Integrin-ECM Interactions Regulate Cadherin-Dependent Cell Adhesion and Are Required for Convergent Extension in Xenopus

AbstractBackground: Convergence extension movements are conserved tissue rearrangements implicated in multiple morphogenetic events. While many of the cell behaviors involved in convergent extension are known, the molecular interactions required for this process remain elusive. However, past evidence suggests that regulation of cell adhesion molecule function is a key step in the progression of these behaviors.Results: Antibody blocking of fibronectin (FN) adhesion or dominant-negative inhibition of integrin β1 function alters cadherin-mediated cell adhesion, promotes cell-sorting behaviors in reaggregation assays, and inhibits medial-lateral cell intercalation and axial extension in gastrulating embryos and explants. Embryo explants were used to demonstrate that normal integrin signaling is required for morphogenetic movements within defined regions but not for cell fate specification. The binding of soluble RGD-containing fragments of fibronectin to integrins promotes the reintegration of dissociated single cells into intact tissues. The changes in adhesion observed are independent of cadherin or integrin expression levels.Conclusions: We conclude that integrin modulation of cadherin adhesion influences cell intercalation behaviors within boundaries defined by extracellular matrix. We propose that this represents a fundamental mechanism promoting localized cell rearrangements throughout development

Integrin α5β1 and Fibronectin Regulate Polarized Cell Protrusions Required for Xenopus Convergence and Extension

SummaryBackgroundIntegrin recognition of fibronectin is required for normal gastrulation including the mediolateral cell intercalation behaviors that drive convergent extension and the elongation of the frog dorsal axis; however, the cellular and molecular mechanisms involved are unclear.ResultsWe report that depletion of fibronectin with antisense morpholinos blocks both convergent extension and mediolateral protrusive behaviors in explant preparations. Both chronic depletion of fibronectin and acute disruptions of integrin α5β1 binding to fibronectin increases the frequency and randomizes the orientation of polarized cellular protrusions, suggesting that integrin-fibronectin interactions normally repress frequent random protrusions in favor of fewer mediolaterally oriented ones. In the absence of integrin α5β1 binding to fibronectin, convergence movements still occur but result in convergent thickening instead of convergent extension.ConclusionsThese findings support a role for integrin signaling in regulating the protrusive activity that drives axial extension. We hypothesize that the planar spatial arrangement of the fibrillar fibronectin matrix, which delineates tissue compartments within the embryo, is critical for promoting productive oriented protrusions in intercalating cells

Shear thickening in densely packed suspensions of spheres and rods confined to few layers

We investigate confined shear thickening suspensions for which the sample thickness is comparable to the particle dimensions. Rheometry measurements are presented for densely packed suspensions of spheres and rods with aspect ratios 6 and 9. By varying the suspension thickness in the direction of the shear gradient at constant shear rate, we find pronounced oscillations in the stress. These oscillations become stronger as the gap size is decreased, and the stress is minimized when the sample thickness becomes commensurate with an integer number of particle layers. Despite this confinement-induced effect, viscosity curves show shear thickening that retains bulk behavior down to samples as thin as two particle diameters for spheres, below which the suspension is jammed. Rods exhibit similar behavior commensurate with the particle width, but they show additional effects when the thickness is reduced below about a particle length as they are forced to align; the stress increases for decreasing gap size at fixed shear rate while the shear thickening regime gradually transitions to a Newtonian scaling regime. This weakening of shear thickening as an ordered configuration is approached contrasts with the strengthening of shear thickening when the packing fraction is increased in the disordered bulk limit, despite the fact that both types of confinement eventually lead to jamming.Comment: 21 pages, 14 figures. submitted to the Journal of Rheolog

