scholarly journals AMPK negatively regulates tensin-dependent integrin activity

2017 ◽  
Vol 216 (4) ◽  
pp. 1107-1121 ◽  
Author(s):  
Maria Georgiadou ◽  
Johanna Lilja ◽  
Guillaume Jacquemet ◽  
Camilo Guzmán ◽  
Maria Rafaeva ◽  
...  
Keyword(s):  
Adhesion Formation ◽  
Cell Spreading ◽  
Fiber Formation ◽  
Β1 Integrin ◽  
Cellular Functions ◽  
Cell Matrix ◽  
Cytoplasmic Tails ◽  
Integrin Ligand ◽  
Fibrillar Adhesion ◽  
Amp Activated Protein Kinase

Tight regulation of integrin activity is paramount for dynamic cellular functions such as cell matrix adhesion and mechanotransduction. Integrin activation is achieved through intracellular interactions at the integrin cytoplasmic tails and through integrin–ligand binding. In this study, we identify the metabolic sensor AMP-activated protein kinase (AMPK) as a β1-integrin inhibitor in fibroblasts. Loss of AMPK promotes β1-integrin activity, the formation of centrally located active β1-integrin– and tensin-rich mature fibrillar adhesions, and cell spreading. Moreover, in the absence of AMPK, cells generate more mechanical stress and increase fibronectin fibrillogenesis. Mechanistically, we show that AMPK negatively regulates the expression of the integrin-binding proteins tensin1 and tensin3. Transient expression of tensins increases β1-integrin activity, whereas tensin silencing reduces integrin activity in fibroblasts lacking AMPK. Accordingly, tensin silencing in AMPK-depleted fibroblasts impedes enhanced cell spreading, traction stress, and fibronectin fiber formation. Collectively, we show that the loss of AMPK up-regulates tensins, which bind β1-integrins, supporting their activity and promoting fibrillar adhesion formation and integrin-dependent processes.

scholarly journals New insights into the phosphorylation of the threonine motif of the β1 integrin cytoplasmic domain

2022 ◽  
Vol 5 (4) ◽  
pp. e202101301
Author(s):  
Ralph T Böttcher ◽  
Nico Strohmeyer ◽  
Jonas Aretz ◽  
Reinhard Fässler
Keyword(s):  
Mass Spectrometry ◽  
Ligand Binding ◽  
Phosphorylation Site ◽  
Cell Spreading ◽  
Mouse Cell ◽  
Β1 Integrin ◽  
Β1 Integrins ◽  
Integrin Ligand ◽  
Human And Mouse ◽  
Major Phosphorylation Site

Integrins require an activation step before ligand binding and signaling that is mediated by talin and kindlin binding to the β integrin cytosolic domain (β-tail). Conflicting reports exist about the contribution of phosphorylation of a conserved threonine motif in the β1-tail (β1-pT788/pT789) to integrin activation. We show that widely used and commercially available antibodies against β1-pT788/pT789 integrin do not detect specific β1-pT788/pT789 integrin signals in immunoblots of several human and mouse cell lysates but bind bi-phosphorylated threonine residues in numerous proteins, which were identified by mass spectrometry experiments. Furthermore, we found that fibroblasts and epithelial cells expressing the phospho-mimicking β1-TT788/789DD integrin failed to activate β1 integrins and displayed reduced integrin ligand binding, adhesion initiation and cell spreading. These cellular defects are specifically caused by the inability of kindlin to bind β1-tail polypeptides carrying a phosphorylated threonine motif or phospho-mimicking TT788/789DD substitutions. Our findings indicate that the double-threonine motif in β1-class integrins is not a major phosphorylation site but if phosphorylated would curb integrin function.

