The collagenous matrix of bovine predentine.

Predentine obtained from bovine teeth by microdissection was solubilized by cyanogen bromide cleavage. The electrophoretic mobility of the resultant peptides was established on polyacrylamide gel and the amino acid composition of several peptides was determined. The data clearly indicated that this collagen is entirely of the Type I genetic species. No differences were detected between the predentine and dentine collagens except that the mature tissue was more highly crosslinked. Nevertheless the amount of stable cross-link formed in the predentine was higher than expected for an immature tissue.     

The orientation of cell divisions determines the shape of Drosophila organs

Organ shape depends on the coordination between cell proliferation and the spatial arrangement of cells during development. Much is known about the mechanisms that regulate cell proliferation, but the processes by which the cells are orderly distributed remain unknown. This can be accomplished either by random division of cells that later migrate locally to new positions (cell allocation) or through polarized cell division (oriented cell division; OCD). Recent data suggest that the OCD is involved in some morphogenetic processes such as vertebrate gastrulation, neural tube closure, and growth of shoot apex in plants; however, little is known about the contribution of OCD during organogenesis. We have analyzed the orientation patterns of cell division throughout the development of wild-type and mutant imaginal discs of Drosophila. Our results show a causal relationship between the orientation of cell divisions in the imaginal disc and the adult morphology of the corresponding organs, indicating a key role of OCD in organ-shape definition. In addition, we find that a subset of planar cell polarity genes is required for the proper orientation of cell division during organ development.     

Local multiple alignment by consensus matrix

A new algorithm for aligning several sequences based on thecalculation of a consensus matrix and the comparison of allthe sequences using this consensus matrix is described. Thisconsensus matrix contains the preference scores of each nucleotideøaminoacid and gaps in every position of the alignment. Two modificationsof the algorithm corresponding to the evolutionary and functionalmeanings of the alignment were developed. The first one solvesthe best-fitting problem without any penalty for end gaps andwith an internal gap penalty function independent on the gaplength. This algorithm should be used when comparing evolutionary-relatedproteins for identifying the most conservative residues. Theother modification of the algorithm finds the most similar segmentsin the given sequences. It can be used for finding those partsof the sequences that are responsible for the same biologicalJunction. In this case the gap penalty function was chosen tobe proportional to the gap length. The result of aligning aminoacid sequences of neutral proteases and a compilation of 65allosteric effectors and substrates of PEP carboxylase are presented.     

Full field strain measurements of collagenous tissue by tracking fiber alignment through vector correlation

Full field strain measurements of biological tissue during loading are often limited to the quantification of fiduciary marker displacements on the tissue surface. These marker measurements can lack the necessary spatial resolution to characterize non-uniform deformation and may not represent the deformation of the load-bearing collagen microstructure. To overcome these potential limitations, a method was developed to track the deformation of the collagen fiber microstructure in ligament tissue. Using quantitative polarized light imaging, fiber alignment maps incorporating both direction and alignment strength at each pixel were generated during facet capsular ligament loading. A grid of virtual markers was superimposed over the tissue in the alignment maps, and the maximization of a vector correlation calculation between fiber alignment maps was used to track marker displacement. Tracking error was quantified through comparisons to the displacements of excised ligament tissue (n=3); separate studies applied uniaxial tension to isolated facet capsular ligament tissue (n=4) to evaluate tracking capabilities during large tissue deformations. The average difference between virtual marker and tissue displacements was 0.07±0.06 pixels. This error in marker location produced principal strain measurements of 1.2±1.6% when markers were spaced 4 pixels apart. During tensile tissue loading, substantial inhomogeneity was detected in the strain field using vector correlation tracking, and the location of maximum strain differed from that produced by standard tracking techniques using coarser meshes. These findings provide a method to directly measure fiber network strains using quantitative fiber alignment data, enabling a better understanding of structure–function relationships in tissues at different length scales.     

