6 Glioma ◽  
Extracellular Matrix Protein ◽  
C6 Glioma Cells ◽  
Dependent Manner ◽  
Type Iii ◽  
Fibronectin Type Iii ◽  
Fibronectin Type

Tenascin (TN) is an extracellular matrix protein found in areas of cell migration during development and expressed at high levels in migratory tumor cells. TN was previously shown to support the attachment and migration of glioma cells in culture. To determine the domains responsible for glioma migration and attachment, we produced recombinant fusion proteins that collectively span the majority of the molecule including its epidermal growth factor-like repeats, fibronectin type III repeats and fibrinogen domain. These domains were tested for their ability to support migration of C6 glioma cells in an aggregate migration assay. A recombinant fusion protein including fibronectin type III (FNIII) repeats 2–6 (TNfn2-6) was the only fragment found to promote migration of C6 glioma cells at levels similar to that promoted by intact TN. Evaluation of smaller segments and individual FNIII repeats revealed that TNfn3 promoted migration and attachment of glioma cells and TNfn6 promoted migration but not attachment. While TNfn3 and TNfn6 promoted migration individually, the presence of both TNfn3 and TNfn6 was required for migration on segments of the FNIII region that included TNfn5. TNfn5 inhibited migration in a dose dependent manner when mixed with TNfn3 and also promoted strong attachment and spreading of C6 glioma cells. TNfn3 and TNfn6 promote cell migration and may function cooperatively to overcome the inhibitory activity of TNfn5. Additional cell attachment studies suggested that both beta1 integrins and heparin may differentially influence the attachment of glioma cells to TN fragments. Together, these findings show that C6 glioma cells integrate their response upon binding to at least three domains within TN.

scholarly journals Characterization of the Roles of Vimentin in Regulating the Proliferation and Migration of HSCs during Hepatic Fibrogenesis

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1184 ◽  
Author(s):  
Pei-Wen Wang ◽  
Tung-Ho Wu ◽  
Tung-Yi Lin ◽  
Mu-Hong Chen ◽  
Chau-Ting Yeh ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Messenger Rna ◽  
Rho Gtpase ◽  
Focal Adhesions ◽  
Cytoskeletal Proteins ◽  
Nuclear Antigen ◽  
Dependent Manner ◽  
Hepatic Fibrogenesis ◽  
And Migration ◽  
Proliferation And Migration

The activation of hepatic stellate cells (HSCs) manifested as proliferation and migration is the pivotal event involved in liver fibrogenesis. The vimentin network, an intermediate filament (IF) system, is one of the critical cascades by which the cell morphology, growth, and motility are modulated. However, the vimentin-mediated cytoskeletal cross talk, as well as the signaling transduction, which further coordinates the cellular responses during hepatic fibrogenesis, is poorly understood. In the current study, both messenger RNA (mRNA) and the vimentin protein were significantly increased in a time-dependent manner in the dimethylnitrosamine (DMN)-exposed liver. In particular, vimentin was highly expressed in the activated HSCs. Again, the overexpressed vimentin was observed in the plasma samples derived from patients with hepatic fibrosis/cirrhosis, suggesting that vimentin may be a key factor in regulating the progression of liver fibrosis. Meanwhile, vimentin knockdown suppressed the migratory propensity, provoked morphological changes, and disturbed the focal adhesions in the HSCs due to the breakdown of associated cytoskeletal proteins. Western blotting showed that vimentin deletion inhibited proliferating cell nuclear antigen (PCNA) and arrested the Rho GTPase family, thereby impairing the HSCs’ growth as well as motility. The phosphorylated extracellular-signal regulated kinase (ERK) and AKT signals were also notably reduced in response to the silence of vimentin. Inhibitors of selected signaling pathways suppressed the migration and differentiation of activated HSCs by regulating specific serine phosphorylated sites on vimentin. Taken together, these findings revealed a novel mechanism of vimentin through which various signaling pathways controlled the proliferation, differentiation, and movement of the HSCs via the ERK/AKT and Rho cascades.

scholarly journals Translocation of Src kinase to the cell periphery is mediated by the actin cytoskeleton under the control of the Rho family of small G proteins.

