scholarly journals The Mechanosensing and Global DNA Methylation of Human Osteoblasts on MEW Fibers

Nanomaterials ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 2943
Author(s):  
Pingping Han ◽  
Cedryck Vaquette ◽  
Abdalla Abdal-hay ◽  
Sašo Ivanovski
Keyword(s):  
Dna Methylation ◽  
Osteogenic Differentiation ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Global Dna Methylation ◽  
Focal Adhesion Complex ◽  
Osteogenic Induction ◽  
Sense And Respond ◽  
Adhesion Complex

Cells interact with 3D fibrous platform topography via a nano-scaled focal adhesion complex, and more research is required on how osteoblasts sense and respond to random and aligned fibers through nano-sized focal adhesions and their downstream events. The present study assessed human primary osteoblast cells’ sensing and response to random and aligned medical-grade polycaprolactone (PCL) fibrous 3D scaffolds fabricated via the melt electrowriting (MEW) technique. Cells cultured on a tissue culture plate (TCP) were used as 2D controls. Compared to 2D TCP, 3D MEW fibrous substrates led to immature vinculin focal adhesion formation and significantly reduced nuclear localization of the mechanosensor-yes-associated protein (YAP). Notably, aligned MEW fibers induced elongated cell and nucleus shape and highly activated global DNA methylation of 5-methylcytosine, 5-hydroxymethylcytosine, and N-6 methylated deoxyadenosine compared to the random fibers. Furthermore, although osteogenic markers (osterix-OSX and bone sialoprotein-BSP) were significantly enhanced in PCL-R and PCL-A groups at seven days post-osteogenic differentiation, calcium deposits on all seeded samples did not show a difference after normalizing for DNA content after three weeks of osteogenic induction. Overall, our study linked 3D extracellular fiber alignment to nano-focal adhesion complex, nuclear mechanosensing, DNA epigenetics at an early point (24 h), and longer-term changes in osteoblast osteogenic differentiation.

scholarly journals Degraded Collagen Fragments Promote Rapid Disassembly of Smooth Muscle Focal Adhesions That Correlates with Cleavage of Pp125FAK, Paxillin, and Talin

1999 ◽  
Vol 147 (3) ◽  
pp. 619-630 ◽  
Author(s):  
Neil O. Carragher ◽  
Bodo Levkau ◽  
Russell Ross ◽  
Elaine W. Raines
Keyword(s):  
Smooth Muscle ◽  
Focal Adhesion ◽  
Type I Collagen ◽  
Focal Adhesions ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Type I ◽  
Focal Adhesion Complex ◽  
Adhesion Complex

Active matrix metalloproteinases and degraded collagen are observed in disease states, such as atherosclerosis. To examine whether degraded collagen fragments have distinct effects on vascular smooth muscle cells (SMC), collagenase-digested type I collagen was added to cultured human arterial SMC. After addition of collagen fragments, adherent SMC lose their focal adhesion structures and round up. Analysis of components of the focal adhesion complex demonstrates rapid cleavage of the focal adhesion kinase (pp125FAK), paxillin, and talin. Cleavage is suppressed by inhibitors of the proteolytic enzyme, calpain I. In vitro translated pp125FAK is a substrate for both calpain I– and II–mediated processing. Mapping of the proteolytic cleavage fragments of pp125FAK predicts a dissociation of the focal adhesion targeting (FAT) sequence and second proline-rich domain from the tyrosine kinase domain and integrin-binding sequence. Coimmunoprecipitation studies confirm that the ability of pp125FAK to associate with paxillin, vinculin, and p130cas is significantly reduced in SMC treated with degraded collagen fragments. Further, there is a significant reduction in the association of intact pp125FAK with the cytoskeletal fraction, while pp125FAK cleavage fragments appear in the cytoplasm in SMC treated with degraded collagen fragments. Integrin-blocking studies indicate that integrin-mediated signals are involved in degraded collagen induction of pp125FAK cleavage. Thus, collagen fragments induce distinct integrin signals that lead to initiation of calpain-mediated cleavage of pp125FAK, paxillin, and talin and dissolution of the focal adhesion complex.

