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We present here a novel form of dynamic adhesion in which both the integrin receptor and the ligand supporting dynamic adhesion have been identified. Laminar flow assays showed that laminin supported attachment of alpha 6 beta 4-positive cells in the presence of fluid shear stress (tau < or = 2 dyn/cm2), indicating that these cells adhered to laminin within a fraction of a second. Further increases in flow rate (3.5 dyn/cm2 < or = tau < or = 100 dyn/cm2) initiated rolling of attached cells in the direction of flow, suggesting that rapidly formed adhesion is reversible and repeatable. Laminin fragment E8, which interacts with alpha 6 integrins, supported dynamic attachment and rolling but extracellular matrix glycoprotein fibronectin did not. In cell lines that express alpha 6 beta 4 but not alpha 6 beta 1 an anti-alpha 6 monoclonal antibody inhibited attachment to laminin in the presence of flow and following 5 minutes of static incubation. Infusion of this antibody onto cells adherent to laminin-coated slides led to rapid detachment of cells from the substratum. An anti-beta 1 monoclonal antibody diminished adhesion strength following static incubation but did not inhibit rapid attachment and flow-initiated rolling. These results indicate that in some alpha 6 beta 4-expressing epithelial and carcinoma cell lines, integrin alpha 6 beta 4 mediates rapidly formed dynamic interactions with laminin.

The Drosophila Dachshund (Dac) gene, cloned as a dominant inhibitor of the hyperactive growth factor mutant, ellipse, encodes a key component of the Retinal Determination Gene Network (RDGN) that governs cell fate. DACH1 (Dachshund homolog 1) regulates gene expression either indirectly as a co-integrator or through direct DNA binding. CAST identified a DACH1 DNA-binding sequence which resembles the FOX (Forkhead Box containing protein) binding site. Genome-wide in-silico promoter analysis of DACH1 binding sites identified gene clusters populating cellular pathways associated with the cell cycle and growth factor signaling. ChIP-Seq mapped DACH1 binding sites to corresponding gene clusters predicted in silico and identified as weight matrix resembling the CAST-defined sequence. DACH1 antagonized FOXM1 target gene expression, promoter occupancy in the context of local chromatin, and contact-independent growth. Further studies were conducted to understand, at a higher level of resolution, the mechanisms governing DACH1-mediated transcriptional repression via DNA-sequence specific binding. DACH1 repressed, while FOX proteins enhanced, both DRE and FOXA responsive element-driven gene expression. Reduced DACH1 expression using a shRNA approach enhanced FOX protein activity. As DACH1 antagonized FOX target gene expression and attenuated FOX signaling, we sought to identify limiting co-integrator proteins governing DACH1 signaling. Proteomic analysis identified transcription elongation regulator 1 (TCERG1) as the transcriptional co-regulator of DACH1 activity. The FF2 domain of TCERG1 was required for DACH1 binding, and the deletion of FF2 abolished DACH1 trans-repression function. The carboxyl terminus of DACH1 was necessary and sufficient for TCERG1 binding. Thus, DACH1 represses gene transcription through direct DNA binding to the promoter region of target genes by recruiting the transcriptional co-regulator, TCERG1. Attenuation of FOX function by the cell fate determination pathway has broad implications given the diverse role of FOX proteins in cellular biology and tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 269. doi:10.1158/1538-7445.AM2011-269

At the onset of inflammation, 20–80% of all leukocytes passing postcapillary venules roll along the endothelium. Recent blocking experiments with antibodies and soluble adhesion receptor molecules, as well as in vitro reconstitution experiments, suggest that leukocyte rolling is mediated by adhesion molecules that belong to the selectin family. What differentiates a selectin-counterreceptor interaction that leads to leukocyte rolling from others that mediate firm adhesion after static incubation but no adhesion when incubated under flow conditions? Here, we explore this question by introducing a quantitative biophysical model that is compatible with the laws of mechanics as applied to rolling leukocytes and the present biochemical and biophysical data on selectin mediated interactions. Our computational experiments point to an adhesion mechanism in which the rate of bond formation is high and the detachment rate low, except at the rear of the contact area where the stretched bonds detach at a high uniform rate. The bond length and bond flexibility play a critical role in enhancing leukocyte rolling at a wide range of fluid shear rates.