1996 ◽  
Vol 135 (6) ◽  
pp. 1551-1564 ◽  
Author(s):  
V J Fincham ◽  
M Unlu ◽  
V G Brunton ◽  
J D Pitts ◽  
J A Wyke ◽  
...  
Keyword(s):  
G Proteins ◽  
Focal Adhesions ◽  
Cytochalasin D ◽  
Mitogen Activated Protein Kinase ◽  
Stress Fibers ◽  
Cell Periphery ◽  
Double Labeling ◽  
Rho Family ◽  
Swiss 3T3 ◽  
Small G Proteins

We have isolated Swiss 3T3 subclones that are resistant to the mitogenic and morphological transforming effects of v-Src as a consequence of aberrant translocation of the oncoprotein under low serum conditions. In chicken embryo and NIH 3T3 fibroblasts under similar conditions, v-Src rapidly translocates from the perinuclear region to the focal adhesions upon activation of the tyrosine kinase, resulting in downstream activation of activator protein-1 and mitogen-activated protein kinase, which are required for the mitogenic and transforming activity of the oncoprotein. Since serum deprivation induces cytoskeletal disorganization in Swiss 3T3, we examined whether regulators of the cytoskeleton play a role in the translocation of v-Src, and also c-Src, in response to biological stimuli. Actin stress fibers and translocation of active v-Src to focal adhesions in quiescent Swiss 3T3 cells were restored by microinjection of activated Rho A and by serum. Double labeling with anti-Src and phalloidin demonstrated that v-Src localized along the reformed actin filaments in a pattern that would be consistent with trafficking in complexes along the stress fibers to focal adhesions. Furthermore, treatment with the actin-disrupting drug cytochalasin D, but not the microtubule-disrupting drug nocodazole, prevented v-Src translocation. In addition to v-Src, we observed that PDGF-induced, Rac-mediated membrane ruffling was accompanied by translocation of c-Src from the cytoplasm to the plasma membrane, an effect that was also blocked by cytochalasin D. Thus, we conclude that translocation of Src from its site of synthesis to its site of action at the cell membrane requires an intact cytoskeletal network and that the small G proteins of the Rho family may specify the peripheral localization in focal adhesions or along the membrane, mediated by their effects on the cytoskeleton.

scholarly journals The lamellipodium is a myosin independent mechanosensor

2017 ◽  
Author(s):  
Patrick W. Oakes ◽  
Tamara C. Bidone ◽  
Yvonne Beckham ◽  
Austin V. Skeeters ◽  
Guillermina R. Ramirez-San Juan ◽  
...  
Keyword(s):  
Cell Attachment ◽  
Myosin Ii ◽  
Adhesion Formation ◽  
Leading Edge ◽  
Cell Spreading ◽  
Substrate Stiffness ◽  
Cell Physiology ◽  
Cell Periphery ◽  
Initial Adhesion ◽  
Kinetics Of

AbstractThe ability of adherent cells to sense changes in the mechanical properties of their extracellular environments is critical to numerous aspects of their physiology. It has been well documented that cell attachment and spreading are sensitive to substrate stiffness. Here we demonstrate that this behavior is actually biphasic, with a transition that occurs around a Young’s modulus of ∼7 kPa. Furthermore, we demonstrate that, contrary to established assumptions, this property is independent of myosin II activity. Rather, we find that cell spreading on soft substrates is inhibited due to reduced nascent adhesion formation within the lamellipodium. Cells on soft substrates display normal leading edge protrusion activity, but these protrusions are not stabilized due to impaired adhesion assembly. Enhancing integrin-ECM affinity through addition of Mn2+ recovers nascent adhesion assembly and cell spreading on soft substrates. Using a computational model to simulate nascent adhesion assembly, we find that biophysical properties of the integrin-ECM bond are optimized to stabilize interactions above a threshold matrix stiffness that is consistent with the experimentally observations. Together these results suggest that myosin II-independent forces in the lamellipodium are responsible for mechanosensation by regulating new adhesion assembly, which in turn, directly controls cell spreading. This myosin II-independent mechanism of substrate stiffness sensing could potentially regulate a number of other stiffness sensitive processes.Significance StatementCell physiology can be regulated by the mechanics of the extracellular environment. Here, we demonstrate that cell spreading is a mechanosensitive process regulated by weak forces generated at the cell periphery and independent of motor activity. We show that stiffness sensing depends on the kinetics of the initial adhesion bonds that are subjected to forces driven by protein polymerization. This work demonstrates how the binding kinetics of adhesion molecules are sensitively tuned to a range of forces that enable mechanosensation.

scholarly journals Recognition of Fibronectin by Penicillium marneffei Conidia via a Sialic Acid-Dependent Process and Its Relationship to the Interaction Between Conidia and Laminin