scholarly journals FAK Structure and Regulation by Membrane Interactions and Force in Focal Adhesions

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 179 ◽  
Author(s):  
Paula Tapial Martínez ◽  
Pilar López Navajas ◽  
Daniel Lietha
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Structural Changes ◽  
Membrane Lipids ◽  
Current Knowledge ◽  
Focal Adhesions ◽  
Structural Features ◽  
Focal Adhesion Complex ◽  
Patient Prognosis ◽  
Adhesion Complex

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase with key roles in the regulation of cell adhesion migration, proliferation and survival. In cancer FAK is a major driver of invasion and metastasis and its upregulation is associated with poor patient prognosis. FAK is autoinhibited in the cytosol, but activated upon localisation into a protein complex, known as focal adhesion complex. This complex forms upon cell adhesion to the extracellular matrix (ECM) at the cytoplasmic side of the plasma membrane at sites of ECM attachment. FAK is anchored to the complex via multiple sites, including direct interactions with specific membrane lipids and connector proteins that attach focal adhesions to the actin cytoskeleton. In migrating cells, the contraction of actomyosin stress fibres attached to the focal adhesion complex apply a force to the complex, which is likely transmitted to the FAK protein, causing stretching of the FAK molecule. In this review we discuss the current knowledge of the FAK structure and how specific structural features are involved in the regulation of FAK signalling. We focus on two major regulatory mechanisms known to contribute to FAK activation, namely interactions with membrane lipids and stretching forces applied to FAK, and discuss how they might induce structural changes that facilitate FAK activation.

scholarly journals Caspase-mediated Cleavage of Focal Adhesion Kinase pp125FAK and Disassembly of Focal Adhesions in Human Endothelial Cell Apoptosis

1998 ◽  
Vol 187 (4) ◽  
pp. 579-586 ◽  
Author(s):  
Bodo Levkau ◽  
Barbara Herren ◽  
Hidenori Koyama ◽  
Russell Ross ◽  
Elaine W. Raines
Keyword(s):  
Extracellular Matrix ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Caspase 3 ◽  
Umbilical Vein ◽  
Focal Adhesions ◽  
Focal Adhesion Complex ◽  
Adhesion Sites ◽  
Induced Apoptosis ◽  
Adhesion Complex

Normal endothelial and epithelial cells undergo apoptosis when cell adhesion and spreading are prevented, implying a requirement for antiapoptotic signals from the extracellular matrix for cell survival. We investigated some of the molecular changes occurring in focal adhesions during growth factor deprivation–induced apoptosis in confluent monolayers of human umbilical vein endothelial cells. Among the first morphologic changes after initiation of the apoptotic process are membrane blebbing, loss of focal adhesion sites, and retraction from the matrix followed by detachment. We observe a specific proteolytic cleavage of focal adhesion kinase (pp125FAK), an important component of the focal adhesion complex, and identify pp125FAK as a novel substrate for caspase-3 and caspase-3–like apoptotic caspases. The initial cleavage precedes detachment, and coincides with loss of pp125FAK and paxillin from focal adhesion sites and their redistribution into the characteristic membrane blebs of apoptotically dying cells. Cleavage of pp125FAK differentially affects its association with signaling and cytoskeletal components of the focal adhesion complex; binding of paxillin, but not pp130Cas (Cas, Crk-associated substrate) and vinculin, to the COOH terminally truncated pp125FAK is abolished. Therefore, caspase-mediated cleavage of pp125FAK may be participating in the disassembly of the focal adhesion complex and actively interrupting survival signals from the extracellular matrix, thus propagating the cell death program.

