Platelet ACKR3/CXCR7 favors antiplatelet lipids over an atherothrombotic lipidome and regulates thromboinflammation.

Platelet ACKR3/CXCR7 surface expression is enhanced and influences prognosis in coronary artery disease (CAD) patients, who exhibit a distinct atherothrombotic platelet lipidome. Current investigation validates the potential of ACKR3/CXCR7 in regulating thromboinflammatory response through its impact on the platelet lipidome. CAD patients with enhanced platelet ACKR3/CXCR7 expression exhibited reduced aggregation. Pharmacological CXCR7 agonist (VUF11207) significantly reduced prothrombotic platelet response in blood from acute coronary syndrome patients ex vivo. CXCR7 agonist administration reduced thrombotic functions and thromboinflammatory plateletleukocyte interactions post-myocardial infarction and arterial injury in vivo. ACKR3/CXCR7 ligation did not affect surface availability of surface receptors, coagulation profile, bleeding time, plasma-dependent thrombin generation (thrombinoscopy), or clot formation (thromboelastography) but counteracted activation-induced phosphatidylserine exposure and procoagulant platelet-assisted thrombin generation. Targeted (micro-UHPLC-ESI-QTrap-MS/MS) and untargeted (UHPLCESI-QTOF-MS/MS) lipidomics analysis revealed that ACKR3/CXCR7 ligation favored generation of antithrombotic lipids (dihomo-γ-linolenic acid [DGLA], 12-hydroxyeicosatrienoic acid [12-HETrE]) over cyclooxygenase-1 (COX-1) or 12-lipoxygenase (12-LOX) metabolized prothrombotic and phospholipase-derived atherogenic lipids in healthy subjects and CAD patients, contrary to antiplatelet therapy. Through 12-HETrE, ACKR3/CXCR7 ligation coordinated with Gαs-coupled prostacyclin receptor to trigger cyclic adenosine monophosphate/protein kinase A-mediated platelet inhibition. ACKR3/CXCR7 ligation reduced generation of lipid agonists and lipid signaling intermediates, which affected calcium mobilization, intracellular signaling, and consequently platelet interaction with physiological matrices and thromboinflammatory secretome. This emphasized its functional dichotomy from prothrombotic CXCR4. Moreover, CXCR7 agonist regulated heparin-induced thrombocytopenia-sera/immunoglobulin G-triggered platelet and neutrophil activation, heparin-induced platelet aggregation, generation of thromboinflammatory lipids, platelet-neutrophil aggregate formation, and thromboinflammatory secretion ex vivo. Therefore, ACKR3/CXCR7 may offer a novel therapeutic strategy in acute/chronic thromboinflammation exaggerated cardiovascular pathologies and CAD.

Cortical cell stiffness is independent of substrate mechanics.

Cortical stiffness is an important cellular property that changes during migration, adhesion and growth. Previous atomic force microscopy (AFM) indentation measurements of cells cultured on deformable substrates have suggested that cells adapt their stiffness to that of their surroundings. Here we show that the force applied by AFM to a cell results in a significant deformation of the underlying substrate if this substrate is softer than the cell. This 'soft substrate effect' leads to an underestimation of a cell's elastic modulus when analysing data using a standard Hertz model, as confirmed by finite element modelling and AFM measurements of calibrated polyacrylamide beads, microglial cells and fibroblasts. To account for this substrate deformation, we developed a 'composite cell-substrate model'. Correcting for the substrate indentation revealed that cortical cell stiffness is largely independent of substrate mechanics, which has major implications for our interpretation of many physiological and pathological processes.

Functional Relevance of the Anaphylatoxin Receptor C3aR for Platelet Function and Arterial Thrombus Formation Marks an Intersection Point Between Innate Immunity and Thrombosis.