Smooth muscle cell rigidity and extracellular matrix organization influence endothelial cell spreading and adhesion formation in coculture

2007 ◽  
Vol 293 (3) ◽  
pp. H1978-H1986 ◽  
Author(s):  
Charles S. Wallace ◽  
Sophie A. Strike ◽  
George A. Truskey
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Endothelial Cell ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Tissue Engineered Blood Vessels ◽  
Focal Adhesion Formation ◽  
Fibrillar Adhesion

Efforts to develop functional tissue-engineered blood vessels have focused on improving the strength and mechanical properties of the vessel wall, while the functional status of the endothelium within these vessels has received less attention. Endothelial cell (EC) function is influenced by interactions between its basal surface and the underlying extracellular matrix. In this study, we utilized a coculture model of a tissue-engineered blood vessel to evaluate EC attachment, spreading, and adhesion formation to the extracellular matrix on the surface of quiescent smooth muscle cells (SMCs). ECs attached to and spread on SMCs primarily through the α5β1-integrin complex, whereas ECs used either α5β1- or αvβ3-integrin to spread on fibronectin (FN) adsorbed to plastic. ECs in coculture lacked focal adhesions, but EC α5β1-integrin bound to fibrillar FN on the SMC surface, promoting rapid fibrillar adhesion formation. As assessed by both Western blot analysis and quantitative real-time RT-PCR, coculture suppressed the expression of focal adhesion proteins and mRNA, whereas tensin protein and mRNA expression were elevated. When attached to polyacrylamide gels with similar elastic moduli as SMCs, focal adhesion formation and the rate of cell spreading increased relative to ECs in coculture. Thus, the elastic properties are only one factor contributing to EC spreading and focal adhesion formation in coculture. The results suggest that the softness of the SMCs and the fibrillar organization of FN inhibit focal adhesions and reduce cell spreading while promoting fibrillar adhesion formation. These changes in the type of adhesions may alter EC signaling pathways in tissue-engineered blood vessels.

AXIAL-SYMMETRIC MODELING AND KINEMATIC ANALYSIS OF SPREADING OF SPARSELY CULTURED FIBROBLASTS

2013 ◽  
Vol 13 (04) ◽  
pp. 1350062
Author(s):  
PEI-JUNG WU ◽  
CHOU-CHING K. LIN ◽  
MING-SHAUNG JU
Keyword(s):  
Focal Adhesion ◽  
Cancer Metastasis ◽  
Adhesion Formation ◽  
Cell Spreading ◽  
Fiber Formation ◽  
Biophysical Model ◽  
Actin Dynamics ◽  
Gillespie Algorithm ◽  
Cultured Fibroblasts ◽  
Cell Protrusion

Cell spreading plays an important role in the modulation of physiological functions such as inflammation and cancer metastasis. The Brownian ratchet model and Bell's model have been used to simulate actin dynamics and bond kinetics for focal adhesion dynamics, respectively. In the present study, these models were modified and two additional subcellular mechanisms, integrin and myosin kinetics, were incoporated. An integrin recruitment function was introduced to determine the size of a focal adhesion associated with the substrate stiffness. The relationship between myosin concentration and the actin protrusion velocity was described by a first-order differential equation. Subcellular processes, including cell protrusion, focal adhesion formation, and stress fiber formation, were integrated into an axial-symmetric biophysical model, while inputs to the model were kinematic data from time-lapse experiments. Numerical simulations of the model using the Gillespie algorithm showed that dynamics of cell spreading can be well described by the model.

scholarly journals Vascular Endothelial-Cadherin Regulates Cytoskeletal Tension, Cell Spreading, and Focal Adhesions by Stimulating RhoA

2004 ◽  
Vol 15 (6) ◽  
pp. 2943-2953 ◽  
Author(s):  
Celeste M. Nelson ◽  
Dana M. Pirone ◽  
John L. Tan ◽  
Christopher S. Chen
Keyword(s):  
Adhesion Formation ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Cell Contact ◽  
Matrix Adhesion ◽  
Cell Matrix ◽  
Focal Adhesion Formation ◽  
Cytoskeletal Tension ◽  
Cell Matrix Adhesion ◽  
Cell Cell

Changes in vascular endothelial (VE)-cadherin–mediated cell-cell adhesion and integrin-mediated cell-matrix adhesion coordinate to affect the physical and mechanical rearrangements of the endothelium, although the mechanisms for such cross talk remain undefined. Herein, we describe the regulation of focal adhesion formation and cytoskeletal tension by intercellular VE-cadherin engagement, and the molecular mechanism by which this occurs. Increasing the density of endothelial cells to increase cell-cell contact decreased focal adhesions by decreasing cell spreading. This contact inhibition of cell spreading was blocked by disrupting VE-cadherin engagement with an adenovirus encoding dominant negative VE-cadherin. When changes in cell spreading were prevented by culturing cells on a micropatterned substrate, VE-cadherin–mediated cell-cell contact paradoxically increased focal adhesion formation. We show that VE-cadherin engagement mediates each of these effects by inducing both a transient and sustained activation of RhoA. Both the increase and decrease in cell-matrix adhesion were blocked by disrupting intracellular tension and signaling through the Rho-ROCK pathway. In all, these findings demonstrate that VE-cadherin signals through RhoA and the actin cytoskeleton to cross talk with cell-matrix adhesion and thereby define a novel pathway by which cell-cell contact alters the global mechanical and functional state of cells.

