Isolation of the intermediate filament protein vimentin by chromatofocusing

A novel, simple and relatively rapid method is described for the isolation of the intermediate-sized filament protein vimentin from eye lens tissue. Chromatofocusing is applied as the sole purification step. The apparent isoelectric point of the protein in 6 M urea and at 22°C is 4.9. Electrophoretic mobility on one- and two-dimensional polyacrylamide gels, solubility in 6 M urea and amino acid composition were used for identification     

Molecular and biological characterization of Streptococcal SpyA-mediated ADP-ribosylation of intermediate filament protein vimentin

The Gram-positive bacterial pathogen Streptococcus pyogenes produces a C3 family ADP-ribosyltransferase designated SpyA (S. pyogenes ADP-ribosyltransferase). Our laboratory has identified a number of eukaryotic protein targets for SpyA, prominent among which are the cytoskeletal proteins actin and vimentin. Because vimentin is an unusual target for modification by bacterial ADP-ribosyltransferases, we quantitatively compared the activity of SpyA on vimentin and actin. Vimentin was the preferred substrate for SpyA (k(cat), 58.5 ± 3.4 min(-1)) relative to actin (k(cat), 10.1 ± 0.6 min(-1)), and vimentin was modified at a rate 9.48 ± 1.95-fold greater than actin. We employed tandem mass spectrometry analysis to identify sites of ADP-ribosylation on vimentin. The primary sites of modification were Arg-44 and -49 in the head domain, with several additional secondary sites identified. Because the primary sites are located in a domain of vimentin known to be important for the regulation of polymerization by phosphorylation, we investigated the effects of SpyA activity on vimentin polymerization, utilizing an in vitro NaCl-induced filamentation assay. SpyA inhibited vimentin filamentation, whereas a catalytic site mutant of SpyA had no effect. Additionally, we demonstrated that expression of SpyA in HeLa cells resulted in collapse of the vimentin cytoskeleton, whereas expression in RAW 264.7 cells impeded vimentin reorganization upon stimulation of this macrophage-like cell line with LPS. We conclude that SpyA modification of vimentin occurs in an important regulatory region of the head domain and has significant functional effects on vimentin assembly.     

Intracellular assembly and sorting of intermediate filament proteins: role of the 42 amino acid lamin insert

Nuclear and cytoplasmic intermediate filament (IF) proteins segregate into two independent cellular networks by mechanisms that are poorly understood. We examined the role of a 42 amino acid (aa) insert unique to vertebrate lamin rod domains in the coassembly of nuclear and cytoplasmic IF proteins by overexpressing chimeric IF proteins in human SW13+ and SW13- cells, which contain and lack endogenous cytoplasmic IF proteins, respectively. The chimeric IF proteins consisted of the rod domain of human nuclear lamin A/C protein fused to the amino and carboxyl-terminal domains of the mouse neurofilament light subunit (NF-L), which contained or lacked the 42 aa insert. Immunofluorescence microscopy was used to follow assembly and targeting of the proteins in cells. Chimeric proteins that lacked the 42 aa insert colocalized with vimentin, whereas those that contained the 42 aa insert did not. When overexpressed in SW13- cells, chimeric proteins containing the 42 aa formed very short or broken cytoplasmic filaments, whereas chimeric proteins that lacked the insert assembled efficiently into long, stable cytoplasmic filaments. To examine the roles of other structural motifs in intracellular targeting, we added two additional sequences to the chimera, a nuclear localization signal (NLS) and a CAAX motif, which are found in nuclear IF proteins. Addition of an NLS alone or an NLS in combination with the CAAX motif to the chimera with the 42 aa insert resulted in cagelike filament that assembled close to the nuclear envelope and nuclear lamina-like targeting, respectively. Our results suggest that the rod domains of eukaryotic nuclear and cytoplasmic IF proteins, which are related to each other, are still compatible upon deletion of the 42 aa insert of coassembly. In addition, NF-L end domains can substitute for the corresponding lamin domains in nuclear lamina targeting.     