Increased transcriptional response to mechanical strain in keloid fibroblasts due to increased focal adhesion complex formation

2005 ◽  
Vol 206 (2) ◽  
pp. 510-517 ◽  
Author(s):  
Zhen Wang ◽  
Kenton D. Fong ◽  
Toan-Thang Phan ◽  
Ivor J. Lim ◽  
Michael T. Longaker ◽  
...  
Keyword(s):  
Complex Formation ◽  
Focal Adhesion ◽  
Mechanical Strain ◽  
Transcriptional Response ◽  
Focal Adhesion Complex ◽  
Adhesion Complex ◽  
Keloid Fibroblasts

scholarly journals PHIP drives glioblastoma motility and invasion by regulating the focal adhesion complex

2020 ◽  
Vol 117 (16) ◽  
pp. 9064-9073
Author(s):  
David de Semir ◽  
Vladimir Bezrookove ◽  
Mehdi Nosrati ◽  
Kara R. Scanlon ◽  
Eric Singer ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Pleckstrin Homology Domain ◽  
Physical Interaction ◽  
Glioblastoma Cells ◽  
Focal Adhesion Complex ◽  
Adhesion Proteins ◽  
Focal Adhesion Proteins ◽  
Tumor Cell Motility ◽  
Adhesion Complex ◽  
Force Transduction

The invasive behavior of glioblastoma is essential to its aggressive potential. Here, we show that pleckstrin homology domain interacting protein (PHIP), acting through effects on the force transduction layer of the focal adhesion complex, drives glioblastoma motility and invasion. Immunofluorescence analysis localized PHIP to the leading edge of glioblastoma cells, together with several focal adhesion proteins: vinculin (VCL), talin 1 (TLN1), integrin beta 1 (ITGB1), as well as phosphorylated forms of paxillin (pPXN) and focal adhesion kinase (pFAK). Confocal microscopy specifically localized PHIP to the force transduction layer, together with TLN1 and VCL. Immunoprecipitation revealed a physical interaction between PHIP and VCL. Targeted suppression of PHIP resulted in significant down-regulation of these focal adhesion proteins, along with zyxin (ZYX), and produced profoundly disorganized stress fibers. Live-cell imaging of glioblastoma cells overexpressing a ZYX-GFP construct demonstrated a role for PHIP in regulating focal adhesion dynamics. PHIP silencing significantly suppressed the migratory and invasive capacity of glioblastoma cells, partially restored following TLN1 or ZYX cDNA overexpression. PHIP knockdown produced substantial suppression of tumor growth upon intracranial implantation, as well as significantly reduced microvessel density and secreted VEGF levels. PHIP copy number was elevated in the classical glioblastoma subtype and correlated with elevated EGFR levels. These results demonstrate PHIP’s role in regulating the actin cytoskeleton, focal adhesion dynamics, and tumor cell motility, and identify PHIP as a key driver of glioblastoma migration and invasion.

scholarly journals Interleukin 1-induced calcium signalling in chondrocytes requires focal adhesions

1997 ◽  
Vol 324 (2) ◽  
pp. 653-658 ◽  
Author(s):  
Laura LUO ◽  
Tony CRUZ ◽  
Christopher McCULLOCH
Keyword(s):  
Signal Transduction ◽  
Focal Adhesion ◽  
Cell Attachment ◽  
Interleukin 1 ◽  
Primary Cultures ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Ion Concentration ◽  
Significant Difference ◽  
In Cells