scholarly journals Osteoblast mineralization requires β1 integrin/ICAP-1–dependent fibronectin deposition

2011 ◽  
Vol 194 (2) ◽  
pp. 307-322 ◽  
Author(s):  
Molly Brunner ◽  
Angélique Millon-Frémillon ◽  
Genevieve Chevalier ◽  
Inaam A. Nakchbandi ◽  
Deane Mosher ◽  
...  
Keyword(s):  
Bone Formation ◽  
Deleterious Effect ◽  
Type I Collagen ◽  
Adhesion Formation ◽  
Type I ◽  
Β1 Integrin ◽  
Osteoblast Proliferation ◽  
Fibrillar Adhesion ◽  
Fibronectin Deposition

The morphogenetic and differentiation events required for bone formation are orchestrated by diffusible and insoluble factors that are localized within the extracellular matrix. In mice, the deletion of ICAP-1, a modulator of β1 integrin activation, leads to severe defects in osteoblast proliferation, differentiation, and mineralization and to a delay in bone formation. Deposition of fibronectin and maturation of fibrillar adhesions, adhesive structures that accompany fibronectin deposition, are impaired upon ICAP-1 loss, as are type I collagen deposition and mineralization. Expression of β1 integrin with a mutated binding site for ICAP-1 recapitulates the ICAP-1–null phenotype. Follow-up experiments demonstrated that ICAP-1 negatively regulates kindlin-2 recruitment onto the β1 integrin cytoplasmic domain, whereas an excess of kindlin-2 binding has a deleterious effect on fibrillar adhesion formation. These results suggest that ICAP-1 works in concert with kindlin-2 to control the dynamics of β1 integrin–containing fibrillar adhesions and, thereby, regulates fibronectin deposition and osteoblast mineralization.

scholarly journals Differential cellular responses to adhesive interactions with galectin-8 and fibronectin coated substrates

2021 ◽  
Author(s):  
Wenhong Li ◽  
Ana Sancho ◽  
Wen-Lu Chung ◽  
Yaron Vinik ◽  
Jürgen Groll ◽  
...  
Keyword(s):  
Cellular Response ◽  
Adhesion Formation ◽  
Rho Kinase ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Complex Matrix ◽  
Focal Adhesion Formation ◽  
Rho Family ◽  
Adhesive Interactions ◽  
Integrin Ligand

The mechanisms underlying the cellular response to extracellular matrices (ECM), consisting of multiple adhesive ligands, are still poorly understood. Here we address this topic by monitoring the differential cellular response to two different extracellular adhesion molecules – fibronectin, a major integrin ligand, and galectin-8, a lectin, that binds β-galactoside residues, as well as to mixtures of the two proteins. Cell spreading on galectin-8 coated substrates results in a larger projected cell area, a more extensive extension of filopodia, yet an inability to form focal adhesions and stress fibers, compared to cell spreading on fibronectin. These differences can be partially reversed by experimental manipulations of small G-proteins of the Rho family and their downstream targets, such as formins, Arp2/3 complex, and Rho kinase. We also show that the physical adhesion of cells to galectin-8 is stronger than the adhesion to fibronectin. Notably, galectin-8 and fibronectin differentially regulate cell spreading and focal adhesion formation, yet they act synergistically to upregulate the number and length of filopodia. The physiological significance of the coherent cellular response to a molecularly complex matrix is discussed.