Cell adhesion plays a fundamental role in the organization of cells in differentiated organs, cell motility, and immune response. A novel micromanipulation method is employed to quantify the direct contribution of surface adhesion receptors to the physical strength of cell adhesion. In this technique, a cell is brought into contact with a glass-supported planar membrane reconstituted with a known concentration of a given type of adhesion molecules. After a period of incubation (5-10 min), the cell is detached from the planar bilayer by pulling away the pipette holding the cell in the direction perpendicular to the glass-supported planar bilayer. In particular, we investigated the adhesion between a Jurkat cell expressing CD2 and a glass-supported planar bilayer containing either the glycosyl-phosphatidylinositol (GPI) or the transmembrane (TM) isoform of the counter-receptor lymphocyte function-associated antigen 3 (LFA-3) at a concentration of 1,000 molecules/microns 2. In response to the pipette force the Jurkat cells that adhered to the planar bilayer containing the GPI isoform of LFA-3 underwent extensive elongation. When the contact radius was reduced by approximately 50%, the cell then detached quickly from its substrate. The aspiration pressure required to detach a Jurkat cell from its substrate was comparable to that required to detach a cytotoxic T cell from its target cell. Jurkat cells that had been separated from the substrate again adhered strongly to the planar bilayer when brought to proximity by micromanipulation. In experiments using the planar bilayer containing the TM isoform of LFA-3, Jurkat cells detached with little resistance to micromanipulation and without changing their round shape.

Background Gene expression is controlled over a wide range at the transcript level through complex interplay between DNA and regulatory proteins, resulting in profiles of gene expression that can be represented as normal, graded, and bimodal (switch-like) distributions. We have previously performed genome-scale identification and annotation of genes with switch-like expression at the transcript level in mouse, using large microarray datasets for healthy tissue, in order to study the cellular pathways and regulatory mechanisms involving this class of genes. We showed that a large population of bimodal mouse genes encoding for cell membrane and extracellular matrix proteins is involved in communication pathways. This study expands on previous results by annotating human bimodal genes, investigating their correspondence to bimodality in mouse orthologs and exploring possible regulatory mechanisms that contribute to bimodality in gene expression in human and mouse. Results Fourteen percent of the human genes on the HGU133A array (1847 out of 13076) were identified as bimodal or switch-like. More than 40% were found to have bimodal mouse orthologs. KEGG pathways enriched for bimodal genes included ECM-receptor interaction, focal adhesion, and tight junction, showing strong similarity to the results obtained in mouse. Tissue-specific modes of expression of bimodal genes among brain, heart, and skeletal muscle were common between human and mouse. Promoter analysis revealed a higher than average number of transcription start sites per gene within the set of bimodal genes. Moreover, the bimodal gene set had differentially methylated histones compared to the set of the remaining genes in the genome. Conclusion The fact that bimodal genes were enriched within the cell membrane and extracellular environment make these genes as candidates for biomarkers for tissue specificity. The commonality of the important roles bimodal genes play in tissue differentiation in both the human and mouse indicates the potential value of mouse data in providing context for human tissue studies. The regulation motifs enriched in the bimodal gene set (TATA boxes, alternative promoters, methlyation) have known associations with complex diseases, such as cancer, providing further potential for the use of bimodal genes in studying the molecular basis of disease.

Background Recent studies have placed gene expression in the context of distribution profiles including housekeeping, graded, and bimodal (switch-like). Single-gene studies have shown bimodal expression results from healthy cell signaling and complex diseases such as cancer, however developing a comprehensive list of human bimodal genes has remained a major challenge due to inherent noise in human microarray data. This study presents a two-component mixture analysis of mouse gene expression data for genes on the Affymetrix MG-U74Av2 array for the detection and annotation of switch-like genes. Two-component normal mixtures were fit to the data to identify bimodal genes and their potential roles in cell signaling and disease progression. Results Seventeen percent of the genes on the MG-U74Av2 array (1519 out of 9091) were identified as bimodal or switch-like. KEGG pathways significantly enriched for bimodal genes included ECM-receptor interaction, cell communication, and focal adhesion. Similarly, the GO biological process "cell adhesion" and cellular component "extracellular matrix" were significantly enriched. Switch-like genes were found to be associated with such diseases as congestive heart failure, Alzheimer's disease, arteriosclerosis, breast neoplasms, hypertension, myocardial infarction, obesity, rheumatoid arthritis, and type I and type II diabetes. In diabetes alone, over two hundred bimodal genes were in a different mode of expression compared to normal tissue. Conclusion This research identified and annotated bimodal or switch-like genes in the mouse genome using a large collection of microarray data. Genes with bimodal expression were enriched within the cell membrane and extracellular environment. Hundreds of bimodal genes demonstrated alternate modes of expression in diabetic muscle, pancreas, liver, heart, and adipose tissue. Bimodal genes comprise a candidate set of biomarkers for a large number of disease states because their expressions are tightly regulated at the transcription level.