Cell flexibility affects the alignment of model myxobacteria

Myxobacteria are social bacteria that exhibit a complex life cycle culminating in the development of multicellular fruiting bodies. The alignment of rod-shaped myxobacteria cells within populations is crucial for development to proceed. It has been suggested that myxobacteria align due to mechanical interactions between gliding cells and that cell flexibility facilitates reorientation of cells upon mechanical contact. However, these suggestions have not been based on experimental or theoretical evidence. Here we created a computational mass-spring model of a flexible rod-shaped cell that glides on a substratum periodically reversing direction. The model was formulated in terms of experimentally measurable mechanical parameters, such as engine force, bending stiffness, and drag coefficient. We investigated how cell flexibility and motility engine type affected the pattern of cell gliding and the alignment of a population of 500 mechanically interacting cells. It was found that a flexible cell powered by engine force at the rear of the cell, as suggested by the slime extrusion hypothesis for myxobacteria motility engine, would not be able to glide in the direction of its long axis. A population of rigid reversing cells could indeed align due to mechanical interactions between cells, but cell flexibility impaired the alignment.     

Native extracellular matrix orientation determines multipotent vascular stem cell proliferation in response to cyclic uniaxial tensile strain and simulated stent indentation

Cardiovascular disease is the leading cause of death worldwide, with multipotent vascular stem cells (MVSC) implicated in contributing to diseased vessels. MVSC are mechanosensitive cells which align perpendicular to cyclic uniaxial tensile strain. Within the blood vessel wall, collagen fibers constrain cells so that they are forced to align circumferentially, in the primary direction of tensile strain. In these experiments, MVSC were seeded onto the medial layer of decellularized porcine carotid arteries, then exposed to 10%, 1 Hz cyclic tensile strain for 10 days with the collagen fiber direction either parallel or perpendicular to the direction of strain. Cells aligned with the direction of the collagen fibers regardless of the orientation to strain. Cells aligned with the direction of strain showed an increased number of proliferative Ki67 positive cells, while those strained perpendicular to the direction of cell alignment showed no change in cell proliferation. A bioreactor system was designed to simulate the indentation of a single, wire stent strut. After 10 days of cyclic loading to 10% strain, MVSC showed regions of densely packed, highly proliferative cells. Therefore, MVSC may play a significant role in in-stent restenosis, and this proliferative response could potentially be controlled by controlling MVSC orientation relative to applied strain.     

Cellulose orientation determines mechanical anisotropy in onion epidermis cell walls

The role of cellulose microfibril orientation in determining cell wall mechanical anisotropy and in the control of the wall plastic versus elastic properties was studied in the adaxial epidermis of onion bulb scales using the constant-load (creep) test. The mean or net cellulose orientation in the outer periclinal wall of the epidermis was parallel to the long axis of the cells. In vitro cell wall extensibility was 30-90% higher in the direction perpendicular to the net microfibril orientation than parallel to it. This was the case for the size of the initial deformation occurring just after the load application and for the rate of time-dependent creep. Loading/unloading experiments confirmed the presence of a real irreversible component in cell wall extension. The plastic component of the time-dependent deformation was higher perpendicular to the net cellulose orientation than parallel to it. An acid buffer (pH 4.5) increased the creep rate by 25-30% but this response was not related to cellulose orientation. The present data provide direct evidence that the net orientation of cellulose microfibrils confers mechanical anisotropy to the walls of seed plants, a characteristic that may be relevant to understanding anisotropic cell growth.     

Incorporation of experimentally-derived fiber orientation into a structural constitutive model for planar collagenous tissues

Structural constitutive models integrate information on tissue composition and structure, avoiding ambiguities in material characterization. However, critical structural information (such as fiber orientation) must be modeled using assumed statistical distributions, with the distribution parameters estimated from fits to the mechanical test data. Thus, full realization of structural approaches continues to be limited without direct quantitative structural information for direct implementation or to validate model predictions. In the present study, fiber orientation information obtained using small angle light scattering (SALS) was directly incorporated into a structural constitutive model based on work by Lanir (J. Biomech., v. 16, pp. 1-12, 1983). Demonstration of the model was performed using existing biaxial mechanical and fiber orientation data for native bovine pericardium (Sacks and Chuong, ABME, v.26, pp. 892-902, 1998). The structural constitutive model accurately predicted the complete measured biaxial mechanical response. An important aspect of this approach is that only a single equibiaxial test to determine the effective fiber stress-strain response and the SALS-derived fiber orientation distribution were required to determine the complete planar biaxial mechanical response. Changes in collagen fiber crimp under equibiaxial strain suggest that, at the meso-scale, fiber deformations follow the global tissue strains. This result supports the assumption of affine strain to estimate the fiber strains. However, future evaluations will have to be performed for tissue subjected to a wider range of strain to more fully validate the current approach.     