Dissecting the 3-D structure of vimentin intermediate filaments by cryo-electron tomography

Vimentin polymerizes via complex lateral interactions of coiled-coil dimers into long, flexible filaments referred to as intermediate filaments (IFs). Intermediate in diameter between microtubules and microfilaments, IFs constitute the third cytoskeletal filament system of metazoan cells. Here we investigated the molecular basis of the 3-D architecture of vimentin IFs by cryo-electron microscopy (cryo-EM) as well as cryo-electron tomography (Cryo-ET) 3-D reconstruction. We demonstrate that vimentin filaments in cross-section exhibit predominantly a four-stranded protofibrilar organization with a right-handed supertwist with a helical pitch of about 96 nm. Compact filaments imaged by cryo-EM appear surprisingly straight and hence appear very stiff. In addition, IFs exhibited an increased flexibility at sites of partial unraveling. This is in strong contrast to chemically fixed, negatively stained preparations of vimentin filaments that generally exhibit smooth bending without untwisting. At some point along the filament unraveling may be triggered and propagates in a cooperative manner so that long stretches of filaments appear to have unraveled rapidly in a coordinated fashion.     

Exploring the mechanical properties of single vimentin intermediate filaments by atomic force microscopy

Intermediate filaments (IFs), together with actin filaments and microtubules, compose the cytoskeleton. Among other functions, IFs impart mechanical stability to cells when exposed to mechanical stress and act as a support when the other cytoskeletal filaments cannot keep the structural integrity of the cells. Here we present a study on the bending properties of single vimentin IFs in which we used an atomic force microscopy (AFM) tip to elastically deform single filaments hanging over a porous membrane. We obtained a value for the bending modulus of non-stabilized IFs between 300 MPa and 400 MPa. Our results together with previous ones suggest that IFs present axial sliding between their constitutive building blocks and therefore have a bending modulus that depends on the filament length. Measurements of glutaraldehyde-stabilized filaments were also performed to reduce the axial sliding between subunits and therefore provide a lower limit estimate of the Young's modulus of the filaments. The results show an increment of two to three times in the bending modulus for the stabilized IFs with respect to the non-stabilized ones, suggesting that the Young's modulus of vimentin IFs should be around 900 MPa or higher.     

Simultaneous labelling of microtubules and fibrillar bundles in tobacco BY-2 cells by the anti-intermediate filament antibody,ME 101

Summary In previous studies on plant cells, antibodies directed against intermediate filaments (IFs) have shown that IF antigens are distributed in one of two quite distinct forms. The first co-distributes with each of the four microtubule arrays (cortical, preprophase band, spindle and phragmoplast), while the second form is associated with cytoplasmic paracrystalline fibrillar bundles (FBs) of 10 nm filaments. Conditions allowing one form to be labelled with antibody have generally proved unsuitable for labelling of the other; this has prevented the simultaneous visualization of the two forms of IF antigen in plants and the study of any possible physical relationships between them at the electron microscopic level. In this paper, we show that ME 101, which recognizes an epitope in the N-terminal portion of all classes of intermediate filaments, stains both forms of plant IF antigen simultaneously in tobacco suspension cells using immunofluorescence or immunogold labelling techniques. These cells contain in their cortex short (ca. l m) fibrillar bundles which stain with ME 101. These bundles appear to be independent of the microtubule-associated epitope which stains in a continuous linear manner with ME 101. When protoplasts are either cleaved open on grids or sequentially extracted with detergents prior to critical point drying, the short fibrillar bundles are specifically labelled by ME 101 tagged with colloidal gold. ME 101 also co-distributed with underlying linear filaments, which appeared to be microtubules. In addition to these structures, the cortex also contains a meshwork of variably-sized fine filaments but these are not labelled with ME 101 nor with an antibody raised against the plant cytoskeleton, which recognizes cytokeratin 8. These results confirm that the fibrillar bundles and the microtubule-associated form of plant IF antigens are present simultaneously rather than experimentally-interconvertible, and that they appear to be physically unconnected.Abbreviations DAPI 4,6-diamidino-2-phenylindole - FB fibrillar bundle - FITC fluorescein isothiocyanate - IF intermediate filaments - MTSB microtubule stabilizing buffer - TBS Tris-buffered-saline     

