The epithelial sodium channel mediates the directionality of galvanotaxis in human keratinocytes

Cellular directional migration in an electric field (galvanotaxis) is one of the mechanisms guiding cell movement in embryogenesis and in skin epidermal repair. The epithelial sodium channel (ENaC), in addition to its function of regulating sodium transport in kidney, has recently been found to modulate cell locomotory speed. Here we tested whether ENaC has an additional function of mediating the directional migration of galvanotaxis in keratinocytes. Genetic depletion of ENaC completely blocks only galvanotaxis and does not decrease migration speed. Overexpression of ENaC is sufficient to drive galvanotaxis in otherwise unresponsive cells. Pharmacologic blockade or maintenance of the open state of ENaC also decreases or increases, respectively, galvanotaxis, suggesting that the channel open state is responsible for the response. Stable lamellipodial extensions formed at the cathodal sides of wild-type cells at the start of galvanotaxis; these were absent in the ENaC knockout keratinocytes, suggesting that ENaC mediates galvanotaxis by generating stable lamellipodia that steer cell migration. We provide evidence that ENaC is required for directional migration of keratinocytes in an electric field, supporting a role for ENaC in skin wound healing.

Phosphorylation of serine 4642 in the C-terminus of plectin by MNK2 and PKA modulates its interaction with intermediate filaments

Plectin is a versatile cytolinker of the plakin family conferring cell resilience to mechanical stress in stratified epithelia and muscles. It acts as a critical organizer of the cytoskeletal system by tethering various intermediate filament (IF) networks through its C-terminal IF-binding domain (IFBD). Mutations affecting the IFBD cause devastating human diseases. Here, we show that serine 4642, which is located in the extreme C-terminus of plectin, is phosphorylated in different cell lines. Phosphorylation of S4642 decreased the ability of plectin IFBD to associate with various IFs, as assessed by immunofluorescence microscopy and cell fractionation studies, as well as in yeast two-hybrid assays. Plectin phosphorylated at S4642 was reduced at sites of IF network anchorage along cell-substrate contacts in both skin and cultured keratinocytes. Treatment of SK-MEL-2 and HeLa cells with okadaic acid increased plectin S4642 phosphorylation, suggesting that protein phosphatase 2A dephosphorylates this residue. Moreover, plectin S4642 phosphorylation was enhanced after cell treatment with EGF, phorbol ester, sorbitol and 8-bromo-cyclic AMP, as well as during wound healing and protease-mediated cell detachment. Using selective protein kinase inhibitors, we identified two different kinases that modulate the phosphorylation of plectin S4642 in HeLa cells: MNK2, which is downstream of the ERK1/2-dependent MAPK cascade, and PKA. Our study indicates that phosphorylation of S4642 has an important regulatory role in the interaction of plectin with IFs and identifies a novel link between MNK2 and the cytoskeleton.

The Caenorhabditis elegans histone hairpin-binding protein is required for core histone gene expression and is essential for embryonic and postembryonic cell division.

As in all metazoans, the replication-dependent histone genes of Caenorhabditis elegans lack introns and contain a short hairpin structure in the 3' untranslated region. This hairpin structure is a key element for post-transcriptional regulation of histone gene expression and determines mRNA 3' end formation, nuclear export, translation and mRNA decay. All these steps contribute to the S-phase-specific expression of the replication-dependent histone genes. The hairpin structure is the binding site for histone hairpin-binding protein that is required for hairpin-dependent regulation. Here, we demonstrate that the C. elegans histone hairpin-binding protein gene is transcribed in dividing cells during embryogenesis and postembryonic development. Depletion of histone hairpin-binding protein (HBP) function in early embryos using RNA-mediated interference leads to an embryonic-lethal phenotype brought about by defects in chromosome condensation. A similar phenotype was obtained by depleting histones H3 and H4 in early embryos, indicating that the defects in hairpin-binding protein-depleted embryos are caused by reduced histone biosynthesis. We have confirmed this by showing that HBP depletion reduces histone gene expression. Depletion of HBP during postembryonic development also results in defects in cell division during late larval development. In addition, we have observed defects in the specification of vulval cell fate in animals depleted for histone H3 and H4, which indicates that histone proteins are required for cell fate regulation during vulval development.

Drosophila valois encodes a divergent WD protein that is required for Vasa localization and Oskar protein accumulation

valois (vls) was identified as a posterior group gene in the initial screens for Drosophila maternal-effect lethal mutations. Despite its early genetic identification, it has not been characterized at the molecular level until now. We show that vls encodes a divergent WD domain protein and that the three available EMS-induced point mutations cause premature stop codons in the vls ORF. We have generated a null allele that has a stronger phenotype than the EMS mutants. The vlsnull mutant shows that vls+ is required for high levels of Oskar protein to accumulate during oogenesis, for normal posterior localization of Oskar in later stages of oogenesis and for posterior localization of the Vasa protein during the entire process of pole plasm assembly. There is no evidence for vls being dependent on an upstream factor of the posterior pathway, suggesting that Valois protein (Vls) instead acts as a co-factor in the process. Based on the structure of Vls, the function of similar proteins in different systems and our phenotypic analysis, it seems likely that vls may promote posterior patterning by facilitating interactions between different molecules.