A wave of EGFR signaling determines cell alignment and intercalation in the Drosophila tracheal placode

Invagination of organ placodes converts flat epithelia into three-dimensional organs. Cell tracing in the Drosophila tracheal placode revealed that, in the 30-minute period before invagination, cells enter mitotic quiescence and form short rows that encircle the future invagination site. The cells in the rows align to form a smooth boundary (;boundary smoothing'), accompanied by a transient increase in myosin at the boundary and cell intercalation oriented in parallel with the cellular rows. Cells then undergo apical constriction and invaginate, followed by radially oriented mitosis in the placode. Prior to invagination, ERK MAP kinase is activated in an outward circular wave, with the wave front often correlating with the smoothing cell boundaries. EGFR signaling is required for myosin accumulation and cell boundary smoothing, suggesting its propagation polarizes the planar cell rearrangement in the tracheal placode, and coordinates the timing and position of intrinsic cell internalization activities.     

Cell division orientation in animals

Cell division orientation during animal development can serve to correctly organize and shape tissues, create cellular diversity or both. The underlying cellular mechanism is regulated spindle orientation. Depending on the developmental context, extrinsic signals or intrinsic cues control the correct orientation of the mitotic spindle. Cell geometry has been known to be another determinant of spindle orientation and recent results have shed new light?on the link between cellular shape and cell division orientation. The importance of controlling spindle orientation is manifested in neurodevelopmental defects such as?microcephaly, tumor initiation as well as defects in tissue architecture and cell fate misspecification. Here, we summarize the role of oriented cell division during animal development and also outline the cellular and molecular mechanisms in selected invertebrate and vertebrate systems.     

Costimulation by extracellular matrix proteins determines the response to TCR ligation

Although ligation of the T-cell antigen receptor (TCR) is central to the responsiveness and antigen specificity of T-cells, it is insufficient to elicit a response. To determine whether the need for costimulation reflects inadequate strength of signal transduction through the TCR or an absolute block of signaling in the absence of a coligand, we studied T-cell activation under serum-free conditions eliminating costimulation by various extracellular matrix proteins which otherwise have an omnipresent and frequently overlooked effect. Engagement of the TCR leads to induction of Fas, but not to measurable IL-2 secretion or apoptosis. Those activation parameters are induced by costimulation through integrin alphaVbeta3. Furthermore, T-cell survival or elimination is determined by the type of ligand binding to this coreceptor with vitronectin, fibronectin, and fibrinogen efficiently inducing apoptosis and IL-2 production while osteopontin and entactin mediate IL-2 secretion comparably without causing programmed cell death. Consistent with the cytokine properties of these ligands, differential costimulation depends on their presentation in soluble rather than immobilized form. The determination of elimination versus survival of activated T-cells by coligation of beta3-integrins may have bearing on the fundamental postthymic mechanisms that shape the T-cell repertoire.     