Seeing is believing! The optical properties of the eye lens are dependent upon a functional intermediate filament cytoskeleton

Beaded filaments are the major cytoskeletal element of the eye lens and they are essential to the optical properties of the eye lens. They were discovered in 1972 by Harry Maisel and Margaret Perry and have since been found to comprise two novel intermediate filament proteins, CP49 and filensin. These proteins possess unique structure features and unusual assembly characteristics, which distinguish them from canonical IF proteins. Whilst CP49 is completely tailless, filensin has a rather short rod domain and extremely large C-terminal tail domain. In vitro, CP49 and filensin do not form IFs on their own. In vitro studies suggest that CP49 and filensin have a distinct coassembly mechanism. Whilst CP49 self-assembles into thick bundles of filaments, filensin only forms short fibrils, but when combined together they form filaments. The generation of gene knockouts by the targeted deletion of Bfsp1 and Bfsp2 that encode filensin and CP49, respectively, have been made to explore the function of beaded filaments in the lens. Our results suggest that the lens-specific beaded filaments are the key cytoskeletal element in organising and maintaining lens fibre cell architecture and are a key factor in determining the optical properties of the lens. We have also found that some common mouse strains contain a natural mutation in Bfsp2 that will effectively generate a CP49 knockout. This finding has important implications for lens research involving other gene knockouts maintained on a 129 background. It has also been observed that mutations in Bfsp2 are the genetic basis of inherited human cataract. Collectively, these data demonstrate that beaded filaments are fundamental to lens function.     

Vmac: a novel protein associated with vimentin-type intermediate filament in podocytes of rat kidney

Vimentin-type intermediate filaments (IFs) play an important role in cytoskeletal organization and cell morphology. We identified here a novel protein associated with vimentin-type IFs and named it vimentin-type IF-associated coiled-coil protein (Vmac). Vmac consists of 171 amino acids with a calculated Mr of 18,844 and has a coiled-coil domain in its N-terminal region and the PDZ-binding tetrapeptide consensus motif in its C-terminal region. Northern blotting showed that the Vmac mRNA was expressed in many rat tissues examined but most abundantly expressed in the kidney. Immunofluorescence microscopy revealed that Vmac was highly concentrated at podocytes of renal glomeruli. Podocytes are highly specialized epithelial cells characterized by a large cell body and numerous foot processes, and express vimentin-type IFs that are distributed in the cell body and the major processes. Immunoelectron microscopy revealed that Vmac was associated with vimentin-type IFs of podocytes. These results indicate that Vmac is a novel protein associated with vimentin-type IF in podocytes of rat kidney.     

A novel interaction of the Golgi complex with the vimentin intermediate filament cytoskeleton

The integration of the vimentin intermediate filament (IF) cytoskeleton and cellular organelles in vivo is an incompletely understood process, and the identities of proteins participating in such events are largely unknown. Here, we show that the Golgi complex interacts with the vimentin IF cytoskeleton, and that the Golgi protein formiminotransferase cyclodeaminase (FTCD) participates in this interaction. We show that the peripherally associated Golgi protein FTCD binds directly to vimentin subunits and to polymerized vimentin filaments in vivo and in vitro. Expression of FTCD in cultured cells results in the formation of extensive FTCD-containing fibers originating from the Golgi region, and is paralleled by a dramatic rearrangements of the vimentin IF cytoskeleton in a coordinate process in which vimentin filaments and FTCD integrate into chimeric fibers. Formation of the FTCD fibers is obligatorily coupled to vimentin assembly and does not occur in vim(-/-) cells. The FTCD-mediated regulation of vimentin IF is not a secondary effect of changes in the microtubule or the actin cytoskeletons, since those cytoskeletal systems appear unaffected by FTCD expression. The assembly of the FTCD/vimentin fibers causes a coordinate change in the structure of the Golgi complex and results in Golgi fragmentation into individual elements that are tethered to the FTCD/vimentin fibers. The observed interaction of Golgi elements with vimentin filaments and the ability of FTCD to specifically interacts with both Golgi membrane and vimentin filaments and promote their association suggest that FTCD might be a candidate protein integrating the Golgi compartment with the IF cytoskeleton.     