BACKGROUND: Platelets have distinct roles in the vascular system in that they are the major mediator of thrombosis, critical for restoration of tissue integrity, and players in vascular inflammatory conditions. In close spatiotemporal proximity, the complement system acts as the first line of defense against invading microorganisms and is a key mediator of inflammation. Whereas the fluid phase cross-talk between the complement and coagulation systems is well appreciated, the understanding of the pathophysiological implications of such interactions is still scant. METHODS: We analyzed coexpression of the anaphylatoxin receptor C3aR with activated glycoprotein IIb/IIIa on platelets of 501 patients with coronary artery disease using flow cytometry; detected C3aR expression in human or murine specimen by polymerase chain reaction, immunofluorescence, Western blotting, or flow cytometry; and examined the importance of platelet C3aR by various in vitro platelet function tests, in vivo bleeding time, and intravital microscopy. The pathophysiological relevance of C3aR was scrutinized with the use of disease models of myocardial infarction and stroke. To approach underlying molecular mechanisms, we identified the platelet small GTPase Rap1b using nanoscale liquid chromatography coupled to tandem mass spectrometry. RESULTS: We found a strong positive correlation of platelet complement C3aR expression with activated glycoprotein IIb/IIIa in patients with coronary artery disease and coexpression of C3aR with glycoprotein IIb/IIIa in thrombi obtained from patients with myocardial infarction. Our results demonstrate that the C3a/C3aR axis on platelets regulates distinct steps of thrombus formation such as platelet adhesion, spreading, and Ca2+ influx. Using C3aR-/- mice or C3-/- mice with reinjection of C3a, we uncovered that the complement activation fragment C3a regulates bleeding time after tail injury and thrombosis. Notably, C3aR-/- mice were less prone to experimental stroke and myocardial infarction. Furthermore, reconstitution of C3aR-/- mice with C3aR+/+ platelets and platelet depletion experiments demonstrated that the observed effects on thrombosis, myocardial infarction, and stroke were specifically caused by platelet C3aR. Mechanistically, C3aR-mediated signaling regulates the activation of Rap1b and thereby bleeding arrest after injury and in vivo thrombus formation. CONCLUSIONS: Overall, our findings uncover a novel function of the anaphylatoxin C3a for platelet function and thrombus formation, highlighting a detrimental role of imbalanced complement activation in cardiovascular diseases.

Combined atomic force microscopy (AFM) and traction force microscopy (TFM) reveals a correlation between viscoelastic material properties and contractile prestress of living cells.

Living cells exhibit a complex mechanical behavior, whose underlying mechanisms are still largely unknown. Emerging from the molecular structure and dynamics of the cytoskeleton, the mechanical behavior comprises "passive" viscoelastic material properties and "active" contractile prestress. To directly investigate the connection between these quantities at the single-cell level, we here present the combination of atomic force microscopy (AFM) with traction force microscopy (TFM). With this combination, we simultaneously measure viscoelastic material parameters (stiffness, fluidity) and contractile prestress of adherent fibroblast and epithelial cells. Although stiffness, fluidity, and contractile prestress greatly vary within a cell population, they are highly correlated: stiffer cells have a lower fluidity and a larger prestress than softer cells. We show that viscoelastic material properties and contractile prestress are both governed by the activity of the actomyosin machinery. Our results underline the connection between a cell's viscoelastic material properties and its contractile prestress and their importance in cell mechanics.

Regulation of oxidized platelet lipidome: implications for coronary artery disease.

AIMS: Hyperlipidaemia enhances susceptibility to thrombosis, while platelet oxidixed LDL (oxLDL) binding in acute coronary syndrome (ACS) correlates with activation status. This study explores the platelet lipidome in symptomatic coronary artery disease (CAD) patients and the functional consequences of the chemokine CXCL12 and its receptors CXCR-4/-7 on lipid uptake in platelets. METHODS AND RESULTS: Platelet-oxLDL detected by flow cytometry was enhanced (P = 0.04) in CAD patients, moderately correlated with platelet CXCR7 surface expression (ρ = 0.39; P < 0.001), while inversely with CXCR4 (ρ = 0.35; P < 0.001). Platelet-oxLDL was elevated (P = 0.01) in ACS patients with angiographic evidence of intracoronary thrombi. Ex vivo analysis of intracoronary thrombi sections revealed oxLDL deposition in platelet-enriched areas verified by immunofluorescence confocal microscopy. LDL-oxLDL uptake enhanced reactive oxygen species, mitochondrial superoxide generation, intraplatelet LDL to oxLDL conversion, and lipid peroxidation, counteracted by SOD2-mimetic MnTMPyP. Lipidomic analysis revealed enhanced intraplatelet-oxidized phospholipids, cholesteryl esters, sphingomyelin, ceramides, di- and triacylglycerols, acylcarnitines in CAD patients compared with age-matched controls as ascertained by liquid chromatography hyphenated to high-resolution mass spectrometry. LDL-oxLDL induced degranulation, αIIbß3-integrin activation, apoptosis, thrombin generation estimated by calibrated automated thrombinoscopy, and shape change verified by live imaging using scanning ion conductance microscopy. Further, LDL-oxLDL enhanced thrombus formation ex vivo and in vivo in mice (ferric chloride-induced carotid artery injury). LDL-oxLDL enhanced platelet CXCL12 release, differentially regulated CXCR4-CXCR7 surface exposure, while CXCL12 prompted LDL-oxLDL uptake and synergistically augmented the LDL-oxLDL-induced pro-oxidative, thrombogenic impact on platelet function. CONCLUSION: An altered platelet lipidome might be associated with thrombotic disposition in CAD, a mechanism potentially regulated by CXCL12-CXCR4-CXCR7 axis.