The cytokine interleukin 1 (IL-1) is an important mediator of connective-tissue destruction in arthritic joints but the mechanisms by which IL-1 mediates signal transduction in chondrocytes is poorly understood. Previous results have indicated that IL-1 receptors co-localize with focal adhesions [Qwarnstrom, Page, Gillis and Dower (1988) J. Biol. Chem. 263, 8261–8269], discrete adhesive domains of cells that function in cell attachment and possibly in signal transduction. We have determined whether focal adhesions restrict IL-1-induced Ca2+ signalling in primary cultures of bovine chondrocytes. In cells grown for 24 h on fibronectin, the basal intracellular Ca2+ ion concentration ([Ca2+]i) was 100±3 nM. Optimal increases of [Ca2+]i above baseline were induced by 10 nM IL-1 (183±30 nM above baseline). There was no significant difference between cells plated on fibronectin or type II collagen (P > 0.2; 233±90 nM above baseline). Ca2+ transients were significantly decreased by the inclusion of 0.5 mM EGTA in the bathing buffer (74±11 nM above baseline), and 1 μM thapsigargin completely blocked Ca2+ transients. Cells plated on poly-(l-lysine) or suspended cells showed no Ca2+ increases, whereas cells grown on fibronectin exhibited IL-1-induced Ca2+ responses that corresponded temporally to the time-dependent cell spreading after plating on fibronectin. Cells plated on poly-(l-lysine) and incubated with fibronectin-coated beads exhibited vinculin staining in association with the beads. In identical cell preparations, IL-1 induced a 136±39 nM increase of [Ca2+]i above baseline in response to 10 nM IL-1β. There were no IL-1-induced Ca2+ increases when cells on poly-(l-lysine) were incubated with fibronectin-coated beads for only 15 min at 37 °C, in cells maintained for 3 h at 4 °C, in cells incubated with BSA beads for 3 h at 37 °C, or in cells pretreated with cytochalasin D. Labelling of IL-1 receptors with 125I-IL-1β showed 3-fold more specific labelling of focal adhesion complexes in cells incubated with fibronectin-coated beads compared with cells incubated with BSA-coated beads, indicating that IL-1 receptor binding or the number of IL-1 receptors was increased in focal adhesions. These results indicate that, in chondrocytes, IL-1-induced Ca2+ signalling is dependent on focal adhesion formation and that focal adhesions recruit IL-1 receptors by redistribution in the cell membrane.

An Analysis of the Cooperative Mechano-Sensitive Feedback Between Intracellular Signaling, Focal Adhesion Development, and Stress Fiber Contractility

2011 ◽  
Vol 78 (4) ◽  
Author(s):  
Amit Pathak ◽  
Robert M. McMeeking ◽  
Anthony G. Evans ◽  
Vikram S. Deshpande
Keyword(s):  
Mechanical Loading ◽  
Focal Adhesion ◽  
Intracellular Signaling ◽  
Feedback Loop ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Stress Fiber ◽  
Mechanical Loads ◽  
Signaling Model ◽  
Wide Range

Cells communicate with their external environment via focal adhesions and generate activation signals that in turn trigger the activity of the intracellular contractile machinery. These signals can be triggered by mechanical loading that gives rise to a cooperative feedback loop among signaling, focal adhesion formation, and cytoskeletal contractility, which in turn equilibrates with the applied mechanical loads. We devise a signaling model that couples stress fiber contractility and mechano-sensitive focal adhesion models to complete this above mentioned feedback loop. The signaling model is based on a biochemical pathway where IP3 molecules are generated when focal adhesions grow. These IP3 molecules diffuse through the cytosol leading to the opening of ion channels that disgorge Ca2+ from the endoplasmic reticulum leading to the activation of the actin/myosin contractile machinery. A simple numerical example is presented where a one-dimensional cell adhered to a rigid substrate is pulled at one end, and the evolution of the stress fiber activation signal, stress fiber concentrations, and focal adhesion distributions are investigated. We demonstrate that while it is sufficient to approximate the activation signal as spatially uniform due to the rapid diffusion of the IP3 through the cytosol, the level of the activation signal is sensitive to the rate of application of the mechanical loads. This suggests that ad hoc signaling models may not be able to capture the mechanical response of cells to a wide range of mechanical loading events.

scholarly journals A synthetic peptide from the heparin-binding domain III (repeats III4-5) of fibronectin promotes stress-fibre and focal-adhesion formation in melanoma cells

2003 ◽  
Vol 371 (2) ◽  
pp. 565-571 ◽  
Author(s):  
José V. MOYANO ◽  
Alfredo MAQUEDA ◽  
Juan P. ALBAR ◽  
Angeles GARCIA-PARDO
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Melanoma Cells ◽  
Small Gtpase ◽  
Binding Domain ◽  
Heparin Binding ◽  
Chondroitin Sulphate Proteoglycans ◽  
Heparin Binding Domain