In vitro assembly properties of mutant and chimeric intermediate filament proteins: insight into the function of sequences in the rod and end domains of IF

The factors and mechanisms regulating assembly of intermediate filament (IF) proteins to produce filaments with their characteristic 10 nm diameter are not fully understood. All IF proteins contain a central rod domain flanked by variable head and tail domains. To elucidate the role that different domains of IF proteins play in filament assembly, we used negative staining and electron microscopy (EM) to study the in vitro assembly properties of purified bacterially expressed IF proteins, in which specific domains of the proteins were either mutated or swapped between a cytoplasmic (mouse neurofilament-light (NF-L) subunit) and nuclear intermediate filament protein (human lamin A). Our results indicate that filament formation is profoundly influenced by the composition of the assembly buffer. Wild type (wt) mouse NF-L formed 10 nm filaments in assembly buffer containing 175 mM NaCl, whereas a mutant deleted of 18 NH2-terminal amino acids failed to assemble under similar conditions. Instead, the mutant assembled efficiently in buffers containing CaCl2 > or = 6 mM forming filaments that were 10 times longer than those formed by wt NF-L, although their diameter was significantly smaller (6-7 nm). These results suggest that the 18 NH2-terminal sequence of NF-L might serve two functions, to inhibit filament elongation and to promote lateral association of NF-L subunits. We also demonstrate that lengthening of the NF-L rod domain, by inserting a 42 aa sequence unique to nuclear IF proteins, does not compromise filament assembly in any noticeable way. Our results suggests that the known inability of nuclear lamin proteins to assemble into 10 nm filaments in vitro cannot derive solely from their longer rod domain. Finally, we demonstrate that the head domain of lamin A can substitute for that of NF-L in filament assembly, whereas substitution of both the head and tail domains of lamins for those of NF-L compromises assembly. Therefore, the effect of lamin A "tail" domain alone, or the synergistic effect of lamin "head" and the "tail" domains together, interferes with assembly into 10-nm filaments.     

Simulating the formation of keratin filament networks by a piecewise-deterministic Markov process

Keratin intermediate filament networks are part of the cytoskeleton in epithelial cells. They were found to regulate viscoelastic properties and motility of cancer cells. Due to unique biochemical properties of keratin polymers, the knowledge of the mechanisms controlling keratin network formation is incomplete. A combination of deterministic and stochastic modeling techniques can be a valuable source of information since they can describe known mechanisms of network evolution while reflecting the uncertainty with respect to a variety of molecular events. We applied the concept of piecewise-deterministic Markov processes to the modeling of keratin network formation with high spatiotemporal resolution. The deterministic component describes the diffusion-driven evolution of a pool of soluble keratin filament precursors fueling various network formation processes. Instants of network formation events are determined by a stochastic point process on the time axis. A probability distribution controlled by model parameters exercises control over the frequency of different mechanisms of network formation to be triggered. Locations of the network formation events are assigned dependent on the spatial distribution of the soluble pool of filament precursors. Based on this modeling approach, simulation studies revealed that the architecture of keratin networks mostly depends on the balance between filament elongation and branching processes. The spatial distribution of network mesh size, which strongly influences the mechanical characteristics of filament networks, is modulated by lateral annealing processes. This mechanism which is a specific feature of intermediate filament networks appears to be a major and fast regulator of cell mechanics.     

Assessing the Cytoskeletal System and its Elements in C6 Glioma Cells and Astrocytes by Atomic Force Microscopy

