Cell adhesion profiling using extracellular matrix protein microarrays

We have developed a microarray-based system for cell adhesion profiling of large panels of cell-adhesive proteins to increase the throughput of in vitro cell adhesion assays, which are currently primarily performed in multiwell plates. Miniaturizing cell adhesion assays to an array format required the development of protocols for the reproducible microspotting of extracellular matrix (ECM) protein solutions and for the handling of cell suspensions during the assay. We generated ECM protein microarrays with high reproducibility in microspot protein content using nitrocellulose-coated glass microslides, combined with piezoelectric microspotting of protein solutions. Protocols were developed that allowed us to use 5000 cells or fewer on an array of 4 x 4 mm consisting of 64 microspots. Using this microarray system, we identified differences of adhesive properties of three cell lines to 14 different ECM proteins. Furthermore, the sensitivity and accuracy of the assays were increased using microarrays with ranges of ECM protein amounts. This microarray system will be particularly useful for extensive comparative cell adhesion profiling studies when only low amounts of adhesive substrate and cells, such as stem cells or cells from biopsies, are available.     

Biochemical and mechanical extracellular matrix properties dictate mammary epithelial cell motility and assembly

Biochemical and mechanical cues of the extracellular matrix have been shown to play important roles in cell-matrix and cell-cell interactions. We have experimentally tested the combined influence of these cues to better understand cell motility, force generation, cell-cell interaction, and assembly in an in vitro breast cancer model. MCF-10A non-tumorigenic mammary epithelial cells were observed on surfaces with varying fibronectin ligand concentration and polyacrylamide gel rigidity. Our data show that cell velocity is biphasic in both matrix rigidity and adhesiveness. The maximum cell migration velocity occurs only at specific combination of substrate stiffness and ligand density. We found cell-cell interactions reduce migration velocity. However, the traction forces cells exert onto the substrate increase linearly with both cues, with cells in pairs exerting higher maximum tractions observed over single cells. A relationship between force and motility shows a maximum in single cell velocity not observed in cell pairs. Cell-cell adhesion becomes strongly favored on softer gels with elasticity ≤ 1250 Pascals (Pa), implying the existence of a compliance threshold that promotes cell-cell over cell-matrix adhesion. Finally on gels with stiffness similar to pre-malignant breast tissue, 400 Pa, cells undergo multicellular assembly and division into 3D spherical aggregates on a 2D surface.     

Cell aggregation and neurite growth in gels of extracellular matrix molecules

Components of the extracellular matrix are believed to guide both nerve cells and neurites to their targets during embryogenesis and, therefore, might be useful for controlling regeneration of nervous tissue in adults. To study the influence of extracellular conditions on neurite outgrowth and cell motility, PC12 cells were suspended in three-dimensional gels containing (i) collagen (0.4 to 2 mg/mL), (ii) collagen (1 mg/mL) with added fibronectin or laminin (1 to 100 mug/mL), and (iii) agarose (7 mg/mL) with added collagen (0.001 to 1 mg/mL). Neurite outgrwoth was stimulated with nerve growth factor (NGF) and both the extent of neurite outgrowth ad cell aggregation were quantitated over 10 to 12 days in culture. The extent of neurite outgrowth was greatest at the lowest collagen concentration tested (0.4 mg/mL) and decreased with increasing concentration. The addition of laminin or fibronectin altered the extent of neurite outgrowth in collagen gels, but the differences were small. Although no neurite growth was observed in pure agarose gels, considerable neurite outgrowth occurred with the addition of small amounts (>/=0.01 mg/mL) of collagen. Mean aggregate size increased more quickly in gels with lower concentrations of collagen. For cells in 1.0 mg/mL collagen, a four- to fivefold increase in aggregate volume was seen between days 2 and 10 o the culture period, whereas the increase in DNA content during this same period was less than twofold, suggesting that the cells were aggregating, not multiplying. These results suggest that the composition of the matrix supporting nerve cells has a significant effect on both neurite outgrowth and cell motility. (c) 1994 John Wiley & Sons, Inc.     

Lymphocyte development of adherence and motility in extracellular matrix during IL-2 stimulation.