An Accurate Model for the Ion Current-Distance Behavior in Scanning Ion Conductance Microscopy Allows for Calibration of Pipet Tip Geometry and Tip-Sample Distance.

The scanning ion conductance microscope (SICM) is an emerging tool for noncontact topography imaging and multiphysical investigation of soft samples in aqueous environments such as living cells. Despite the increasing popularity of SICM, several aspects of the imaging process are still unknown; for example, there is still no accurate description of the behavior of the ion current for a varying tip-sample distance. To predict this ion current-distance behavior, we provide a new numerical model based on finite element modeling. The model allows, for the first time, accurately determining the tip-sample distance during an SICM experiment. Furthermore, we present a nondestructive method for calibrating the pipet tip geometry by fitting the numerical model to the experimental ion current-distance data and verify this method using pipets with opening radii between 30 and 300 nm.

Comparative morphology analysis of live blood platelets using scanning ion conductance and robotic dark-field microscopy.

Many conventional microscopy techniques for investigating platelet morphology such as electron or fluorescence microscopy require highly invasive treatment of the platelets such as fixation, drying and metal coating or staining. Here, we present two unique but entirely different microscopy techniques for direct morphology analysis of live, unstained platelets: scanning ion conductance microscopy (SICM) and robotic dark-field microscopy (RDM). We demonstrate that both techniques allow for a quantitative evaluation of the morphological features of live adherent platelets. We show that their morphology can be quantified by both techniques using the same geometric parameters and therefore can be directly compared. By imaging the same identical platelets subsequently with SICM and RDM, we found that area, perimeter and circularity of the platelets are directly correlated between SICM and dark-field microscopy (DM), while the fractal dimension (FD) differed between the two microscopy techniques. We show that SICM and RDM are both valuable tools for the ex vivo investigation of the morphology of live platelets, which might contribute to new insights into the physiological and pathophysiological role of platelet spreading.

Lateral Resolution and Image Formation in Scanning Ion Conductance Microscopy.

The scanning ion conductance microscope (SICM) is a powerful tool for imaging the topography of soft samples in an aqueous environment. Despite the rising popularity of the SICM, the image formation process and the fundamental limit of the lateral resolution are still a matter of debate. Using microfabricated samples, we investigated the imaging of small cylindrical particles, elongated objects, and topography steps and present the first direct comparison of numerical and experimental data. For the lateral resolution we considered two alternative definitions: the distance at which two small particles can clearly be resolved from each other in an image, and the apparent full width at half-maximum of small particles. For both definitions, we found a lateral resolution of about 3 times the inner opening radius of the pipet. We further validated this resolution limit in measurements on supported lipid bilayers and a polycarbonate sample using pipets with opening radii down to 8 nm.

Comparison of Atomic Force Microscopy and Scanning Ion Conductance Microscopy for Live Cell Imaging.

Atomic force microscopy (AFM) and scanning ion conductance microscopy (SICM) are excellent and commonly used techniques for imaging the topography of living cells with high resolution. We present a direct comparison of AFM and SICM for imaging microvilli, which are small features on the surface of living cells, and for imaging the shape of whole cells. The imaging quality on microvilli increased significantly after cell fixation for AFM, whereas for SICM it remained constant. The apparent shape of whole cells in the case of AFM depended on the imaging force, which deformed the cell. In the case of SICM, cell deformations were avoided, owing to the contact-free imaging mechanism. We estimated that the lateral resolution on living cells is limited by the cell's elastic modulus for AFM, while it is not for SICM. By long-term, time-lapse imaging of microvilli dynamics, we showed that the imaging quality decreased with time for AFM, while it remained constant for SICM.