Object To investigate how the characteristic structure of the cytoskeleton in glioma cells is associated with invasiveness. Methods The whole cytoskeletal system was characterized by atomic force microscopy (AFM), while single cytoskeletal elements were exhibited by AFM and using cytoskeletal protein inhibitors to inhibit microfilaments or/and microtubules and displayed by immunofluorescence microscopy. The fluorescence intensity of F-actin was measured by flow cytometry and the structural difference between C6 glioma cells and astrocytes was studied. Results Cytoskeletons in both cells presented network structures, however, the C6 glioma cells showed an irregular edge root and their microfilaments were creber and dense. Intermediate filaments were extensive network structure with non-polarized multipoint connections. The microtubules were relatively big and long and formed tight bundles with close connections between bundles. Astrocytes had a regular and smooth edge, with sparse microfilaments, while the intermediate filaments were dense and interwoven and the microtubules were dense bundled, but only loosely connected each other. Besides, the fluorescence intensity of F-actin was significantly higher in C6 glioma cells (202.54 ± 11.06) than in the astrocytes (62.64 ± 10.23), P < 0.01. Conclusion Whole cytoskeleton and its elements of C6 cells were disclosed of characteristic structures associated with invasiveness. Meanwhile, the content of F-actin could be used as a parameter for measuring cell invasiveness.     

Nanomechanics of lipid bilayers by force spectroscopy with AFM: A perspective

Lipid bilayers determine the architecture of cell membranes and regulate a myriad of distinct processes that are highly dependent on the lateral organization of the phospholipid molecules that compose the membrane. Indeed, the mechanochemical properties of the membrane are strongly correlated with the function of several membrane proteins, which demand a very specific, highly localized physicochemical environment to perform their function. Several mesoscopic techniques have been used in the past to investigate the mechanical properties of lipid membranes. However, they were restricted to the study of the ensemble properties of giant bilayers. Force spectroscopy with AFM has emerged as a powerful technique able to provide valuable insights into the nanomechanical properties of supported lipid membranes at the nanometer/nanonewton scale in a wide variety of systems. In particular, these measurements have allowed direct measurement of the molecular interactions arising between neighboring phospholipid molecules and between the lipid molecules and the surrounding solvent environment. The goal of this review is to illustrate how these novel experiments have provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Here we report in detail the main discoveries achieved by force spectroscopy with AFM on supported lipid bilayers, and we also discuss on the exciting future perspectives offered by this growing research field.     

Expression of fetal-type intermediate filaments by 17-day-old rat Sertoli cells cultured on reconstituted basement membrane

Summary The expression of cytokeratin- and vimentin-type intermediate filaments was studied by means of immunohistochemistry in Sertoli cells cultured on two types of reconstituted basement membrane in two-compartment culture chambers. In situ, the Sertoli cells of 17-day-old rats contained only vimentin intermediate filaments. During culture, a gradual reorganization of intermediate filaments accompanied by an increased cytokeratin immunoreactivity was observed. After 6 days, Sertoli cells contained both cytokeratin and vimentin, and the same cytokeratin type as in fetal and newborn testis was revealed by electrophoresis and immunoblotting. The present study shows that the isolation and culture of Sertoli cells causes, even in an improved culture system qualitative changes in the expression of intermediate filament proteins.     

Thiolutin inhibits endothelial cell adhesion by perturbing Hsp27 interactions with components of the actin and intermediate filament cytoskeleton

Thiolutin is a dithiole synthesized by Streptomyces sp. that inhibits endothelial cell adhesion and tumor growth. We show here that thiolutin potently inhibits developmental angiogenesis in zebrafish and vascular outgrowth from tissue explants in 3D cultures. Thiolutin is a potent and selective inhibitor of endothelial cell adhesion accompanied by rapid induction of HSPB1 (Hsp27) phosphorylation. The inhibitory effects of thiolutin on endothelial cell adhesion are transient, potentially due to a compensatory increase in Hsp27 protein levels. Accordingly, heat shock induction of Hsp27 limits the anti-adhesive activity of thiolutin. Thiolutin treatment results in loss of actin stress fibers, increased cortical actin as cells retract, and decreased cellular F-actin. Mass spectrometric analysis of Hsp27 binding partners following immunoaffinity purification identified several regulatory components of the actin cytoskeleton that associate with Hsp27 in a thiolutin-sensitive manner including several components of the Arp2/3 complex. Among these, ArpC1a is a direct binding partner of Hsp27. Thiolutin treatment induces peripheral localization of phosphorylated Hsp27 and Arp2/3. Hsp27 also associates with the intermediate filament components vimentin and nestin. Thiolutin treatment specifically ablates Hsp27 interaction with nestin and collapses nestin filaments. These results provide new mechanistic insights into regulation of cell adhesion and cytoskeletal dynamics by Hsp27.     

