A mechanoresponsive cadherin-keratin complex directs polarized protrusive behavior and collective cell migration

Collective cell migration requires maintenance of adhesive contacts between adjacent cells, coordination of polarized cell protrusions, and generation of propulsive traction forces. We demonstrate that mechanical force applied locally to C-cadherins on single Xenopus mesendoderm cells is sufficient to induce polarized cell protrusion and persistent migration typical of individual cells within a collectively migrating tissue. Local tension on cadherin adhesions induces reorganization of the keratin intermediate filament network toward these stressed sites. Plakoglobin, a member of the catenin family, is localized to cadherin adhesions under tension and is required for both mechanoresponsive cell behavior and assembly of the keratin cytoskeleton at the rear of these cells. Local tugging forces on cadherins occur in vivo through interactions with neighboring cells, and these forces result in coordinate changes in cell protrusive behavior. Thus, cadherin-dependent force-inducible regulation of cell polarity in single mesendoderm cells represents an emergent property of the intact tissue.     

Ion transport across the frog olfactory mucosa: the basal and odorant-stimulated states

The Ussing method was adapted to study the basal electrolyte transfer as well as the events that occur upon odorant stimulation in frog olfactory mucosa. The unstimulated short-circuit current was due mainly to a furosemide-sensitive ion transport system on the apical side of the olfactory mucosa. This current was not amiloride sensitive. The current-voltage relationship of the unstimulated state was linear. That of the odorant-evoked current was non-linear and amiloride-sensitive. Ouabain caused collapse of both the unstimulated and odorant-stimulated short-circuit current. In this case, voltage-clamping the tissue to non-zero values restored the odorant-evoked current with polarity depending on that of the clamping voltage. This suggested that the direction of the current is determined by that of the sodium electrochemical potential difference. Our results indicate that the unstimulated short-circuit current occurs through an apical sodium cotransport system, while the odorant-evoked current is due to odorant-activated, passive sodium channels that are amiloride sensitive.     

Cooperativity between Plasmodium falciparum adhesive proteins for invasion into erythrocytes

Plasmodium falciparum is the most virulent of the Plasmodium species infective to humans. Different P. falciparum strains vary in their dependence on erythrocyte receptors for invasion and their ability to switch in their utilization of different receptor repertoires. Members of the reticulocyte-binding protein-like (RBL) family of invasion ligands are postulated to play a central role in defining ligand–receptor interactions, known as invasion pathways. Here we report the targeted gene disruption of PfRh2b and PfRh2a in W2mef, a parasite strain that is heavily dependent on sialic-acid receptors for invasion, and show that the PfRh2b ligand is functional in this parasite background. Like the parental line, parasites lacking either PfRh2a or PfR2b can switch to a sialic acid-independent invasion pathway. However, both of the switched lines exhibit a reduced efficiency for invasion into sialic acid-depleted cells, suggesting a role for both PfRh2b and PfRh2a in invasion via sialic acid-independent receptors. We also find a strong selective pressure for the reconstitution of PfRh2b expression at the expense of PfRh2a. Our results reveal the importance of genetic background in ligand–receptor usage by P. falciparum parasites, and suggest that the co-ordinate expression of PfRh2a, PfRh2b together mediate efficient sialic acid-independent erythrocyte invasion.     

Xenopus ADAM 13 is a metalloprotease required for cranial neural crest-cell migration.

BACKGROUND: Cranial neural-crest (CNC) cells originate from the lateral edge of the anterior neuroepithelium and migrate to form parts of the peripheral nervous system, muscles, cartilage, and bones of the face. Neural crest-cell migration involves the loss of adhesion from the surrounding neuroepithelium and a corresponding increase in cell adhesion to the extracellular matrix (ECM) present in migratory pathways. While proteolytic activity is likely to contribute to the regulation of neural crest-cell adhesion and migration, the role of a neural crest-specific protease in these processes has yet to be demonstrated. We previously showed that CNC cells express ADAM 13, a cell surface metalloprotease/disintegrin. Proteins of this family are known to act in cell-cell adhesion and as sheddases. ADAMs have also been proposed to degrade the ECM, but this has not yet been shown in a physiological context. RESULTS: Using a tissue transplantation technique, we show that Xenopus CNC cells overexpressing wild-type ADAM 13 migrate along the same hyoid, branchial, and mandibular pathways used by normal CNC cells. In contrast, CNC cell grafts that express protease-defective ADAM 13 fail to migrate along the hyoid and branchial pathways. In addition, ectopic expression of wild-type ADAM 13 results in a gain-of-function phenotype in embryos, namely the abnormal positioning of trunk neural-crest cells. We further show that explanted embryonic tissues expressing wild-type, but not protease-defective, ADAM 13 display decreased cell-matrix adhesion. Purified ADAM 13 can cleave fibronectin, and tissue culture cells that express wild-type, but not protease-defective, ADAM 13 can remodel a fibronectin substrate. CONCLUSIONS: Our findings support the hypothesis that the protease activity of ADAM 13 plays a critical role in neural crest-cell migration along defined pathways. We propose that the ADAM 13-dependent modification of ECM and/or other guidance molecules is a key step in the directed migration of the CNC.     