Natural Resources in 2025 - Implications for Development : Opportunity of a trunaround: An expert reassessment

In recent years developing countries have faced highly dynamic changes affecting their natural resource base and their potential for development. Taking into account these changes in the development context, InfoResources initiated a critical reassessment of the results of InfoResources Trends 2005 and again invited experts from around the world to assess trends that least developed countries are likely to be facing by 2025. The unanimous signal conveyed by the international experts for this assessment is alarming: The degradation of natural resources is progressing. By 2025 it will reach a point where livelihoods in least developing countries will be significantly threatened and an increasing number of agro-ecosystems will lose their capacity to deliver important services. Expected positive social trends will not suffice as leverage to reverse the degradation of natural resources and thus alleviate poverty and hunger. However, the present reassessment clearly reveals that a change in thinking and a shift in paradigms have begun to take place. However, a turnaround can only succeed if the emerging awareness of the need to reorient policy-making and the economy is followed by concrete action. It will be crucial that policies and institutions regain regulating power over greedy economic forces. This reassessment does not claim to be comprehensive. However, the present publication, which synthesises the experts’ inputs, aims at providing food for thought and initiating discussions.

Proximodistal identity during vertebrate limb regeneration is regulated by Meis homeodomain proteins

The mechanisms by which cells obtain instructions to precisely re-create the missing parts of an organ remain an unresolved question in regenerative biology. Urodele limb regeneration is a powerful model in which to study these mechanisms. Following limb amputation, blastema cells interpret the proximal-most positional identity in the stump to reproduce missing parts faithfully. Classical experiments showed the ability of retinoic acid (RA) to proximalize blastema positional values. Meis homeobox genes are involved in RA-dependent specification of proximal cell identity during limb development. To understand the molecular basis for specifying proximal positional identities during regeneration, we isolated the axolotl Meis homeobox family. Axolotl Meis genes are RA-regulated during both regeneration and embryonic limb development. During limb regeneration, Meis overexpression relocates distal blastema cells to more proximal locations, whereas Meis knockdown inhibits RA proximalization of limb blastemas. Meis genes are thus crucial targets of RA proximalizing activity on blastema cells.

Pericellular fibronectin is required for RhoA-dependent responses to cyclic strain in fibroblasts

To test the hypothesis that the pericellular fibronectin matrix is involved in mechanotransduction, we compared the response of normal and fibronectin-deficient mouse fibroblasts to cyclic substrate strain. Normal fibroblasts seeded on vitronectin in fibronectin-depleted medium deposited their own fibronectin matrix. In cultures exposed to cyclic strain, RhoA was activated, actin-stress fibers became more prominent, MAL/MKL1 shuttled to the nucleus, and mRNA encoding tenascin-C was induced. By contrast, these RhoA-dependent responses to cyclic strain were suppressed in fibronectin knockdown or knockout fibroblasts grown under identical conditions. On vitronectin substrate, fibronectin-deficient cells lacked fibrillar adhesions containing alpha5 integrin. However, when fibronectin-deficient fibroblasts were plated on exogenous fibronectin, their defects in adhesions and mechanotransduction were restored. Studies with fragments indicated that both the RGD-synergy site and the adjacent heparin-binding region of fibronectin were required for full activity in mechanotransduction, but not its ability to self-assemble. In contrast to RhoA-mediated responses, activation of Erk1/2 and PKB/Akt by cyclic strain was not affected in fibronectin-deficient cells. Our results indicate that pericellular fibronectin secreted by normal fibroblasts is a necessary component of the strain-sensing machinery. Supporting this hypothesis, induction of cellular tenascin-C by cyclic strain was suppressed by addition of exogenous tenascin-C, which interferes with fibronectin-mediated cell spreading.

Tenascin-C and tenascin-W in whisker follicle stem cell niches: possible roles in regulating stem cell proliferation and migration

The whisker follicle has CD34-positive stem cells that migrate from their niche near the bulge along the glassy membrane to the whisker bulb, where they participate in the formation of the whisker shaft. Using immunohistochemistry we found the glycoprotein tenascin-C in the fibrous capsule of mouse whisker follicles, along the glassy membrane and in the trabecular region surrounding keratin-15-negative, CD34-positive stem cells. The related glycoprotein tenascin-W is found in the CD34-positive stem cell niche, in nearby trabeculae, and along the glassy membrane. Tenascin-W is also found in the neural stem cell niche of nearby hair follicles. The formation of stress fibers and focal adhesion complexes in CD34-positive whisker-derived stem cells cultured on fibronectin was inhibited by both tenascin-C and tenascin-W, which is consistent with a role for these glycoproteins in promoting the migration of these cells from the niche to the whisker bulb. Tenascin-C, but not tenascin-W, increased the proliferation of whisker follicle stem cells in vitro. Thus, the CD34-positive whisker follicle stem cell niche contains both tenascin-C and tenascin-W, and these glycoproteins may play a role in directing the migration and proliferation of these stem cells.