1999 ◽  
Vol 67 (10) ◽  
pp. 5200-5205 ◽  
Author(s):  
A. J. Hamilton ◽  
L. Jeavons ◽  
S. Youngchim ◽  
N. Vanittanakom
Keyword(s):  
Sialic Acid ◽  
Matrix Protein ◽  
Penicillium Marneffei ◽  
Extracellular Matrix Protein ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Acetylneuraminic Acid ◽  
Microtiter Plates ◽  
Dependent Process ◽  
Common Receptor

ABSTRACT Adhesion of Penicillium marneffei conidia to the extracellular matrix protein laminin via a sialic acid-dependent process has previously been demonstrated. This study describes the interaction of P. marneffei conidia with fibronectin and examines the relationship of this process to the recognition of laminin via conidia. Immunofluorescence microscopy demonstrated that fibronectin bound to the surface of conidia and to phialides, but not to hyphae, in a pattern similar to that reported for laminin. Conidia were able to bind to fibronectin immobilized on microtiter plates in a concentration-dependent manner. However, binding to fibronectin (at any given concentration of protein and conidia) was less than that to laminin under equivalent conditions. Soluble fibronectin and antifibronectin antibody inhibited adherence of conidia to fibronectin in the plate adherence assay; soluble laminin also caused pronounced inhibition. Various monosaccharides and several peptides had no effect on adherence to fibronectin. However, N-acetylneuraminic acid abolished adherence to fibronectin, indicating that the interaction was mediated through a sialic acid-dependent process; the latter parallels observations of laminin binding by conidia. Fibronectin binding (and binding of laminin) was considerably reduced by prolonged preincubation of conidia with chymotrypsin, suggesting the protein nature of the binding site. Conidia from older cultures were more adherent to both immobilized fibronectin and laminin than conidia from younger cultures. Ligand affinity binding demonstrated the presence of a 20-kDa protein with the ability to bind both fibronectin and laminin. There would therefore appear to be a common receptor for the binding of fibronectin and laminin on the surface of P. marneffei, and the interaction described here maybe important in mediating attachment of the fungus to host tissue.

scholarly journals A protein microarray-based in vitro transglutaminase assay platform for epitope mapping and peptide discovery

2021 ◽  
Author(s):  
Chen Liu
Keyword(s):  
Large Scale ◽  
Matrix Protein ◽  
Tissue Transglutaminase ◽  
Protein Microarray ◽  
Scale Up ◽  
Alpha Synuclein ◽  
Extracellular Matrix Protein ◽  
Dependent Manner ◽  
Scanning Method

Transglutaminases (TGs) are a family of crosslinking enzymes catalyzing the formation of intra- and inter-molecular glutamine-lysine isopeptide bonds in a calcium dependent manner. Activation of transglutaminases is pathogenically associated with severe human diseases including neurodegenerations, cardiovascular diseases, and autoimmune diseases. Although continuous efforts determining the enzymes substrate preference have been witnessed, a high-throughput assay platform with the omic efficiency is still missing for the global identification of substrate-specific TG modification sites. In this study we report a protein microarray-based in vitro TG assay platform for rapid and large-scale (up to 30000 reactions per chip) determination of the glutamine (Q)-bearing TG modification motifs. With this platform we identified the Q16 in superoxide dismutase 1 and Q109 in alpha-synuclein as the modification sites for tissue transglutaminase (TG2), the most ubiquitous member of the enzyme family. Of particular interest, we found a close match between the modification motif and published vaccine epitope sequence in alpha-synuclein. Our data collectively suggest the glutamine and its follow-up five residues on the C terminal compose a minimal determinant motif for TG2 modification. To screen for site-specific interfering peptides and assist gene editing-based protein engineering, we developed an onchip amino-acid scanning method for the optimization of TG2 modification motifs. Using this approach we optimized the TG2 modification motif QQIV in the extracellular matrix protein fibronectin and obtained 14 variants with significantly higher TG2 reactivity that might serve as the competitive inhibitor peptides and 1 with lower reactivity. We further confirmed the efficacy of this approach using 12-mer peptides, the longest ones that could be synthesized on the chip. Taken together, our synthetic assay platform will be able to not only deliver a precise epitope blueprint for personalized immunotherapy and vaccination but also provide proof-of-concept and directional studies for TG-based peptide discovery and protein design.

scholarly journals Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts

2004 ◽  
Vol 164 (3) ◽  
pp. 427-439 ◽  
Author(s):  
Go Totsukawa ◽  
Yue Wu ◽  
Yasuharu Sasaki ◽  
David J. Hartshorne ◽  
Yoshihiko Yamakita ◽  
...  
Keyword(s):  
Cell Migration ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Myosin Ii ◽  
Focal Adhesions ◽  
Cell Periphery ◽  
Membrane Ruffling ◽  
Regulatory Myosin Light Chain ◽  
Membrane Protrusions ◽  
Mlc Phosphorylation