Imaging viscoelastic properties of live cells by AFM: power-law rheology on the nanoscale.

We developed force clamp force mapping (FCFM), an atomic force microscopy (AFM) technique for measuring the viscoelastic creep behavior of live cells with sub-micrometer spatial resolution. FCFM combines force-distance curves with an added force clamp phase during tip-sample contact. From the creep behavior measured during the force clamp phase, quantitative viscoelastic sample properties are extracted. We validate FCFM on soft polyacrylamide gels. We find that the creep behavior of living cells conforms to a power-law material model. By recording short (50-60 ms) force clamp measurements in rapid succession, we generate, for the first time, two-dimensional maps of power-law exponent and modulus scaling parameter. Although these maps reveal large spatial variations of both parameters across the cell surface, we obtain robust mean values from the several hundreds of measurements performed on each cell. Measurements on mouse embryonic fibroblasts show that the mean power-law exponents and the mean modulus scaling parameters differ greatly among individual cells, but both parameters are highly correlated: stiffer cells consistently show a smaller power-law exponent. This correlation allows us to distinguish between wild-type cells and cells that lack vinculin, a dominant protein of the focal adhesion complex, even though the mean values of viscoelastic properties between wildtype and knockout cells did not differ significantly. Therefore, FCFM spatially resolves viscoelastic sample properties and can uncover subtle mechanical signatures of proteins in living cells.

Effect of sample slope on image formation in scanning ion conductance microscopy.

Scanning ion conductance microscopy (SICM) is a scanning probe technique that allows investigating surfaces of complex, convoluted samples such as living cells with minimal impairment. This technique monitors the ionic current through the small opening of an electrolyte-filled micro- or nanopipet that is approached toward a sample, submerged in an electrolyte. The conductance drops in a strongly distance-dependent manner. For SICM imaging, the assumption is made that positions of equal conductance changes correspond to equal tip-sample distances and thus can be utilized to reconstruct the sample surface. Here, we examined this assumption by investigating experimental approach curves toward silicone droplets, as well as finite element modeling of the imaging process. We found that the assumption is strictly true only for perpendicular approaches toward a horizontal sample and otherwise overestimates the sample height by up to several pipet opening radii. We developed a method to correct this overestimation and applied it to correct images of fixed cellular structures and living entire cells.

Bacterial interactions with proteins and cells relevant to the development of life-threatening endocarditis studied by use of a quartz-crystal microbalance.

Implant-related infections are a major challenge in clinical routine because of severe complications, for example infective endocarditis (IE). The purpose of this study was to investigate the real-time interaction of S. gordonii with proteins and cells important in the development of IE, in a flow system, by use of a quartz-crystal microbalance (QCM). Acoustic sensors were biologically modified by preconditioning with sterile saliva, platelet-poor plasma (PPP), or platelet-rich plasma (PRP), followed then by perfusion of a bacterial suspension. After perfusion, additional fluorescence and scanning electron microscopic (SEM) studies were performed. The surface structure of S. gordonii was analyzed by atomic force microscopy (AFM). Compared with S. gordonii adhesion on the abiotic sensor surface following normal mass loading indicated by a frequency decrease, adhesion on saliva, PPP, or PRP-conditioned sensors resulted in an increase in frequency. Furthermore, adhesion induced slightly increased damping signals for saliva and PPP-coated sensors but a decrease upon bacterial adhesion to PRP, indicating the formation of a more rigid biofilm. Microscopic analysis confirmed the formation of dense and vital bacterial layers and the aggregation of platelets and bacteria. In conclusion, our study shows that the complex patterns of QCM output data observed are strongly dependent on the biological substrate and adhesion mechanisms of S. gordonii. Overall, QCM sheds new light on the pathways of such severe infections as IE.

Measuring the Shape, Stiffness, and Interface Tension of Droplets with the Scanning Ion Conductance Microscope.