The cysteine-rich domain regulates ADAM protease function in vivo

ADAMs are membrane-anchored proteases that regulate cell behavior by proteolytically modifying the cell surface and ECM. Like other membrane-anchored proteases, ADAMs contain candidate “adhesive” domains downstream of their metalloprotease domains. The mechanism by which membrane-anchored cell surface proteases utilize these putative adhesive domains to regulate protease function in vivo is not well understood. We address this important question by analyzing the relative contributions of downstream extracellular domains (disintegrin, cysteine rich, and EGF-like repeat) of the ADAM13 metalloprotease during Xenopus laevis development. When expressed in embryos, ADAM13 induces hyperplasia of the cement gland, whereas ADAM10 does not. Using chimeric constructs, we find that the metalloprotease domain of ADAM10 can substitute for that of ADAM13, but that specificity for cement gland expansion requires a downstream extracellular domain of ADAM13. Analysis of finer resolution chimeras indicates an essential role for the cysteine-rich domain and a supporting role for the disintegrin domain. These and other results reveal that the cysteine-rich domain of ADAM13 cooperates intramolecularly with the ADAM13 metalloprotease domain to regulate its function in vivo. Our findings thus provide the first evidence that a downstream extracellular adhesive domain plays an active role in regulating ADAM protease function in vivo. These findings are likely relevant to other membrane-anchored cell surface proteases

Conservation and divergence of ADAM family proteins in the Xenopus genome

Background Members of the disintegrin metalloproteinase (ADAM) family play important roles in cellular and developmental processes through their functions as proteases and/or binding partners for other proteins. The amphibian Xenopus has long been used as a model for early vertebrate development, but genome-wide analyses for large gene families were not possible until the recent completion of the X. tropicalis genome sequence and the availability of large scale expression sequence tag (EST) databases. In this study we carried out a systematic analysis of the X. tropicalis genome and uncovered several interesting features of ADAM genes in this species. Results Based on the X. tropicalis genome sequence and EST databases, we identified Xenopus orthologues of mammalian ADAMs and obtained full-length cDNA clones for these genes. The deduced protein sequences, synteny and exon-intron boundaries are conserved between most human and X. tropicalis orthologues. The alternative splicing patterns of certain Xenopus ADAM genes, such as adams 22 and 28, are similar to those of their mammalian orthologues. However, we were unable to identify an orthologue for ADAM7 or 8. The Xenopus orthologue of ADAM15, an active metalloproteinase in mammals, does not contain the conserved zinc-binding motif and is hence considered proteolytically inactive. We also found evidence for gain of ADAM genes in Xenopus as compared to other species. There is a homologue of ADAM10 in Xenopus that is missing in most mammals. Furthermore, a single scaffold of X. tropicalis genome contains four genes encoding ADAM28 homologues, suggesting genome duplication in this region. Conclusions Our genome-wide analysis of ADAM genes in X. tropicalis revealed both conservation and evolutionary divergence of these genes in this amphibian species. On the one hand, all ADAMs implicated in normal development and health in other species are conserved in X. tropicalis. On the other hand, some ADAM genes and ADAM protease activities are absent, while other novel ADAM proteins in this species are predicted by this study. The conservation and unique divergence of ADAM genes in Xenopus probably reflect the particular selective pressures these amphibian species faced during evolution.National Institutes of Health. Department of Health and Human Services (Ruth L. Kirschstein postdoctoral fellowship)National Institutes of Health. Department of Health and Human Services (5T32GA09109)American Heart Association (postdoctoral fellowship)March of Dimes Birth Defects Foundation (grant 1-FY10-399)March of Dimes Birth Defects Foundation (grant F405-140)National Institutes of Health (U.S.) (HD26402)National Institutes of Health (U.S.) (DE14365

Shear thickening and jamming in densely packed suspensions of different particle shapes

We investigated the effects of particle shape on shear thickening in densely packed suspensions. Rods of different aspect ratios and non-convex hooked rods were fabricated. Viscosity curves and normal stresses were measured using a rheometer for a wide range of packing fractions for each shape. Suspensions of each shape exhibit qualitatively similar Discontinuous Shear Thickening. The logarithmic slope of the stress/shear-rate relation increases dramatically with packing fraction and diverges at a critical packing fraction phi_c which depends on particle shape. The packing fraction dependence of the viscosity curves for different convex shapes can be collapsed when the packing fraction is normalized by phi_c. Intriguingly, viscosity curves for non-convex particles do not collapse on the same set as convex particles, showing strong shear thickening over a wider range of packing fraction. The value of phi_c is found to coincide with the onset of a yield stress at the jamming transition, suggesting the jamming transition also controls shear thickening. The yield stress is found to correspond with trapped air in the suspensions, and the scale of the stress can be attributed to interfacial tension forces which dramatically increase above phi_c due to the geometric constraints of jamming. The relationship between shear and normal stresses is found to be linear in both the shear thickening and jammed regimes, indicating that the shear stresses come from friction. In the limit of zero shear rate, normal stresses pull the rheometer plates together due to the surface tension of the liquid below phi_c, but push the rheometer plates apart due to jamming above phi_c.Comment: 13 pages, 13 figures. published in Physical Review