Cell-cell adhesion plays a fundamental role in tissue and organ development, cell mediated immunity and blood flow. In the present study a micro-mechanical model of specific adhesion is presented. Analytical expressions are derived for the adhesive energy density (Γ) at zero speed of peeling for the cases of immobile (trapped) as well as laterally mobile bonds. It is shown that Γ increases in both cases with the increasing density of bonds and with the binding of affinity of unstressed bonds. In the case of laterally mobile bonds Γ also increases with the extent of peeling. The analytical results are shown to be valid whether or not one takes into account of the bending stiffness of adhering membranes. It is also shown that Γ does not depend on the functional form of bond elasticity. The effect of the speed of peeling on the number density distribution of attached bonds is considered next. Numerical solutions for the energy required to separate conjugated cell pairs are presented. The theoretical predictions are then used to analyze experimental data on red cell aggregation and adhesion between a cytotoxic-T cell and its target cell. The results show that the binding affinity of unstressed bonds and their number density before conjugation can be obtained from data on slow peeling of cell-pairs. The information on the diffusivity of bonds, their stiffness and their rates of attachment and detachment are more difficult to obtain, requiring a set of experiments with increasing rates of separation (conjugation) of cell-pairs.

The boundary integral equation method is a numerical technique extensively applied in the solution of boundary value problems from many different engineering fields. The starting point of the method is the formulation of an integral equation which gives the variable at any point in terms of single and double layer potentials whose densities are the values of the variable and its derivatives on the boundary. The method consists of the numerical solution of this integral equation when the field point is taken to lie on the boundary. The present paper extends the formulation of the method to Stokes flows of a collection of particles in infinite circular cylinders. This is achieved by developing matrix Green's functions for Stokes flows in cylindrical boundaries. The results are found to be in good agreement with the results of Wang and Skalak1 and Tozeren.3

Recent experiments on cell-cell adhesion indicate that (i) bonds between two cells migrate towards the region of conjugation during peeling, and (ii) cell membrane tension increases with the rate of peeling. Analytical methods that would allow the derivation of intrinsic parameters of adhesion from such experimental data have yet to be explored. In this study we introduce a specific adhesion model to investigate the complex interaction between rate of peeling, lateral diffusivity and the rate of detachment of bonds. Our analysis show that adhesive energy density increases with the rate of peeling and with lateral diffusivity of the bonds but decreases with increasing rate of detachment. The rate dependency of the adhesive energy is due to the modulation of the number density distribution of bonds by the speed of peeling.

The erythrocyte membrane is modeled as a two-dimensional viscoelastic continuum that evolves under the application of stress. The present analysis of the erythrocyte membrane is motivated by the recent development of knowledge about its molecular structure. The constitutive equations proposed in the present analysis explain in a consistent manner the data on both the deformation and recovery phases of the micropipette experiment. The rheological equations of the present study are applied in a later section to the analysis of a plane membrane deformation that is quantitatively similar to the tank-treading motion of the erythrocytes in a shear field. The computations yield useful information on how the membrane viscosity becomes a more dominant feature in tank-treading motion. The material constants appearing in the proposed constitutive equations may be useful indications of the biochemical state of the membrane in health and disease.

The influence of stress on the growth and remodeling of a soft biological tissue is considered. For this purpose, the soft tissue is idealized as a fiber network. The stress-free lengths of the fibers composing the network are not fixed as in an inert elastic solid, but are assumed to evolve as a result of growth and stress adaptation. Similarly, the topology of the fiber network may also evolve under the application of stress. A set of constitutive equations are proposed which relate the tissue stress to the deformation of the tissue as well as to its growth and microstructure. It is shown that distinctly different growth patterns which may arise during initial growth or wound healing can be modeled by the proposed mathematical analysis.

