RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes

RhoA is a small guanosine-5'-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell-cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA-null keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell-cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC-mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA-null cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA-null keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.     

An increased receptive field of olfactory receptor Or43a in the antennal lobe of Drosophila reduces benzaldehyde-driven avoidance behavior

Most animals orient themselves in their environment through the perception of olfactory cues. In order to gain insight into the principles of olfactory processing in Drosophila, we misexpressed olfactory receptor Or43a in additional olfactory receptor neurons of the third antennal segment using enhancer trap line GH320. The behavioral response of GH320/UAS-or43a flies was changed upon benzaldehyde application. Using the T-maze assay, misexpressing flies performed a reduced avoidance reaction to benzaldehyde as compared with wild type. This reduction of avoidance could be mimicked in wild type flies by exposing them to a mixture of benzaldehyde and ethyl acetate. We therefore conclude that the application of benzaldehyde, an identified ligand of Or43a, resulted in activation of a number of glomeruli in transformed flies in addition to glomerulus DA4, which is the regular target of Or43a expressing neurons. Our results demonstrate the relevance of specific olfactory sensory input and subsequent processing in the antennal lobe for Drosophila behavior.     

Conserving pool-breeding amphibians in human-dominated landscapes through local implementation of Best Development Practices

Seasonal forest pools in the northeastern USA are unique ecosystems whose functions are intimately associated with adjacent upland habitats. This connection, coupled with their small size and ephemeral surface water, has made conservation of pool resources challenging. Seasonal pools provide optimal breeding habitat for animals adapted to temporary waters including ambystomatid salamanders (Ambystoma spp.), wood frogs (Rana sylvatica LeConte), and some invertebrates and plants. To date, wetland conservation efforts have been primarily limited to 2 pathways: land use regulation and preservation. Although both of these pathways have the potential to conserve some pool resources, they are often insufficient to maintain an array of pools in the landscape that support local population dynamics of amphibians. We propose a third pathway – local land-use planning – that can complement regulatory and preservation efforts. This suite of strategies, embodied in our Best Development Practices (BDPs), recognizes that not all pools will be conserved; local governances will need to develop priorities for conservation. The BDPs encourage local governances to (1) proactively identify their pool resources, (2) rank those pools according to their relative ecological value, and (3) establish management procedures and apply recommended guidelines in accordance with the relative rankings. We recommend that pools be ranked using biological criteria (e.g., presence of listed species, presence of breeding species, and egg mass abundance) and on the availability and quality of adjacent terrestrial habitat. We recommend 3 management zones: the pool depression, the pool envelope (i.e., land within 30 m of the pool), and the critical terrestrial habitat (i.e., 30–230 m from the pool). Residential, industrial, and commercial development, which may compromise pool habitat (e.g., through building and road construction, site clearing, stormwater management, and lighting), should follow the recommended guidelines presented in Appendix 1 of this paper. Planning at the watershed level, using such tools as overlay zones, wetland ordinances, and easements, should lead to more effective, long-term management of, at a minimum, the most ecologically important seasonal forest pool resources and will provide developers with clear development guidelines. This process is already being successfully implemented in a number of New England towns.     

Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes

Neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE are members of a family of proteins that use the Arp2/3 complex to stimulate actin assembly in actin-based motile processes. By entering into distinct macromolecular complexes, they act as convergent nodes of different signalling pathways. The role of WAVE in generating lamellipodial protrusion during cell migration is well established. Conversely, the precise cellular functions of N-WASP have remained elusive. Here, we report that Abi1, an essential component of the WAVE protein complex, also has a critical role in regulating N-WASP-dependent function. Consistently, Abi1 binds to N-WASP with nanomolar affinity and, cooperating with Cdc42, potently induces N-WASP activity in vitro. Molecular genetic approaches demonstrate that Abi1 and WAVE, but not N-WASP, are essential for Rac-dependent membrane protrusion and macropinocytosis. Conversely, Abi1 and N-WASP, but not WAVE, regulate actin-based vesicular transport, epidermal growth factor receptor (EGFR) endocytosis, and EGFR and transferrin receptor (TfR) cell-surface distribution. Thus, Abi1 is a dual regulator of WAVE and N-WASP activities in specific processes that are dependent on actin dynamics.     

PDGF-BB enhances alpha1beta1 integrin-mediated activation of the ERK/AP-1 pathway involved in collagen matrix remodeling by rat mesangial cells

Platelet-derived growth factor-BB (PDGF-BB) has been implicated in the pathogenesis of progressive glomerulonephritis (GN). Previous studies have reported that PDGF-BB stimulates mesangial cells (MCs)-induced collagen matrix remodeling through enhancement of alpha1beta1 integrin-dependent migratory activity. To determine the cell signaling pathway responsible for abnormal MC-related mesangial matrix remodeling in progressive GN, we studied the involvement of the extracellular signal-regulated kinase (ERK)/activator protein-1 (AP-1) pathway in PDGF-BB-enhanced collagen gel contraction. Western blotting and gel shift assay revealed that MC-induced gel contraction resulted in ERK activation in parallel with that of AP-1 binding, peaking at 4 h and lasting at least for 24 h. Application of the MEK inhibitor, U0126, and the c-jun/AP-1 inhibitor, curcumin, inhibited gel contraction and AP-1 activity, respectively, dose dependently. PDGF-BB enhanced not only gel contraction but ERK phosphorylation and AP-1 activity by MCs. Marked inhibitory effects on PDGF-BB-induced gel contraction and ERK/AP-1 activity were observed in the presence of either function blocking anti-alpha1- or anti-beta1-integrin antibody or U0126. Consistently, AP-1-inactive MCs expressing a dominant-negative mutant of c-jun showed a significant decrease of PDGF-BB-induced gel contraction as compared with mock-transfected MCs. Finally, migration assay showed that ERK/AP-1 activity is required for PDGF-BB-stimulated alpha1beta1 integrin-dependent MC migration to collagen I. These results indicated that PDGF-BB enhances alpha1beta1 integrin-mediated collagen matrix reorganization through the activation of the ERK/AP-1 pathway that is crucial for MC migration. We conclude that the ERK/AP-1 pathway plays an important role in PDGF-BB-induced alpha1beta1 integrin-dependent collagen matrix remodeling; therefore, the inhibition of its pathway may provide a novel approach to regulate abnormal collagen matrix remodeling in progressive GN.     