Decrease in rat taste receptor cell intracellular pH is the proximate stimulus in sour taste transduction

Taste receptor cells (TRCs)respond to acid stimulation, initiating perception of sour taste.Paradoxically, the pH of weak acidic stimuli correlates poorly with theperception of their sourness. A fundamental issue surrounding sourtaste reception is the identity of the sour stimulus. We tested thehypothesis that acids induce sour taste perception by penetratingplasma membranes as H⁺ ions or as undissociated moleculesand decreasing the intracellular pH (pH_i) of TRCs. Our datasuggest that taste nerve responses to weak acids (acetic acid andCO₂) are independent of stimulus pH but strongly correlatewith the intracellular acidification of polarized TRCs. Taste nerveresponses to CO₂ were voltage sensitive and were blockedwith MK-417, a specific blocker of carbonic anhydrase. Strong acids(HCl) decrease pH_i in a subset of TRCs that contain apathway for H⁺ entry. Both the apical membrane and theparacellular shunt pathway restrict H⁺ entry such that alarge decrease in apical pH is translated into a relatively smallchange in TRC pH_i within the physiological range. Weconclude that a decrease in TRC pH_i is the proximate stimulus in rat sour taste transduction.

     

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.     

Prolonged cell cycle arrest in irradiated human diploid skin fibroblasts: the role of nutrient deprivation

Ionizing radiation has been reported to cause an irreversible cell cycle arrest in normal human diploid fibroblasts. However, colony survival assays show that even at high doses of gamma radiation, human diploid fibroblasts do not irreversibly arrest, and that a dose-dependent fraction is capable of continued cycling. In this study, we resolve the apparent discrepancy between colony survival assays and the observed radiation-induced prolonged arrest. Using flow cytometry analysis, we have confirmed that human diploid fibroblasts do exhibit a prolonged cell cycle arrest in both G(1) and G(2)/M phases of the cell cycle. However, a single replacement of fresh growth medium stimulated a fraction of the arrested population of cells to transiently re-enter the cell cycle. Daily medium changes stimulated these irradiated human diploid fibroblasts to continue cycling until they were contact-inhibited. Thus the fraction of human diploid fibroblasts which survive radiation exposure and are capable of cycling appears to permanently arrest as a result of nutrient insufficiency. Western blot analysis demonstrated a radiation-induced elevation in TP53 (formerly known as p53) protein levels within 2 h postirradiation, followed by a decrease to levels comparable to those in unirradiated controls. The TP53 and CDKN1A (formerly known as p21) protein levels were indistinguishable after 24 h and remained elevated for a 6-day period of observation in both control and irradiated cultures. Our studies indicate that human diploid fibroblasts are capable of re-entering the cell cycle after exposure to ionizing radiation and that this re-entry is dependent on a constant supply of nutrients provided by fresh medium changes. The fraction of cells capable of resuming cell cycling is consistent with the surviving fraction of cells in colony assays.     

Optimising and Communicating Options for the Control of Invasive Plant Disease When There Is Epidemiological Uncertainty

Although local eradication is routinely attempted following introduction of disease into a new region, failure is commonplace. Epidemiological principles governing the design of successful control are not well-understood. We analyse factors underlying the effectiveness of reactive eradication of localised outbreaks of invading plant disease, using citrus canker in Florida as a case study, although our results are largely generic, and apply to other plant pathogens (as we show via our second case study, citrus greening). We demonstrate how to optimise control via removal of hosts surrounding detected infection (i.e. localised culling) using a spatially-explicit, stochastic epidemiological model. We show how to define optimal culling strategies that take account of stochasticity in disease spread, and how the effectiveness of disease control depends on epidemiological parameters determining pathogen infectivity, symptom emergence and spread, the initial level of infection, and the logistics and implementation of detection and control. We also consider how optimal culling strategies are conditioned on the levels of risk acceptance/aversion of decision makers, and show how to extend the analyses to account for potential larger-scale impacts of a small-scale outbreak. Control of local outbreaks by culling can be very effective, particularly when started quickly, but the optimum strategy and its performance are strongly dependent on epidemiological parameters (particularly those controlling dispersal and the extent of any cryptic infection, i.e. infectious hosts prior to symptoms), the logistics of detection and control, and the level of local and global risk that is deemed to be acceptable. A version of the model we developed to illustrate our methodology and results to an audience of stakeholders, including policy makers, regulators and growers, is available online as an interactive, user-friendly interface at http://www.webidemics.com/. This version of our model allows the complex epidemiological principles that underlie our results to be communicated to a non-specialist audience.