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A F Horwitz

Publications and source records attributed to A F Horwitz.

At least 19 recordsLinked to original sources
PubMed2004

Spatial mapping of integrin interactions and dynamics during cell migration by image correlation microscopy.

Image correlation microscopy methodology was extended and used to determine retrospectively the density, dynamics and interactions of alpha5-integrin in migrating cells. Alpha5-integrin is present in submicroscopic clusters containing 3-4 integrins before it is discernibly organized. The integrin in nascent adhesions, as identified by the presence of paxillin, is approximately 1.4 times more concentrated, approximately 4.5 times more clustered and much less mobile than in surrounding regions. Thus, while integrins are clustered throughout the cell, they differ in nascent adhesions and appear to initiate adhesion formation, despite their lack of visible organization. In more mature adhesions where the integrin is visibly organized there are approximately 900 integrins microm(-2) (about fivefold higher than surrounding regions). Interestingly, alpha5-integrin and alpha-actinin, but not paxillin, reside in a complex throughout the cell, where they diffuse and flow together, even in regions where they are not organized. During adhesion disassembly some integrins diffuse away slowly, alpha-actinin undergoes a directed movement at speeds similar to actin retrograde flow (0.29 microm min(-1)), while all of the paxillin diffuses away rapidly.

Actinin↗
PubMed2001

The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL).

The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinDeltaLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin-PKL interaction. In cells overexpressing paxillinDeltaLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

ADP-Ribosylation Factors↗
PubMed2001

Differential dynamics of alpha 5 integrin, paxillin, and alpha-actinin during formation and disassembly of adhesions in migrating cells.

To investigate the mechanisms by which adhesions form and disperse in migrating cells, we expressed alpha 5 integrin, alpha-actinin, and paxillin as green fluorescent protein (GFP) fusions. All localized with their endogenous counterparts and did not perturb migration when expressed at moderate levels. alpha 5-GFP also rescued the adhesive defects in CHO B2 cells, which are alpha 5 integrin deficient. In ruffling cells, alpha 5-GFP and alpha-actinin--GFP localized prominently at the leading edge in membrane protrusions. Of the three GFP fusion proteins that we examined, paxillin was the first component to appear visibly organized in protrusive regions of the cell. When a new protrusion formed, the paxillin appeared to remodel from older to newer adhesions at the leading edge. alpha-Actinin subsequently entered adhesions, which translocated toward the cell center, and inhibited paxillin turnover. The new adhesions formed from small foci of alpha-actinin--GFP and paxillin-GFP, which grew in size. Subsequently, alpha 5 integrin entered the adhesions to form visible complexes, which served to stabilize the adhesions. alpha 5-GFP also resided in endocytic vesicles that emanated from the leading edge of protrusions. Integrin vesicles at the cell rear moved toward the cell body. As cells migrated, alpha 5 vesicles also moved from a perinuclear region to the base of the lamellipodium. The alpha 5 vesicles colocalized with transferrin receptor and FM 4-64 dye. After adhesions broke down in the rear, alpha 5-GFP was found in fibrous structures behind the cell, whereas alpha-actinin--GFP and paxillin-GFP moved up the lateral edge of retracting cells as organized structures and then dissipated.

Actinin↗
PubMed2000

The role of transient ERK2 signals in fibronectin- and insulin-mediated DNA synthesis.

Both the extracellular matrix and growth factors jointly regulate cell cycle progression via a complex network of signaling pathways. Applying quantitative assays and analysis, we demonstrate here that concurrent stimulation of Chinese hamster ovary (CHO) cells with fibronectin (Fn) and insulin elicits a DNA synthesis response that reveals a synergy far more complex than a simple additive enhancement of response magnitude. CHO cell adhesion to higher Fn density shifts the sensitivity of the DNA synthesis response to insulin concentration from smoothly graded to sharply 'switch-like' and dramatically decreases the insulin concentration required for half-maximal response by about 1000-fold. Conversely, treatment with insulin has a milder and less complex effect on the response to varying Fn concentrations. Governing this DNA synthesis response is a common requirement for a transient, cell area-independent extracellular signal-regulated kinase 2 (ERK2) signal. Moreover, we show that the time-integrated value of this 'pulse' signal provides an appropriate metric for quantifying the dependence of DNA synthesis on the degree of ERK2 activation. Indeed, in the absence of insulin, the adhesion-mediated response is linearly proportional to ERK2 activation over a broad range of stimulatory Fn and MEK inhibitor amounts. However, in the presence of both Fn and insulin, total integrated ERK2 activity (the sum of Fn- and insulin-mediated signals) no longer serves as a predictor of DNA synthesis, demonstrating that the signaling crosstalk underlying response synergism does not converge at ERK2 activation. Instead, adhesion to higher Fn density enhances insulin stimulation of DNA synthesis, not by increasing insulin-mediated ERK2 activation, but via parallel elevation of at least one other insulin-mediated signal such as IRS-1 phosphorylation.