Synthetic Studies in Phytochrome Chemistry

An account is given of the author’s several approaches to the synthesis of the parent chromophore of phytochrome (1), a protein-bound linear tetrapyrrole derivative that controls photomorphogenesis in higher plants. These studies culminated in enantioselective syntheses of both (2R)- and (2S)-phytochromobilin (4), as well as several 13C-labeled derivatives designed to probe the site of Z,E-isomerization during photoexcitation. When reacted in vitro, synthetic 2R-4 and recombinant-derived phytochrome apoprotein N-C produced a protein-bound chromophore with identical difference spectra to naturally occurring 1

Generality of shear thickening in suspensions

Suspensions are of wide interest and form the basis for many smart fluids. For most suspensions, the viscosity decreases with increasing shear rate, i.e. they shear thin. Few are reported to do the opposite, i.e. shear thicken, despite the longstanding expectation that shear thickening is a generic type of suspension behavior. Here we resolve this apparent contradiction. We demonstrate that shear thickening can be masked by a yield stress and can be recovered when the yield stress is decreased below a threshold. We show the generality of this argument and quantify the threshold in rheology experiments where we control yield stresses arising from a variety of sources, such as attractions from particle surface interactions, induced dipoles from applied electric and magnetic fields, as well as confinement of hard particles at high packing fractions. These findings open up possibilities for the design of smart suspensions that combine shear thickening with electro- or magnetorheological response.Comment: 11 pages, 9 figures, accepted for publication in Nature Material

Multiscale computational analysis of Xenopus laevis morphogenesis reveals key insights of systems-level behavior

<p>Abstract</p> <p>Background</p> <p>Tissue morphogenesis is a complex process whereby tissue structures self-assemble by the aggregate behaviors of independently acting cells responding to both intracellular and extracellular cues in their environment. During embryonic development, morphogenesis is particularly important for organizing cells into tissues, and although key regulatory events of this process are well studied in isolation, a number of important systems-level questions remain unanswered. This is due, in part, to a lack of integrative tools that enable the coupling of biological phenomena across spatial and temporal scales. Here, we present a new computational framework that integrates intracellular signaling information with multi-cell behaviors in the context of a spatially heterogeneous tissue environment.</p> <p>Results</p> <p>We have developed a computational simulation of mesendoderm migration in the <it>Xenopus laevis </it>explant model, which is a well studied biological model of tissue morphogenesis that recapitulates many features of this process during development in humans. The simulation couples, via a JAVA interface, an ordinary differential equation-based mass action kinetics model to compute intracellular Wnt/β-catenin signaling with an agent-based model of mesendoderm migration across a fibronectin extracellular matrix substrate. The emergent cell behaviors in the simulation suggest the following properties of the system: maintaining the integrity of cell-to-cell contact signals is necessary for preventing fractionation of cells as they move, contact with the Fn substrate and the existence of a Fn gradient provides an extracellular feedback loop that governs migration speed, the incorporation of polarity signals is required for cells to migrate in the same direction, and a delicate balance of integrin and cadherin interactions is needed to reproduce experimentally observed migratory behaviors.</p> <p>Conclusion</p> <p>Our computational framework couples two different spatial scales in biology: intracellular with multicellular. In our simulation, events at one scale have quantitative and dynamic impact on events at the other scale. This integration enables the testing and identification of key systems-level hypotheses regarding how signaling proteins affect overall tissue-level behavior during morphogenesis in an experimentally verifiable system. Applications of this approach extend to the study of tissue patterning processes that occur during adulthood and disease, such as tumorgenesis and atherogenesis.</p

Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies

Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction-limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (~70 nm) imaging of cells and mammalian tissues on conventional microscopes.United States. National Institutes of Health (1R01GM104948)United States. National Institutes of Health (1DP1NS087724)United States. National Institutes of Health ( NIH 1R01EY023173)United States. National Institutes of Health (1U01MH106011