To extravasate into normal and neoplastic tissue, lymphocytes must migrate through the subendothelial basement membrane and underlying interstitium, structures rich in extracellular matrix (ECM). We have performed a time-course study of the development of motility in ECM by murine lymphocytes during in vitro exposure to high titers of IL-2 (1000 Cetus units/ml). This protocol generates immunotherapeutic lymphocyte populations expressing lymphokine-activated killer activity. Spontaneous motility was measured in three-dimensional gels of type I (interstitial) collagen or Matrigel, a model basement membrane. A newly developed assay permitted not only the measurement of distance traveled by the leading cell front, but also the separation of lymphocytes on the basis of three types of behavior. The motile fraction consisted of lymphocytes that penetrated beneath the ECM gel surface during an 18-h migration period. There were also two nonmotile fractions: the nonadherent fraction, which failed to bind to the gel surface; and the adherent fraction, which bound but did not penetrate during the assay period. During a 3- to 5-day exposure to high titer IL-2, both adherence and motility increased significantly. In type I collagen, cells of the NK lineage developed greater surface adherence and less motility than cells of the T lineage. The surface-adherent fraction expressed higher lymphokine-activated killer and NK activity than did the nonadherent or motile fractions. Under prolonged IL-2 stimulation (7 to 12 days), there was a decline in the percentage of cells exhibiting motility in both types of ECM, and an increase in the percentage of surface-adherent cells. The findings indicate that the behavior of an IL-2-stimulated lymphocyte population in ECM is profoundly influenced by the duration of IL-2 exposure. Furthermore, lack of lymphocyte motility may reflect two different behaviors, nonadherence and adherence without motility. The nonadherent and surface-adherent populations may differ in phenotypic distribution and function. The motility system described in this report will be useful in separating and studying the mechanisms that produce lymphocyte adherence and motility, and in understanding the in vivo implications of these behaviors.     

Cell adhesion to extracellular matrix regulates the life cycle of integrins.

The expression of alpha 5 beta 1 integrin on the surface of fibroblasts requires adhesion to substratum. We have examined the basis for this adhesion-dependent surface expression by comparing the life cycle of integrins in parallel cultures of adherent and nonadherent cells. Results of biosynthetic labeling experiments in NRK fibroblasts showed that the synthesis and biosynthetic processing of the beta 1 integrin subunit proceed in the absence of cell attachment; however, when examining the behavior of preexisting cell surface integrins, we observed that the alpha beta 1 integrins are internalized and degraded when adhesion to substratum is blocked. A kinetic analysis of integrin internalization in cycloheximide-treated NRK cells showed that each of the fibroblast integrins we examined (in both the beta 1 and beta 3 families) are lost from the cell surface after detachment from substratum. Thus, the default integrin life cycle in fibroblasts involves continuous synthesis, processing, transport to the cell surface, and internalization/degradation. Interestingly, studies with NIH-3T3 cells expressing alpha 1 beta 1 integrin showed that the loss of cell-surface alpha 5 beta 1 integrin is blocked by adhesion of cells to dishes coated with type IV collagen (a ligand for alpha 1 beta 1 integrin) as well as fibronectin. Similarly, adhesion of these cells to dishes coated with type IV collagen stabilizes the surface expression of alpha 5 beta 1 as well as alpha 1 beta 1 integrin. We propose that the adhesion of fibroblasts to extracellular matrix protein alters the integrin life cycle and permits retention of these proteins at the cell surface where they can play important roles in transmitting adhesion-dependent signals.     

Neutrophil motility in extracellular matrix gels: mesh size and adhesion affect speed of migration.

Polymorphonuclear leukocyte (PMN) migration through tissue extracellular space is an essential step in the inflammatory response, but little is known about the factors influencing PMN migration through gels of extracellular matrix (ECM). In this study, PMN migration within reconstituted gels containing collagen type I or collagen type I supplemented with laminin, fibronectin, or heparin was measured by quantitative direct visualization, resulting in a random motility coefficient (mum a quantitative index for rate of cell dispersion) for the migrating cell population. The random motility coefficient in unsupplemented collagen (0.4 mg/ml) gels was approximately 9 x 10(-9) cm2/s. Supplementing gels with heparin or fibronectin produced a significant decrease in mu, even at the lowest concentrations studied (1 microgram/ml fibronectin or 0.4 microgram/ml heparin). At least 100 micrograms/ml of laminin, or 20% of the total gel protein, was required to produce a similar decrease in mu. Scanning electron microscopy revealed two different gel morphologies: laminin or fibronectin appeared to coat the 150-nm collagen fibers whereas heparin appeared to induce fiber bundle formation and, therefore, larger interstitial spaces. The decrease in mu observed in heparin-supplemented gels correlated with the increased mesh size of the fiber network, but the difference observed in mu for fibronectin- and laminin-supplemented gels did not correlate with either mesh size or the mechanical properties of the gel, as determined by rheological measurements. However, PMNs adhered to fibronectin-coated surfaces in greater numbers than to collagen- or laminin-coated surfaces, suggesting that changes in cell adhesion to protein fibers can also produce significant changes in cell motility within an ECM gel.     