Genomic splice-site analysis reveals frequent alternative splicing close to the dominant splice site

Alternative pre-mRNA splicing may be the most efficient and widespread mechanism to generate multiple protein isoforms from single genes. Here, we describe the genomic analysis of one of the most frequent types of alternative pre-mRNA splicing, alternative 5'- and 3'-splice-site selection. Using an EST-based alternative splicing database recording >47,000 alternative splicing events, we determined the frequency and location of alternative 5'- and 3'-splice sites within the human genome. The most common alternative splice sites used in the human genome are located within 6 nucleotides (nt) of the dominant splice site. We show that the EST database overrepresents alternative splicing events that maintain the reading frame, thus supporting the concept that RNA quality-control steps ensure that mRNAs that encode for potentially harmful protein products are destroyed and do not serve as templates for translation. The most frequent location for alternative 5'-splice sites is 4 nt upstream or downstream from the dominant splice site. Sequence analysis suggests that this preference is a consequence of the U1 snRNP binding sequence at the 5'-splice site, which frequently contains a GU dinucleotide 4 nt downstream from the dominant splice site. Surprisingly, approximately 50% of duplicated 3'-YAG splice junctions are subject to alternative splicing. This high probability of alternative 3'-splice-site activation in close proximity of the dominant 3'-splice site suggests that the second step of the splicing may be prone to violate splicing fidelity.     

The structure of the mammalian signal recognition particle (SRP) receptor as prototype for the interaction of small GTPases with Longin domains

The eukaryotic signal recognition particle (SRP) and its receptor (SR) play a central role in co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum. The SR is a heterodimeric complex assembled by the two GTPases SRalpha and SRbeta, which is membrane-anchored. Here we present the 2.45-A structure of mammalian SRbeta in its Mg2+ GTP-bound state in complex with the minimal binding domain of SRalpha termed SRX. SRbeta is a member of the Ras-GTPase superfamily closely related to Arf and Sar1, while SRX belongs to the SNARE-like superfamily with a fold also known as longin domain. SRX binds to the P loop and the switch regions of SRbeta-GTP. The binding mode and structural similarity with other GTPase-effector complexes suggests a co-GAP (GTPase-activating protein) function for SRX. Comparison with the homologous yeast structure and other longin domains reveals a conserved adjustable hydrophobic surface within SRX which is of central importance for the SRbeta-GTP:SRX interface. A helix swap in SRX results in the formation of a dimer in the crystal structure. Based on structural conservation we present the SRbeta-GTP:SRX structure as a prototype for conserved interactions in a variety of GTPase regulated targeting events occurring at endomembranes.     

Cascading predator control interacts with productivity to determine the trophic level of biomass accumulation in a benthic food web

Large-scale exploitation of higher trophic levels by humans, together with global-scale nutrient enrichment, highlights the need to explore interactions between predator loss and resource availability. The hypothesis of exploitation ecosystems suggests that top–down and bottom–up control alternate between trophic levels, resulting in a positive relationship between primary production and the abundance of every second trophic level. Specifically, in food webs with three effective trophic levels, primary producers and predators should increase with primary production, while in food webs with two trophic levels, only herbivores should increase. We provided short-term experimental support for these model predictions in a natural benthic community with three effective trophic levels, where the number of algal recruits, but not the biomass of gastropod grazers, increased with algal production. In contrast, when the food web was reduced to two trophic levels by removing larger predators, the number of algal recruits was unchanged while gastropod grazer biomass increased with algal production. Predator removal only affected the consumer-controlled early life-stages of algae, indicating that both the number of trophic levels and the life-stage development of the producer trophic level determine the propagation of trophic cascades in benthic systems. Our results support the hypothesis that predators interact with resource availability to determine food-web structure.     

A heat wave and dispersal cause dominance shift and decrease biomass in experimental metacommunities

In experimental metacommunities with marine benthic microalgae, we tested whether heat stress changes effects of connectivity and habitat heterogeneity on metacommunity structure and functioning, by manipulating a simulated heat wave, dispersal frequency and a light intensity gradient. We found that all measures of mean local and regional diversity and community biomass significantly declined after the heat wave and showed no sign of recovery. Additionally, dispersal decreased diversity and increased dominance in both the heat stressed and control communities. Together the heat wave and high dispersal frequency induced a dominance shift by spreading a temperature tolerant but low yielding species from its source patches with low light intensity across the metacommunity, an effect that increased with time. Although different species became dominant at high dispersal frequency with and without the heat wave, the shift towards a temperature tolerant species was not sufficient to maintain total community biomass. Thus, short‐term disturbance may cause longer‐term loss of ecosystem function due to dominance shifts in the composition of communities. This study illustrates the importance of employing multispecies approaches when attempting to predict responses of communities to environmental changes.