Animals↗
PubMed1999

Quantitative relationship among integrin-ligand binding, adhesion, and signaling via focal adhesion kinase and extracellular signal-regulated kinase 2.

Because integrin-mediated signals are transferred through a physical architecture and synergistic biochemical network whose properties are not well defined, quantitative relationships between extracellular integrin-ligand binding events and key intracellular responses are poorly understood. We begin to address this by quantifying integrin-mediated FAK and ERK2 responses in CHO cells for varied alpha(5)beta(1) expression level and substratum fibronectin density. Plating cells on fibronectin-coated surfaces initiated a transient, biphasic ERK2 response, the magnitude and kinetics of which depended on integrin-ligand binding properties. Whereas ERK2 activity initially increased with a rate proportional to integrin-ligand bond number for low fibronectin density, the desensitization rate was independent of integrin and fibronectin amount but proportional to the ERK2 activity level with an exponential decay constant of 0.3 (+/- 0.08) min(-1). Unlike the ERK2 activation time course, FAK phosphorylation followed a superficially disparate time course. However, analysis of the early kinetics of the two signals revealed them to be correlated. The initial rates of FAK and ERK2 signal generation exhibited similar dependence on fibronectin surface density, with both rates monotonically increasing with fibronectin amount until saturating at high fibronectin density. Because of this similar initial rate dependence on integrin-ligand bond formation, the disparity in their time courses is attributed to differences in feedback regulation of these signals. Whereas FAK phosphorylation increased to a steady-state level as new integrin-ligand bond formation continued during cell spreading, ERK2 activity was decoupled from the integrin-ligand stimulus and decayed back to a basal level. Accordingly, we propose different functional metrics for representing these two disparate dynamic signals: the steady-state tyrosine phosphorylation level for FAK and the integral of the pulse response for ERK2. These measures of FAK and ERK2 activity were found to correlate with short term cell-substratum adhesivity, indicating that signaling via FAK and ERK2 is proportional to the number of integrin-fibronectin bonds.

Animals↗
PubMed1999

A rapid and sensitive quantitative kinase activity assay using a convenient 96-well format.

Activation of protein kinases in response to growth factor and extracellular matrix stimulation has been implicated in regulating a number of cell functions including differentiation, gene expression, migration, and proliferation. An improved quantitative assay for measuring protein kinase activity is crucial to the detailed study of this important category of signaling proteins and their role in regulating cell behavior. We describe a modified in vitro kinase activity assay that is both sensitive and quantitative. It offers several advantages when compared to the traditional immunoprecipitation/kinase assay: (i) high sensitivity that reduces the required amount of cell lysate by an order of magnitude, (ii) an immunoseparation technique utilizing antibody immobilization onto the surface of microtiter wells that replaces the cumbersome immunoprecipitation method, (iii) a 96-well plate configuration that eases handling of multiple samples and increases throughput of the assay, and (iv) the use of 96-well filter plates that greatly reduces radioactive liquid waste generation. While we implement this technique in a case study for measuring the activity of extracellular signal-regulated kinase 2 (ERK2), this assay can be extended to studying other protein kinases by using an appropriate antibody and in vitro substrate for the kinase of interest.

Animals↗
PubMed1999

Quantitative changes in integrin and focal adhesion signaling regulate myoblast cell cycle withdrawal.