Imaging and probing liquid-liquid interfaces at the micro- and nanoscale are of high relevance, for example, in materials science, surface chemistry, and microfluidics. However, existing imaging techniques are limited in resolution, average over large sample areas, or interact with the sample. Here, we present a method to quantify the shape, stiffness, and interface tension of liquid droplets with the scanning ion conductance microscope (SICM), providing submicrometer resolution and the ability to perform noncontact mechanical measurements. We show that we can accurately image the three-dimensional shape of micrometer-sized liquid droplets made of, for example, decane, hexane, or different oils. We then introduce numerical models to quantitatively obtain their stiffness and interface tension from SICM data. We verified our method by measuring the interface tension of decane droplets changing under the influence of surfactants at different concentrations. Finally, we use SICM to resolve the dissolution dynamics of decane droplets, showing that droplet shape exhibits different dissolution modes and stiffness continuously increases while the interface tension remains constant. We thereby demonstrate that SICM is a useful method to investigate liquid-liquid interfaces on the microscale with applications in materials or life sciences.

Chorein sensitivity of actin polymerization, cell shape and mechanical stiffness of vascular endothelial cells.

BACKGROUND/AIMS: Endothelial cell stiffness plays a key role in endothelium-dependent control of vascular tone and arterial blood pressure. Actin polymerization and distribution of microfilaments is essential for mechanical cell stiffness. Chorein, a protein encoded by the VPS13A gene, defective in chorea-acanthocytosis (ChAc), is involved in neuronal cell survival as well as cortical actin polymerization of erythrocytes and blood platelets. Chorein is expressed in a wide variety of further cells, yet nothing is known about the impact of chorein on cells other than neurons, erythrocytes and platelets. The present study explored whether chorein is expressed in human umbilical vein endothelial cells (HUVECs) and addressed the putative role of chorein in the regulation of cytoskeletal architecture, stiffness and survival of those cells. METHODS: In HUVECs with or without silencing of the VPS13A gene, VPS13A mRNA expression was determined utilizing quantitative RT-PCR, cytoskeletal organization visualized by confocal microscopy, G/F actin ratio and phosphorylation status of focal adhesion kinase quantified by western blotting, cell death determined by flow cytometry, mechanical properties studied by atomic force microscopy (AFM) and cell morphology analysed by scanning ion conductance microscopy (SICM). RESULTS: VPS13A mRNA expression was detectable in HUVECs. Silencing of the VPS13A gene attenuated the filamentous actin network, decreased the ratio of soluble G-actin over filamentous F-actin, reduced cell stiffness and changed cell morphology as compared to HUVECs silenced with negative control siRNA. These effects were paralleled by a significant decrease in FAK phosphorylation following VPS13A silencing. Moreover, silencing of the VPS13A gene increased caspase 3 activity and induced necrosis in HUVECs. CONCLUSIONS: Chorein is a novel regulator of cytoskeletal architecture, cell shape, mechanical stiffness and survival of vascular endothelial cells.

Contour and persistence length of Corynebacterium diphtheriae pili by atomic force microscopy.

Many bacteria are characterized by nanoscale ultrastructures, for example S-layers, flagella, fimbriae, or pili. The last two are especially important for attachment to different abiotic and biotic surfaces and for host-pathogen interactions. In this study, we investigated the geometric and elastic properties of pili of different Corynebacterium diphtheriae strains by atomic force microscopy (AFM). We performed quantitative contour-length analysis of bacterial pili and found that the visible contour length of the pili can be described by a log-normal distribution. Our data revealed significant strain-specific variations in the mean visible contour length of the pili, ranging from 260 to 1,590 nm. To estimate their full contour length, which is not directly accessible from the AFM images, we developed a simple correction model. Using this model, we determined the mean full contour length as 510-2,060 nm. To obtain the persistence length we used two different methods of analysis, one based on the end-to-end distance of the pili and one based on the bending angles of short segments. In comparison, the bending angle analysis proved to be more precise and resulted in persistence lengths in the narrow range of 220-280 nm, with no significant strain-specific variations. This is small compared with some other bacterial polymers, for example type IV pili, F-pili, or flagella.

Mechanics of migrating platelets investigated with scanning ion conductance microscopy.