The intermediate filament protein vimentin binds specifically to a recombinant integrin alpha2/beta1 cytoplasmic tail complex and co-localizes with native alpha2/beta1 in endothelial cell focal adhesions

Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short alpha and beta cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin alpha2beta1 is a major collagen receptor but to date, only few proteins have been shown to interact with the alpha2 cytoplasmic tail or with the alpha2beta1 complex. In order to identify novel binding partners of a alpha2beta1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-alpha2 and GST-Jun alpha2 bound His-tagged calreticulin while GST-beta1 and GST-Fos beta1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun alpha2/GST-Fos beta1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with alphavbeta3-positive focal contacts. Here, we provide evidence that this interaction also occurs with alpha2beta1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen.     

Modulation of repulsive forces between neurofilaments by sidearm phosphorylation

Recent studies have advanced the notion that the axonal organization of neurofilaments (NFs) is based on mutual steric repulsion between the unstructured "sidearm" domains of adjacent NFs. Here, we present experimental evidence that these repulsive forces are modulated by the degree of sidearm phosphorylation. When NFs are sedimented into a gelatinous pellet, pellet volume falls with increasing ionic strength and enzymatic dephosphorylation; sedimentation of phosphorylated NFs in the presence of divalent cations also dramatically reduces pellet volume. Further, atomic force microscopy imaging of isolated mammalian NFs reveals robust exclusion of colloidal particles from the NF backbone that is reduced at high ionic strength and attenuated when the filaments are enzymatically dephosphorylated. Phosphate-phosphate repulsion on the NF sidearm appears to modulate NF excluded volume in a graded fashion, thereby controlling axonal NF organization through interfilament forces.     

Most genes encoding cytoplasmic intermediate filament (IF) proteins of the nematode Caenorhabditis elegans are required in late embryogenesis

Intestinal cells of C. elegans show an unexpectedly high complexity of cytoplasmic intermediate filament (IF) proteins. Of the 11 known IF genes six are coexpressed in the intestine, i.e. genes B2, C1, C2, D1, D2, and E1. Specific antibodies and GFP-promoter constructs show that genes B2, D1, D2, and E1 are exclusively expressed in intestinal cells. Using RNA interference (RNAi) by microinjection at 25 degrees C rather than at 20 degrees C we observe for the first time lethal phenotypes for C1 and D2. RNAi at 25 degrees C also shows that the known A1 phenotype occurs already in the late embryo after microinjection and is also observed by feeding which was not the case at 20 degrees C. Thus, RNAi at 25 degrees C may also be useful for the future analysis of other nematode genes. Finally, we show that triple RNAi at 20 degrees C is necessary for the combinations B2, D1, E1 and B2, D1, D2 to obtain a phenotype. Together with earlier results on genes A1, A2, A3, B1, and C2 RNAi phenotypes are now established for all 11IF genes except for the A4 gene. RNAi phenotypes except for A2 (early larval lethality) and C2 (adult phenotype) relate to the late embryo. We conclude that in C. elegans cytoplasmic IFs are required for tissue integrity including late embryonic stages. This is in strong contrast to the mouse, where ablation of IF genes apparently does not affect the embryo proper.     

The identification and localization of two intermediate filament proteins in the tunic of Styela plicata (Tunicata, Styelidae)

The intermediate filament (IF) proteins Styela C and Styela D from the tunicate Styela (Urochordata) are co-expressed in all epidermal cells and they are thought to behave as type I and type II keratins. These two IF proteins, Styela C and Styela D, were identified in immunoblots of proteins isolated from the tunic of Styela plicata. The occurrence and distribution of these proteins within the tunic of this ascidian was examined by means of immunofluorescence and immunoperoxidase techniques, using anti-Styela C and anti-Styela D antibodies. In addition, immuno-electron microscopy of the tunic showed that the two proteins are located in the cuticle layer and in the tunic matrix. These results represent the first data about the presence of IF proteins in the tunic of adult ascidian S. plicata. The possible involvement of these IF proteins in reinforcing the integrity of the tunic, that represents the interface between the animal body and the external environment, is discussed.     

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.