We previously demonstrated contrasting roles for integrin alpha subunits and their cytoplasmic domains in controlling cell cycle withdrawal and the onset of terminal differentiation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A.F. Horwitz. 1996. J. Cell Biol. 133:169-184). Ectopic expression of the integrin alpha5 or alpha6A subunit in primary quail myoblasts either decreases or enhances the probability of cell cycle withdrawal, respectively. In this study, we addressed the mechanisms by which changes in integrin alpha subunit ratios regulate this decision. Ectopic expression of truncated alpha5 or alpha6A indicate that the alpha5 cytoplasmic domain is permissive for the proliferative pathway whereas the COOH-terminal 11 amino acids of alpha6A cytoplasmic domain inhibit proliferation and promote differentiation. The alpha5 and alpha6A cytoplasmic domains do not appear to initiate these signals directly, but instead regulate beta1 signaling. Ectopically expressed IL2R-alpha5 or IL2R-alpha6A have no detectable effect on the myoblast phenotype. However, ectopic expression of the beta1A integrin subunit or IL2R-beta1A, autonomously inhibits differentiation and maintains a proliferative state. Perturbing alpha5 or alpha6A ratios also significantly affects activation of beta1 integrin signaling pathways. Ectopic alpha5 expression enhances expression and activation of paxillin as well as mitogen-activated protein (MAP) kinase with little effect on focal adhesion kinase (FAK). In contrast, ectopic alpha6A expression suppresses FAK and MAP kinase activation with a lesser effect on paxillin. Ectopic expression of wild-type and mutant forms of FAK, paxillin, and MAP/erk kinase (MEK) confirm these correlations. These data demonstrate that (a) proliferative signaling (i.e., inhibition of cell cycle withdrawal and the onset of terminal differentiation) occurs through the beta1A subunit and is modulated by the alpha subunit cytoplasmic domains; (b) perturbing alpha subunit ratios alters paxillin expression and phosphorylation and FAK and MAP kinase activation; (c) quantitative changes in the level of adhesive signaling through integrins and focal adhesion components regulate the decision of myoblasts to withdraw from the cell cycle, in part via MAP kinase.

Animals↗
PubMed1998

Identification of a domain on the integrin alpha5 subunit implicated in cell spreading and signaling.

The alpha5 beta1 integrin is a cell surface receptor for fibronectin implicated in several cellular activities including cell proliferation, differentiation, and migration. The primary site at which the alpha5 beta1 integrin interacts with fibronectin is the RGD (Arg-Gly-Asp) amino acid sequence. In general, the sites on the integrin alpha subunits involved in ligand binding are not well characterized. Based on previous cross-linking studies, sequence alignment, predicted conformation, and intron-exon boundaries, we identified a 144-residue region (positions 223-367) on the alpha5 subunit as a putative binding region and divided it into four subdomains named domains I, II, III, and IV. Chimeric receptors were prepared in which sequences on the alpha5 subunit were exchanged with the corresponding sequences on the alpha6 subunit, which is specific for laminin and does not bind via an RGD sequence. The mutated human alpha5 integrin gene was transfected into CHO B2 cells, which are deficient in alpha5 expression. Only chimeras of domain III or IV express on the cell surface. Both of these chimeras decreased the adhesion, spreading, focal adhesion assembly, and migration on fibronectin. The adhesion of the chimeric receptors to fibronectin remained sensitive to the RGD peptide, and antibodies that inhibit interaction with the fibronectin synergy site and RGD loop remain inhibitory for the chimeras, indicating that our chimeras do not inhibit binding to either the RGD or synergy sites. Finally, the affinity of soluble fibronectin to cells via the alpha5 beta1 receptor decreased only about 3-fold. This decrease is substantially less than the observed effects on migration and spreading, which were not altered by changes in substrate concentration. Thus, the alteration in binding sites does not easily account for the changes in cell spreading and focal adhesion assembly. The tyrosine phosphorylation and focal adhesion assembly that are seen when cells expressing the wild type alpha5 receptor adhere to fibronectin were inhibited in cells expressing the chimeric receptors. Therefore, our results suggest that the chimeras of these domains likely interrupt alpha5-mediated conformational signaling.

Amino Acid Sequence↗
PubMed1998

Immunopurification of a sarcomeric junctional protein complex containing GAPDH.