We examined the role of regulatory myosin light chain (MLC) phosphorylation of myosin II in cell migration of fibroblasts. Myosin light chain kinase (MLCK) inhibition blocked MLC phosphorylation at the cell periphery, but not in the center. MLCK-inhibited cells did not assemble zyxin-containing adhesions at the periphery, but maintained focal adhesions in the center. They generated membrane protrusions all around the cell, turned more frequently, and migrated less effectively. In contrast, Rho-associated kinase (ROCK) inhibition blocked MLC phosphorylation in the center, but not at the periphery. ROCK-inhibited cells assembled zyxin-containing adhesions at the periphery, but not focal adhesions in the center. They moved faster and more straight. On the other hand, inhibition of myosin phosphatase increased MLC phosphorylation and blocked peripheral membrane ruffling, as well as turnover of focal adhesions and cell migration. Our results suggest that myosin II activated by MLCK at the cell periphery controls membrane ruffling, and that the spatial regulation of MLC phosphorylation plays critical roles in controlling cell migration of fibroblasts.

scholarly journals Simulated Microgravity Influences VEGF, MAPK, and PAM Signaling in Prostate Cancer Cells

2020 ◽  
Vol 21 (4) ◽  
pp. 1263 ◽  
Author(s):  
Trine Engelbrecht Hybel ◽  
Dorothea Dietrichs ◽  
Jayashree Sahana ◽  
Thomas J. Corydon ◽  
Mohamed Z. Nassef ◽  
...  
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Cell Culture ◽  
Extracellular Matrix ◽  
Matrix Protein ◽  
Simulated Microgravity ◽  
Focal Adhesions ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Matrix Protein ◽  
Vegf Mrna

Prostate cancer is one of the leading causes of cancer mortality in men worldwide. An unusual but unique environment for studying tumor cell processes is provided by microgravity, either in space or simulated by ground-based devices like a random positioning machine (RPM). In this study, prostate adenocarcinoma-derived PC-3 cells were cultivated on an RPM for time periods of 3 and 5 days. We investigated the genes associated with the cytoskeleton, focal adhesions, extracellular matrix, growth, survival, angiogenesis, and metastasis. The gene expression of signaling factors of the vascular endothelial growth factor (VEGF), mitogen-activated protein kinase (MAPK), and PI3K/AKT/mTOR (PAM) pathways was investigated using qPCR. We performed immunofluorescence to study the cytoskeleton, histological staining to examine the morphology, and a time-resolved immunofluorometric assay to analyze the cell culture supernatants. When PC-3 cells were exposed to simulated microgravity (s-µg), some cells remained growing as adherent cells (AD), while most cells detached from the cell culture flask bottom and formed multicellular spheroids (MCS). After 3-day RPM exposure, PC-3 cells revealed significant downregulation of the VEGF, SRC1, AKT, MTOR, and COL1A1 gene expression in MCS, whereas FLT1, RAF1, MEK1, ERK1, FAK1, RICTOR, ACTB, TUBB, and TLN1 mRNAs were not significantly changed. ERK2 and TLN1 were elevated in AD, and FLK1, LAMA3, COL4A5, FN1, VCL, CDH1, and NGAL mRNAs were significantly upregulated in AD and MCS after 3 days. After a 5-day culture in s-µg, the PC-3 cells showed significant downregulations of VEGF mRNA in AD and MCS, and FN1, CDH1, and LAMA3 in AD and SCR1 in MCS. In addition, we measured significant upregulations in FLT1, AKT, ERK1, ERK2, LCN2, COL1A1, TUBB, and VCL mRNAs in AD and MCS, and increases in FLK1, FN1, and COL4A5 in MCS as well as LAMB2, CDH1, RAF1, MEK1, SRC1, and MTOR mRNAs in AD. FAK1 and RICTOR were not altered by s-µg. In parallel, the secretion rate of VEGFA and NGAL proteins decreased. Cytoskeletal alterations (F-actin) were visible, as well as a deposition of collagen in the MCS. In conclusion, RPM-exposure of PC-3 cells induced changes in their morphology, cytoskeleton, and extracellular matrix protein synthesis, as well as in their focal adhesion complex and growth behavior. The significant upregulation of genes belonging to the PAM pathway indicated their involvement in the cellular changes occurring in microgravity.

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