Ephrin-B1 transduces signals to activate integrin-mediated migration, attachment and angiogenesis.

Ephrin-B/EphB family proteins are implicated in bidirectional signaling and were initially defined through the function of their ectodomain sequences in activating EphB receptor tyrosine kinases. Ephrin-B1-3 are transmembrane proteins sharing highly conserved C-terminal cytoplasmic sequences. Here we use a soluble EphB1 ectodomain fusion protein (EphB1/Fc) to demonstrate that ephrin-B1 transduces signals that regulate cell attachment and migration. EphB1/Fc induced endothelial ephrin-B1 tyrosine phosphorylation, migration and integrin-mediated (alpha(v)beta(3) and alpha(5)beta(1)) attachment and promoted neovascularization, in vivo, in a mouse corneal micropocket assay. Activation of ephrin-B1 by EphB1/Fc induced phosphorylation of p46 JNK but not ERK-1/2 or p38 MAPkinases. By contrast, mutant ephrin-B1s bearing either a cytoplasmic deletion (ephrin-B1DeltaCy) or a deletion of four C-terminal amino acids (ephrin-B1DeltaPDZbd) fail to activate p46 JNK. Transient expression of intact ephin-B1 conferred EphB1/Fc migration responses on CHO cells, whereas the ephrin-B1DeltaCy and ephrin-B1DeltaPDZbd mutants were inactive. Thus ephrin-B1 transduces 'outside-in' signals through C-terminal protein interactions that affect integrin-mediated attachment and migration.

Spatio-temporal Rho GTPase signaling - where are we now?

Rho-family GTPases are molecular switches that transmit extracellular cues to intracellular signaling pathways. Their regulation is likely to be highly regulated in space and in time, but most of what is known about Rho-family GTPase signaling has been derived from techniques that do not resolve these dimensions. New imaging technologies now allow the visualization of Rho GTPase signaling with high spatio-temporal resolution. This has led to insights that significantly extend classic models and call for a novel conceptual framework. These approaches clearly show three things. First, Rho GTPase signaling dynamics occur on micrometer length scales and subminute timescales. Second, multiple subcellular pools of one given Rho GTPase can operate simultaneously in time and space to regulate a wide variety of morphogenetic events (e.g. leading-edge membrane protrusion, tail retraction, membrane ruffling). These different Rho GTPase subcellular pools might be described as 'spatio-temporal signaling modules' and might involve the specific interaction of one GTPase with different guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and effectors. Third, complex spatio-temporal signaling programs that involve precise crosstalk between multiple Rho GTPase signaling modules regulate specific morphogenetic events. The next challenge is to decipher the molecular circuitry underlying this complex spatio-temporal modularity to produce integrated models of Rho GTPase signaling.

Endemic pemphigus foliaceus: towards understanding autoimmune mechanisms of disease development

Fogo selvagem is an endemic form of pemphigus foliaceus (EPF) found in Brazil. Environmental and genetic factors are thought to contribute to the disease, which is associated with pathogenic IgG4 autoantibodies against the desmosomal cadherin desmoglein 1. In this issue, as an additional framework to understand autoimmune mechanisms in EPF, Flores et al. have investigated whether fogo selvagem patients and healthy individuals from endemic areas develop autoantibody responses against other desmosomal cadherins and E-cadherin.

Concentration-dependent isoflurane effects on withdrawal reflexes in pigs and the role of the stimulation paradigm

In this prospective two-phase experimental trial, 10 pigs were anaesthetized twice with isoflurane only. In the first phase, the individual minimum alveolar concentration (MAC) was determined and in the second phase the effects on withdrawal reflexes of increasing end-tidal isoflurane concentrations (from 1.6% to 2.8%) were assessed. Single, 10 and 60 repeated electrical stimulations were used to evoke withdrawal reflexes which were recorded and quantified by electromyography. Recruitment curves for reflex amplitude for increasing stimulation intensities and isoflurane concentrations were constructed. Isoflurane MAC was 1.9+/-0.3%. Reflexes evoked by repeated stimulation were suppressed at isoflurane concentrations significantly higher than those which suppressed complex movements during MAC determination (P=0.014 and P=0.006 for 10 and 60 repeated stimuli respectively). Isoflurane up to 2.8% was still not able to abolish reflex activity evoked by repeated stimulations in all pigs. Single stimulation reflexes were suppressed at significantly lower concentrations than repeated stimulation reflexes (P=0.008 and P=0.004 for 10 and 60 repeated stimuli, respectively). Reflex amplitude was significantly correlated with isoflurane concentration (P<0.001, r=-0.85) independent of the individual MAC. The findings indicate that the level at which isoflurane suppresses withdrawal reflexes is dependent on the stimulation paradigm (single vs. repeated electrical stimulation), and there is limited value in expressing reflex withdrawal suppression in terms of MAC as purposeful and reflex movements are independently affected by isoflurane in individual animals.