We have isolated a monoclonal antibody, P4B2, which localizes to multiple anchorage junctions, namely, a subset of focal adhesions, the Z-disk of muscle, and neuromuscular junctions. Immunopurification of the antigen to this antibody from chicken brain tissue yielded a complex of three prominent proteins with mobilities of 36, 30, and 18 kDa. Amino acid sequencing of the purified proteins identified the 36-kDa protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The other two protein bands were heterogeneous, containing proteins found in the synaptic vesicle fusion core complex. Immunolocalization of P4B2 antigen in developing cultured muscle cells showed that the antigen is incorporated into Z-lines soon after the sarcomeric architecture was positive for alpha-actinin. Together, the data indicate the P4B2 antigen is part of a unique GAPDH-containing protein complex that may be involved in reinforcement of established cytoskeletal structures.

Amino Acid Sequence↗
PubMed1998

Integrin and cadherin synergy regulates contact inhibition of migration and motile activity.

Integrin receptors play a central role in cell migration through their roles as adhesive receptors for both other cells and extracellular matrix components. In this study, we demonstrate that integrin and cadherin receptors coordinately regulate contact-mediated inhibition of cell migration. In addition to promoting proliferation (Sastry, S., M. Lakonishok, D. Thomas, J. Muschler, and A. Horwitz. 1996. J. Cell Biol. 133:169-184), ectopic expression of the alpha5 integrin in cultures of primary quail myoblasts promotes a striking contact-mediated inhibition of cell migration. Myoblasts ectopically expressing alpha5 integrin (alpha5 myoblasts) move normally when not in contact, but upon contact, they show inhibition of migration and motile activity (i.e., extension and retraction of membrane protrusions). As a consequence, these cells tend to grow in aggregates and do not migrate to close a wound. This phenotype is also seen with ectopic expression of beta1 integrin, paxillin, or activated FAK (CD2 FAK) and therefore appears to result from enhanced integrin-mediated signaling. The contact inhibition observed in the alpha5 myoblasts is mediated by N-cadherin, whose expression is upregulated more than fivefold. Perturbation studies using low calcium conditions, antibody inhibition, and ectopic expression of wild-type and mutant N-cadherins all implicate N-cadherin in the contact inhibition of migration. Ectopic expression of N-cadherin also produces cells that show inhibited migration upon contact; however, they do not show suppressed motile activity, suggesting that integrins and cadherins coordinately regulate motile activity. These observations have potential importance to normal and pathologic processes during embryonic development and tumor metastasis.

Animals↗
PubMed1998

Physical and biochemical regulation of integrin release during rear detachment of migrating cells.

Cell migration can be considered as a repeated cycle of membrane protrusion and attachment, cytoskeletal contraction and rear detachment. At intermediate and high levels of cell-substratum adhesiveness, cell speed appears to be rate-limited by rear detachment, specifically by the disruption of cytoskeleton-adhesion receptor-extracellular matrix (ECM) linkages. Often, cytoskeletal linkages fracture to release integrin adhesion receptors from the cell. Cell-extracellular matrix bonds may also dissociate, allowing the integrins to remain with the cell. To investigate molecular mechanisms involved in fracturing these linkages and regulating cell speed, we have developed an experimental system to track integrins during the process of rear retraction in Chinese hamster ovary (CHO) cells. Integrin expression level was varied by transfecting CHO B2 cells, which express very little endogenous alpha5 integrin, with a plasmid containing human alpha5 integrin cDNA and sorting the cells into three populations with different alpha5 expression levels. Receptor/ligand affinity was varied using CHO cells transfected with either alphaIIbbeta3 or alphaIIbbeta3(beta1-2), a high affinity variant. alphaIIbbeta3(beta1-2) is activated to a higher affinity state with an anti-LIBS2 antibody. Fluorescent probes were conjugated to non-adhesion perturbing anti-integrin antibodies, which label integrins in CHO cells migrating on a matrix-coated glass coverslip. The rear retraction area was determined using phase contrast microscopy and integrins initially in this area were tracked by fluorescence microscopy and a cooled CCD camera. We find that rear retraction rate appears to limit cell speed at intermediate and high adhesiveness, but not at low adhesiveness. Upon rear retraction, the amount of integrin released from the cell increases as extracellular matrix concentration, receptor level and receptor-ligand affinity increase. In fact, integrin release is a constant function of cell-substratum adhesiveness and the number of cell-substratum bonds. In the adhesive regime where rear detachment limits the rate of cell migration, cell speed has an inverse relationship to the amount of integrin released at the rear of the cell. At high cell-substratum adhesiveness, calpain, a Ca2+-dependent protease, is also involved in release of cytoskeletal linkages during rear retraction. Inhibition of calpain results in decreased integrin release from the cell membrane, and consequently a decrease in cell speed, during migration. These observations suggest a model for rear retraction in which applied tension and calpain-mediated cytoskeletal linkage cleavage are required at high adhesiveness, but only applied tension is required at low adhesiveness.