In the present study, an analytical method is developed to deduce the constitutive equations of fibers embedded in a thick shell from the time-variant pressure volume curves obtained by experimental procedures. It is assumed that the spherical shell under consideration is composed of a fiber reinforced material and undergoes radial deflection, modeling the behavior of some biological shells such as urinary bladder. The fiber stress is expressed as a function of fiber strain, rate of strain and the degree of biochemical activation. The function form is chosen such that equations of mechanical equilibrium can be integrated analytically to yield chamber pressure as a function of chamber volume, time rate of change of volume and activation. Arbitrary coefficients appearing in the fiber stress-equation are also present in the resultant time-variant pressure-volume relation. These coefficients can be determined by curve-fitting commonly used clinical data such as cystometry measurements.

The deformation of a portion of erythrocyte during aspirational entry into a micropipette has been analyzed on the basis of a constant area deformation of an infinite plane membrane into a cylindrical tube. Consideration of the equilibrium of the membrane at the tip of the pipette has generated the relation between the aspirated length and the dimensionless time during deformational entry as well as during relaxation after the removal of aspiration pressure. Experimental studies on deformation and relaxation of normal human erythrocytes were performed with the use of micropipettes and a video dimension analyzer which allowed the continuous recording of the time-courses. The deformation consisted of an initial rapid phase with a membrane viscosity (range 0.6 x 10(-4) to 4 x 10(-4) dyn.s/cm) varying inversely with the degree of deformation and a later slow phase with a high membrane viscosity (mean 2.06 x 10(-2) dyn.s/cm) which was not correlated with the degree of deformation. The membrane viscosity of the recovery phase after 20 s of deformation (mean 5.44 x 10(-4) dyn.s/cm) was also independent of the degree of deformation. When determined after a short period of deformation (e.g., 2 s), however, membrane viscosity of the recovery phase became lower and agreed with that of the deformation phase. These results suggest that the rheological properties of the membrane can undergo dynamic changes depending on the extent and duration of deformation, reflecting molecular rearrangement in response to membrane strain.

It is shown that the mean intercept length distribution for planar fibre networks or for materials composed of a set of plates is not in general elliptic and cannot be expressed analytically in terms of a second-order tensor. However, our numerical computations indicate that the polar plot of the mean intercept length at the angle of measurement may become nearly ellipsoidal as the microstructure (fibres or plates) become less discretely organized, but yet remain orthotropic. The equations presented in this study may be used to obtain fibre (plate) orientation density functions from the experimental data on mean intercept length distribution.

The several widely different values of the elastic modulus of the human red blood cell membrane which have been reported in the literature are incorporated into a single strain energy function consisting of two terms. One term gives the small stresses and low elastic modulus which is observed when the red cell membrane is deformed at constant area. The second term contributes a large isotropic stress dependent on the change of area. The strain energy function is applied to the process of sphering of red blood cells in a hypotonic solution. It is shown that a nearly perfect sphere can result even though the red blood cell membrane is homogeneous in all areas of the cell. Results pertinent to sieving and micropipette experiments are also explored.

A micromanipulation method is used to determine the adhesive energy density (gamma) between pairs of cytotoxic T cells (F1) and their target cells (JY: HLA-A2-B7-DR4,W6). gamma is defined as the energy per unit area that must be supplied to reduce the region of contact between a conjugated cell pair. Our analysis of the data indicates that the force applied by the micropipette on the cell is not uniformly distributed throughout the contact region as we had previously assumed (Sung, K. L. P., L. A. Sung, M. Crimmins, S. J. Burakoff, and S. Chien. 1986. Science (Wash. DC). 234: 1405–1408), but acts only at the edges of the contact region. We show that gamma is not constant during peeling but increases with decreasing contact area of the conjugated cell pairs F1-JY, F1-F1, and JY-JY in contrast to the constancy of gamma for typical engineering adhesives. This finding supports the notion that the cross-linking protein molecules slide towards the conjugated area across the leading edge of the separation while remaining attached to both cells. Our mathematical analysis shows that the elastic energy stored in the cross-links by the membrane tensions balances the diffusive forces that act against cross-bridge migration. The binding affinity between F1-JY is found to be approximately 15–20 times larger than the corresponding affinity for F1-F1. The number of binding sites of F1 for attachment to JY is approximately the same for binding F1 to another F1 and vary between 10(5) and 10(6).