Cell adhesion to fibronectin results in formation of actin stress fibres and focal adhesions. In fibroblasts, this response requires two co-operative signals provided by interactions of the RGD sequence with α5β1 integrin and the heparin-binding domain II (Hep II) domain with syndecan-4. Within Hep II, this activity was mapped to repeat III13 and to the peptide FN-C/H-V(WQPPRARITGY, repeat III14). We previously described that the synthetic heparin-binding peptide/III5 (HBP/III5) (WTPPRAQITGYRLTVGLTRR, repeat III5) binds heparin and mediates cell adhesion via chondroitin sulphate proteoglycans. We have now studied whether HBP/III5 co-operates with α5β1 and drives a full cytoskeletal response in melanoma cells. SKMEL-178 cells attached and spread on the RGD-containing FNIII7–FNIII10 (FNIII7–10) fragment, but did not form stress fibres or focal adhesions. Co-immobilization of HBP/III5 with FNIII7–10 or adding soluble HBP/III5 to cells prespread on FNIII7–10, effectively induced these structures. Cell transfection with dominant-negative N19RhoA, a member of the small GTPase family, abolished the HBP/III5 effect. Both chondroitinase and heparitinase diminished focal adhesions, indicating that both types of proteoglycans bound HBP/III5 in melanoma cells. We have mapped the active sequence of HBP/III5 to YRLTVGLTRR, which is a novel sequence in fibronectin with focal-adhesion-promoting activity. The last two arginine (R) residues of this sequence are required for activity, since their replacement by alanine completely abrogated the HBP/III5 cytoskeletal effect. Moreover, this sequence is also active in the context of large fibronectin fragments. Our results establish that the Hep III region provides co-operative signals to α5β1 for the progression of the cytoskeletal response and that these include activation of RhoA.

scholarly journals Restructuring of Focal Adhesion Plaques by Pi 3-Kinase

2000 ◽  
Vol 150 (3) ◽  
pp. 627-642 ◽  
Author(s):  
Jeffrey A. Greenwood ◽  
Anne B. Theibert ◽  
Glenn D. Prestwich ◽  
Joanne E. Murphy-Ullrich
Keyword(s):  
Extracellular Matrix ◽  
Focal Adhesion ◽  
Adhesive Strength ◽  
Focal Adhesions ◽  
Lipid Product ◽  
Phosphoinositide 3 Kinase ◽  
Triton X ◽  
Adhesion Plaque ◽  
Adhesion Complex

Focal adhesions are an elaborate network of interconnecting proteins linking actin stress fibers to the extracellular matrix substrate. Modulation of the focal adhesion plaque provides a mechanism for the regulation of cellular adhesive strength. Using interference reflection microscopy, we found that activation of phosphoinositide 3-kinase (PI 3-kinase) by PDGF induces the dissipation of focal adhesions. Loss of this close apposition between the cell membrane and the extracellular matrix coincided with a redistribution of α-actinin and vinculin from the focal adhesion complex to the Triton X-100–soluble fraction. In contrast, talin and paxillin remained localized to focal adhesions, suggesting that activation of PI 3-kinase induced a restructuring of the plaque rather than complete dispersion. Furthermore, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5)-P3), a lipid product of PI 3-kinase, was sufficient to induce restructuring of the focal adhesion plaque. We also found that PtdIns (3,4,5)-P3 binds to α-actinin in PDGF-treated cells. Further evidence demonstrated that activation of PI 3-kinase by PDGF induced a decrease in the association of α-actinin with the integrin β subunit, and that PtdIns (3,4,5)-P3 could disrupt this interaction in vitro. Modification of focal adhesion structure by PI 3-kinase and its lipid product, PtdIns (3,4,5)-P3, has important implications for the regulation of cellular adhesive strength and motility.

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