Animals↗
PubMed1997

Regulation of cell migration by the calcium-dependent protease calpain.

Integrin receptors play an important role during cell migration by mediating linkages and transmitting forces between the extracellular matrix and the actin cytoskeleton. The mechanisms by which these linkages are regulated and released during migration are not well understood. We show here that cell-permeable inhibitors of the calcium-dependent protease calpain inhibit both beta1 and beta3 integrin-mediated cell migration. Calpain inhibition specifically stabilizes peripheral focal adhesions, increases adhesiveness, and decreases the rate of cell detachment. Furthermore, these inhibitors alter the fate of integrin receptors at the rear of the cell during migration. A Chinese hamster ovary cell line expressing low levels of calpain I also shows reduced migration rates with similar morphological changes, further implicating calpain in this process. Taken together, the data suggest that calpain inhibition modulates cell migration by stabilizing cytoskeletal linkages and decreasing the rate of retraction of the cell's rear. Inhibiting calpain-mediated proteolysis may therefore be a potential therapeutic approach to control pathological cell migration such as tumor metastasis.

Animals↗
PubMed1997

Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness.

Migration of cells in higher organisms is mediated by adhesion receptors, such as integrins, that link the cell to extracellular-matrix ligands, transmitting forces and signals necessary for locomotion. Whether cells will migrate or not on a given substratum, and also their speed, depends on several variables related to integrin-ligand interactions, including ligand levels, integrin levels, and integrin-ligand binding affinities. These and other factors affect the way molecular systems integrate to effect and regulate cell migration. Here we show that changes in cell migration speed resulting from three separate variables-substratum ligand level, cell integrin expression level, and integrin-ligand binding affinity-are all quantitatively predictable through the changes they cause in a single unifying parameter: short-term cell-substratum adhesion strength. This finding is consistent with predictions of a mathematical model for cell migration. The ligand concentration promoting maximum migration speed decreases reciprocally as integrin expression increases. Increases in integrin-ligand affinity similarly result in maximal migration at reciprocally lower ligand concentrations. The maximum speed attainable, however, remains unchanged as ligand concentration, integrin expression, or integrin-ligand affinity vary, suggesting that integrin coupling with intracellular motors remains unaltered.

Animals↗
PubMed1996

Mutations in the cytoplasmic domain of the integrin beta1 chain indicate a role for endocytosis factors in bacterial internalization.

Mutations that result in defective beta1-integrin focal adhesion formation were analyzed for effects on bacterial internalization. Mutations in the cytoplasmic domain of the beta1 chain that disrupt the sequence NPIY resulted in integrins deficient in bacterial uptake. Other mutations in the beta1 chain that reduced cytoskeletal association showed enhanced bacterial uptake. Replacement of the NPIY sequence of the beta1 subunit by the endocytosis internalization sequence PPGY resulted in integrin receptors highly proficient in bacterial internalization, yet severely defective in focal contact localization. Electron microscopy indicated that coated structures associated specifically with bacteria-binding beta1-integrins, with an apparent recruitment of coated pits from ventral cell surfaces to apical surfaces corresponding to nascent bacterial phagosomes. Clathrin inhibition studies indicated a role for the adaptor molecule AP2 as well as clathrin in integrin-mediated bacterial internalization. These results indicate that association of beta1-integrins with the cytoskeleton at focal contacts interferes with integrin-mediated bacterial internalization. Also, although actin polymerization is required for bacterial uptake, clathrin is probably involved in bacterial uptake promoted by beta-1-integrins.

Amino Acid Sequence↗