Regulation of the RhoA pathway in human endothelial cell spreading on type IV collagen: role of calcium influx

1999 ◽  
Vol 112 (19) ◽  
pp. 3205-3213 ◽  
Author(s):  
L. Masiero ◽  
K.A. Lapidos ◽  
I. Ambudkar ◽  
E.C. Kohn
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Focal Adhesion ◽  
Type Iv Collagen ◽  
Cell Spreading ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Type Iv ◽  
Stress Fiber Formation

We have shown that nonvoltage-operated Ca(2+) entry regulates human umbilical vein endothelial cell adhesion, migration, and proliferation on type IV collagen. We now demonstrate a requirement for Ca(2+) influx for activation of the RhoA pathway during endothelial cell spreading on type IV collagen. Reorganization of actin into stress fibers was complete when the cells where fully spread at 90 minutes. No actin organization into stress fibers was seen in endothelial cells plated on type I collagen, indicating a permissive effect of type IV collagen. CAI, a blocker of nonvoltage-operated Ca(2+) channels, prevented development of stress fiber formation in endothelial cells on type IV collagen. This permissive effect was augmented by Ca(2+) influx, as stimulated by 0. 5 microM thapsigargin or 0.1 microM ionomycin, yielding faster development of actin stress fibers. Ca(2+) influx and actin rearrangement in response to thapsigargin and ionomycin were abrogated by CAI. Activated, membrane-bound RhoA is a substrate for C3 exoenzyme which ADP-ribosylates and inactivates RhoA, preventing actin stress fiber formation. Pretreatment of endothelial cells with C3 exoenzyme prevented basal and thapsigargin-augmented stress fiber formation. While regulation of Ca(2+) influx did not alter RhoA translocation, it reduced in vitro ADP-ribosylation of RhoA (P(2)<0. 05), suggesting Ca(2+) influx is needed for RhoA activation during spreading on type IV collagen; no Ca(2+) regulated change in RhoA was seen in HUVECs spreading on type I collagen matrix. Blockade of Ca(2+) influx of HUVEC spread on type IV collagen also reduced tyrosine phosphorylation of p190Rho-GAP and blocked thapsigargin-enhanced binding of p190Rho-GAP to focal adhesion kinase. Thus, Ca(2+) influx is necessary for RhoA activation and for linkage of the RhoA/stress fiber cascade to the focal adhesion/focal adhesion kinase pathway during human umbilical vein endothelial cell spreading on type IV collagen.

Nanoporosity Stimulates Cell Spreading and Focal Adhesion Formation in Cells with Mutated Paxillin

2020 ◽  
Vol 12 (13) ◽  
pp. 14924-14932
Author(s):  
Dainelys Guadarrama Bello ◽  
Aurélien Fouillen ◽  
Antonella Badia ◽  
Antonio Nanci
Keyword(s):  
Focal Adhesion ◽  
Adhesion Formation ◽  
Cell Spreading ◽  
Focal Adhesion Formation ◽  
In Cells

AMP-activated protein kinase: new regulation, new roles?

2012 ◽  
Vol 445 (1) ◽  
pp. 11-27 ◽  
Author(s):  
David Carling ◽  
Claire Thornton ◽  
Angela Woods ◽  
Matthew J. Sanders
Keyword(s):  
Protein Kinase ◽  
Living Cells ◽  
Cellular Functions ◽  
Disease States ◽  
Kinase Cascade ◽  
Amp Activated Protein Kinase ◽  
Obesity And Cancer ◽  
Hydrolysis Of ◽  
Protein Kinase Cascade

The hydrolysis of ATP drives virtually all of the energy-requiring processes in living cells. A prerequisite of living cells is that the concentration of ATP needs to be maintained at sufficiently high levels to sustain essential cellular functions. In eukaryotic cells, the AMPK (AMP-activated protein kinase) cascade is one of the systems that have evolved to ensure that energy homoeostasis is maintained. AMPK is activated in response to a fall in ATP, and recent studies have suggested that ADP plays an important role in regulating AMPK. Once activated, AMPK phosphorylates a broad range of downstream targets, resulting in the overall effect of increasing ATP-producing pathways whilst decreasing ATP-utilizing pathways. Disturbances in energy homoeostasis underlie a number of disease states in humans, e.g. Type 2 diabetes, obesity and cancer. Reflecting its key role in energy metabolism, AMPK has emerged as a potential therapeutic target. In the present review we examine the recent progress aimed at understanding the regulation of AMPK and discuss some of the latest developments that have emerged in key areas of human physiology where AMPK is thought to play an important role.

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