Regulation of fibroblast migration on collagenous matrix by a cell surface peptidase complex

The invasion of migratory cells through connective tissues involves metallo- and serine types of cell surface proteases. We show that formation of a novel protease complex, consisting of the membrane-bound prolyl peptidases seprase and dipeptidyl peptidase IV (DPPIV), at invadopodia of migratory fibroblasts is a prerequisite for cell invasion and migration on a collagenous matrix. Seprase and DPPIV form a complex on the cell surface that elicits both gelatin binding and gelatinase activities localized at invadopodia of cells migrating on collagenous fibers. The protease complex participates in the binding to gelatin and localized gelatin degradation, cellular migration, and monolayer wound closure. Serine protease inhibitors can block the gelatinase activity and the localized gelatin degradation by cells. Antibodies to the gelatin-binding domain of DPPIV reduce the cellular abilities of the proteases to degrade gelatin but do not affect cellular adhesion or spreading on type I collagen. Furthermore, expression of the seprase-DPPIV complex is restricted to migratory cells involved in wound closure in vitro and in connective tissue cells during closure of gingival wounds but not in differentiated tissue cells. Thus, we have identified cell surface proteolytic activities, which are non-metalloproteases, seprase and DPPIV, that are responsible for the tissue-invasive phenotype.     

Sequence alignment with an appropriate substitution matrix.

A widely used algorithm for computing an optimal local alignment between two sequences requires a parameter set with a substitution matrix and gap penalties. It is recognized that a proper parameter set should be selected to suit the level of conservation between sequences. We describe an algorithm for selecting an appropriate substitution matrix at given gap penalties for computing an optimal local alignment between two sequences. In the algorithm, a substitution matrix that leads to the maximum alignment similarity score is selected among substitution matrices at various evolutionary distances. The evolutionary distance of the selected substitution matrix is defined as the distance of the computed alignment. To show the effects of gap penalties on alignments and their distances and help select appropriate gap penalties, alignments and their distances are computed at various gap penalties. The algorithm has been implemented as a computer program named SimDist. The SimDist program was compared with an existing local alignment program named SIM for finding reciprocally best-matching pairs (RBPs) of sequences in each of 100 protein families, where RBPs are commonly used as an operational definition of orthologous sequences. SimDist produced more accurate results than SIM on 50 of the 100 families, whereas both programs produced the same results on the other 50 families. SimDist was also used to compare three types of substitution matrices in scoring 444,461 pairs of homologous sequences from the 100 families.     

The extracellular matrix guides the orientation of the cell division axis

The cell division axis determines the future positions of daughter cells and is therefore critical for cell fate. The positioning of the division axis has been mostly studied in systems such as embryos or yeasts, in which cell shape is well defined. In these cases, cell shape anisotropy and cell polarity affect spindle orientation. It remains unclear whether cell geometry or cortical cues are determinants for spindle orientation in mammalian cultured cells. The cell environment is composed of an extracellular matrix (ECM), which is connected to the intracellular actin cytoskeleton via transmembrane proteins. We used micro-contact printing to control the spatial distribution of the ECM on the substrate and demonstrated that it has a role in determining the orientation of the division axis of HeLa cells. On the basis of our analysis of the average distributions of actin-binding proteins in interphase and mitosis, we propose that the ECM controls the location of actin dynamics at the membrane, and thus the segregation of cortical components in interphase. This segregation is further maintained on the cortex of mitotic cells and used for spindle orientation.     

Duration of ischaemia determines matrix metalloproteinase-2 activation in the reperfused rabbit heart

It has been hypothesised that activation of matrix metalloproteinase-2 (MMP-2) contributes to reversible myocardial dysfunction (stunning) following short-term ischaemia and reperfusion. Gelatin zymography was used to measure release of both pro-MMP-2 (72 kDa) and MMP-2 (62 kDa), into the coronary effluent from isolated, perfused rabbit hearts during 90 min aerobic perfusion (control), or low-flow ischaemia (15 or 60 min at 1 mL/min), followed by 60 min reperfusion. In controls, pro-MMP-2 was detected in the coronary effluent throughout the first 30 min of aerobic perfusion, but MMP-2 was not detected. In contrast, MMP-2 was detected in the coronary effluent during reperfusion after both 15 and 60 min ischaemia. However, while left ventricular systolic function was impaired after both 15 min and 60 min ischaemia, a significant increase in the release of MMP-2 was only detected in hearts following 60 min ischaemia. The dissociation between mechanical function and MMP-2 levels suggest that MMP-2 does not contribute to myocardial stunning in this model, but may contribute to myocardial dysfunction following prolonged ischaemia.