Platelets are small blood cells involved in hemostasis, wound healing, and immune response. After adhesion and spreading, platelets can migrate at sites of injury inducing an early immune response to inflammation or infection. Platelet migration requires fibrinogen-integrin binding and fibrinogen depletion from the substrate inducing a self-generated ligand gradient guiding the direction of migration. This type of cellular motion is referred to as haptotactic migration. The underlying mechanisms of haptotactic platelet migration have just recently been discovered, but the connection to platelet mechanics has remained unknown yet. Using scanning ion conductance microscopy (SICM), we investigated the three-dimensional morphology and mechanics of platelets during haptotactic migration for the first time. Migrating platelets showed a polarized, anisotropic shape oriented in the direction of migration. This polarization goes hand in hand with a characteristic subcellular stiffness distribution showing a region of increased stiffness at the leading edge. Moreover, the mechanical properties of the leading edge revealed a highly dynamic stiffening and softening process with rapid changes of the elastic modulus by a factor of up to 5× per minute. Inhibition of actin polymerization stopped the dynamic stiffening and softening process and halted the migration. By combining SICM with confocal fluorescence microscopy, we found that the increased stiffness and mechanical dynamics at the leading edge coincided with an increased volumetric F-actin density. Our data provide a connection between platelet mechanics and the cytoskeletal contribution to the migration process of platelets.

Homophilic Interaction Between Transmembrane-JAM-A and Soluble JAM-A Regulates Thrombo-Inflammation: Implications for Coronary Artery Disease.

Genetic predisposition through F11R-single-nucleotide variation (SNV) influences circulatory soluble junctional adhesion molecule-A (sJAM-A) levels in coronary artery disease (CAD) patients. Homozygous carriers of the minor alleles (F11R-SNVs rs2774276, rs790056) show enhanced levels of thrombo-inflammatory sJAM-A. Both F11R-SNVs and sJAM-A are associated with worse prognosis for recurrent myocardial infarction in CAD patients. Platelet surface-associated JAM-A correlate with platelet activation markers in CAD patients. Activated platelets shed transmembrane-JAM-A, generating proinflammatory sJAM-A and JAM-A-bearing microparticles. Platelet transmembrane-JAM-A and sJAM-A as homophilic interaction partners exaggerate thrombotic and thrombo-inflammatory platelet monocyte interactions. Therapeutic strategies interfering with this homophilic interface may regulate thrombotic and thrombo-inflammatory platelet response in cardiovascular pathologies where circulatory sJAM-A levels are elevated.

Comparison of scanning ion conductance microscopy with atomic force microscopy for cell imaging.

We present the first direct comparison of scanning ion conductance microscopy (SICM) with atomic force microscopy (AFM) for cell imaging. By imaging the same fibroblast or myoblast cell with both technologies in series, we highlight their advantages and disadvantages with respect to cell imaging. The finite imaging force applied to the sample in AFM imaging results in a coupling of mechanical sample properties into the measured sample topography. For soft samples such as cells this leads to artifacts in the measured topography and to elastic deformation, which we demonstrate by imaging whole fixed cells and cell extensions at high resolution. SICM imaging, on the other hand, has a noncontact character and can provide the true topography of soft samples at a comparable resolution.

Spatial correlation of cell stiffness and traction forces in cancer cells measured with combined SICM and TFM.

The mechanical properties of cancer cells at the single-cell and the subcellular level might be the key for answering long-standing questions in the diagnosis and treatment of cancer. However, the subcellular distribution of two main mechanical properties, cell stiffness and traction forces, has been investigated only rarely and qualitatively yet. Here, we present the first direct combination of scanning ion conductance microscopy (SICM) and traction force microscopy (TFM), which we used to identify a correlation between the local stiffness and the local traction force density in living cells. We found a correlation in normal breast epithelial cells, but no correlation in cancerous breast epithelial cells. This indicates that the interplay between cell stiffness and traction forces is altered in cancer cells as compared to healthy cells, which might give new insight in the research field of cancer cell mechanobiology.

The influence of Pyk2 on the mechanical properties in fibroblasts.

The cell surface receptor integrin is involved in signaling mechanical stresses via the focal adhesion complex (FAC) into the cell. Within FAC, the focal adhesion kinase (FAK) and Pyk2 are believed to act as important scaffolding proteins. Based on the knowledge that many signal transducing molecules are transiently immobilized within FAC connecting the cytoskeleton with integrins, we applied magnetic tweezer and atomic force microscopic measurements to determine the influence of FAK and Pyk2 in cells mechanically. Using mouse embryonic fibroblasts (MEF; FAK(+/+), FAK(-/-), and siRNA-Pyk2 treated FAK(-/-) cells) provided a unique opportunity to describe the function of FAK and Pyk2 in more detail and to define their influence on FAC and actin distribution.