In the present study we investigate the transient conjugation of cell pairs by using a mathematical model. Macromolecules responsible for adhesion (bonds) are assumed to exist in two reversible states, attached and unattached, and exert a force elastic in nature only when they cross-link the two cell surfaces (attached state). Bonds form a link between the two cell surfaces only in the attached form. The unattached bridges are assumed laterally mobile in the plane of the cell membrane. Lateral mobility of attached bonds may be limited by structures on the undersurface of the cell membrane. Using this model we show that the bond density distribution between a cytotoxic T-cell (F-1) and a cancer cell (JY:HLA-A2-B7-DR4, W6) approaches equilibrium within 10 min, the incubation period used in experiments by Sung, K.L.P., L.A. Sung, M. Crimmins, S.J. Burakoff, and S. Chien (1986. Science [Wash. DC]. 234:1405–1408). If the diffusion coefficient of attached bonds is set equal to zero in the computations the model predictions indicate accumulation of bonds at the edge of conjugation. This prediction is consistent with present experimental data on lectin-induced red blood cell aggregation (Vayo, M., R. Skalak, P. Brunn, S. Usami, and S. Chien. 1987. Fed. Proc. 46:1043). It is concluded that significant features of micromanipulation data on specific adhesion can be explained by the diffusivity properties of bonds responsible for adhesion.

An analytical model is developed to study the interaction between the left ventricle and vascular system. Ventricular pressure is expressed as a function of the chamber volume, volumetric strain rate, and the degree of activation. A three-element Wind-kessel model is employed to represent the hydraulic properties of the vascular system. Conditions of interaction between the left ventricle and the vascular system are formulated in mathematical terms. Numerical solutions are obtained for the mechanical events occurring during a cardiac cycle as a function of time. The time variations of aortic pressure and ventricular volume predicted by the model compare well with the experimental results of Sunagawa and co-workers [Am. J. Physiol. 243 (Heart Circ. Physiol. 12): H346-H350, 1982, and Am. J. Physiol. 245 (Heart Circ. Physiol. 14): H773-H780, 1983]. Furthermore, the application of the present model to the experimental data has allowed the derivation of the intrinsic contractility parameters in these experiments. The unique features of this analytical model are that 1) it provides the time-variant pressure and volume curves of the left ventricle in relation to the aorta, 2) it generates information on the effects of heart rate on these hemodynamic parameters, and 3) it allows the derivation of intrinsic contractility parameters from experimental data.

A simple model of a double-headed crossbridge is introduced to explain the retardation of force decay after an imposed stretch in skeletal muscle fibers under equilibrium conditions. The critical assumption in the model is that once one of the heads of a crossbridge is attached to one of the available actin sites, the attachment of the second head will be restricted to a level of strain determined by the attachment of the first head. The crossbridge structure, namely the connection of both heads of a crossbridge to the same tail region, is assumed to impose this constraint on the spatial configurations of crossbridge heads. The unique feature of the model is the prediction that, in the presence of a ligand (PPi, ADP, AMP-PNP) and absence of Ca2+, the halftime of force decay is many times larger than the inverse rate of detachment of a crossbridge head measured in solution. This prediction is in agreement with measured values of half-times of force decay in fibers under similar conditions (Schoenberg, M., and E. Eisenberg. 1985. Biophys. J. 48:863-871f). It is predicted that a crossbridge head is more likely to re-attach to its previously strained position than remain unattached while the other head is attached, leading to the slow decay of force. Our computations also show that the apparent cooperativity in crossbridge binding observed in experiments (Brenner, B., L. C. Yu, L. E. Greene, E. Eisenberg, and M. Schoenberg. 1986. Biophys. J. 50:1101-1108) can be partially accounted by the double-headed crossbridge attachment. Our model predictions fit the aforementioned data best when the crossbridge stiffness does not change significantly with the dissociation of one of the two attached heads. This observation suggests that crossbridge stiffness is determined either by the extensibility (flexibility) of the double helical tail region or its junction to the thick filament backbone.