Cell motility assays

This report summarises practical aspects to measuring cell motility in culture. The methods described here were discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop organised by John Masters and Gareth E Jones that was held at University College London on 19th April 2007.     

An extracellular matrix, calmodulin-binding protein from Dictyostelium with EGF-like repeats that enhance cell motility

CyrA is a novel cysteine-rich protein with four EGFL repeats that was isolated using the calmodulin (CaM) binding overlay technique (CaMBOT), suggesting it is a CaM-binding protein (CaMBP). The full-length 63 kDa cyrA is cleaved into two major C-terminal fragments, cyrA-C45 and cyrA-C40. A putative CaM-binding domain was detected and both CaM-agarose binding and CaM immunoprecipitation verified that cyrA-C45 and cyrA-C40 each bind to CaM in both a Ca²⁺-dependent and -independent manner. cyrA-C45 was present continuously throughout growth and development but was secreted at high levels during the multicellular slug stage of Dictyostelium development. At this time, cyrA localizes to the extracellular matrix (ECM). ECM purification verified the presence of cyrA-C45. An 18 amino acid peptide (DdEGFL1) from the first EGFL repeat sequence of cyrA (EGFL1) that is present in both cyrA-C45 and -C40 enhances both random cell motility and cAMP-mediated chemotaxis. Here we reveal that the dose-dependent enhancement of motility by DdEGFL1 is related to the time of cell starvation. Addition of DdEGFL1 also inhibits cyrA proteolysis. The status of cyrA as an extracellular CaMBP was further clarified by the demonstration that CaM is secreted during development. Antagonism of CaM with W7 resulted in enhanced cyrA proteolysis suggesting a functional role for extracellular CaM in protecting CaMBPs from proteolysis. cyrA is the first extracellular CaMBP identified in Dictyostelium and since it is an ECM protein with EGF-like repeats that enhance cell motility and it likely also represents the first matricellular protein identified in a lower eukaryote.     

Brain extracellular matrix

The extracellular matrix of the adult brain tissue has a uniquecomposition. The striking feature of this matrix is the prominenceof lecticans, proteoglycans that contain a lectin domain anda hyaluronic acid-binding domain. Hyaluronic acid and tenascinfamily adhesive/anti-adhesive proteins are also abundant. Matrixproteins common in other tissues are nearly absent in adultbrain. The brain extracellular matrix appears to have trophiceffects on neuronal cells and affect neurite outgrowth. Theunique composition of this matrix may be responsible for theresistance of brain tissue toward invasion by tumors of non-neuronalorigin. extracellular matrix lectican versican review     

Cell response to RGD density in cross-linked artificial extracellular matrix protein films

This study examines the adhesion, spreading, and migration of human umbilical vein endothelial cells on cross-linked films of artificial extracellular matrix (aECM) proteins. The aECM proteins described here were designed for application in small-diameter grafts and are composed of elastin-like structural repeats and fibronectin cell-binding domains. aECM-RGD contains the RGD sequence derived from fibronectin; the negative control protein aECM-RDG contains a scrambled cell-binding domain. The covalent attachment of poly(ethylene glycol) (PEG) to aECM substrates reduced nonspecific cell adhesion to aECM-RDG-PEG but did not preclude sequence-specific adhesion of endothelial cells to aECM-RGD-PEG. Variation in ligand density was accomplished by the mixing of aECM-RGD-PEG and aECM-RDG-PEG prior to cross-linking. Increasing the density of RGD domains in cross-linked films resulted in more robust cell adhesion and spreading but did not affect cell migration speed. Control of cell-binding domain density in aECM proteins can thus be used to modulate cell adhesion and spreading and will serve as an important design tool as these materials are further developed for use in surgery, tissue engineering, and regenerative medicine.     

Cell motility and the formation of lamellipodia

A report on work presented at the 39th Annual Meeting of the American Society for Cell Bilogy, Washington DC, December 11-15, 1999