Twin‐arginine translocation system (tat) mutants of Salmonella are attenuated due to envelope defects,not respiratory defects

The twin‐arginine translocation system (Tat) transports folded proteins across the cytoplasmic membrane and is critical to virulence in Salmonella and other pathogens. Experimental and bioinformatic data indicate that 30 proteins are exported via Tat in Salmonella Typhimurium. However, there are no data linking specific Tat substrates with virulence. We inactivated every Tat‐exported protein and determined the virulence phenotype of mutant strains. Although a tat mutant is highly attenuated, no single Tat‐exported substrate accounts for this virulence phenotype. Rather, the attenuation is due primarily to envelope defects caused by failure to translocate three Tat substrates, the N‐acetylmuramoyl‐l ‐alanine amidases, AmiA and AmiC, and the cell division protein, SufI. Strikingly, neither the amiA amiC nor the sufI mutations alone conferred any virulence defect. Although AmiC and SufI have previously been localized to the divisome, the synthetic phenotypes observed are the first to suggest functional overlap. Many Tat substrates are involved in anaerobic respiration, but we show that a mutant completely deficient in anaerobic respiration retains full virulence in both the oral and systemic phases of infection. Similarly, an obligately aerobic mutant is fully virulent. These results suggest that in the classic mouse model of infection, S. Typhimurium is replicating only in aerobic environments.     

<Emphasis Type="Italic">Dictyostelium</Emphasis> puromycin-sensitive aminopeptidase A is a nucleoplasmic nucleomorphin-binding protein that relocates to the cytoplasm during mitosis

Nucleomorphin (NumA1) is a nucleolar/nucleoplasmic protein linked to cell cycle in Dictyostelium. It interacts with puromycin-sensitive aminopeptidase A (PsaA) which in other organisms is a Zn²⁺-metallopeptidase thought to be involved in cell cycle progression and is involved in several human diseases. Here, we have shown that Dictyostelium PsaA contains domains characteristic of the M1 family of Zn²⁺-metallopeptidases: a GAMEN motif and a Zn²⁺-binding domain. PsaA colocalized with NumA1 in the nucleoplasm in vegetative cells and was also present to a lesser extent in the cytoplasm. The same localization pattern was observed in cells from slugs, however, in fruiting bodies PsaA was only detected in spore nuclei. During mitosis PsaA redistributed mainly throughout the cytoplasm. It possesses a functional nuclear localization signal (⁶⁸⁰RKRF⁶⁸³) necessary for nuclear entry. To our knowledge, this is the first nuclear localization signal identified in a Psa from any organism. Treatment with Ca²⁺ chelators or calmodulin antagonists indicated that neither Ca²⁺ nor calmodulin is involved in PsaA localization. These results are interpreted in terms of the inter-relationship between NumA1 and PsaA in cell function in Dictyostelium.     

Functional changes in the snail statocyst system elicited by microgravity

Background

The mollusk statocyst is a mechanosensing organ detecting the animal''s orientation with respect to gravity. This system has clear similarities to its vertebrate counterparts: a weight-lending mass, an epithelial layer containing small supporting cells and the large sensory hair cells, and an output eliciting compensatory body reflexes to perturbations.

Methodology/Principal Findings

In terrestrial gastropod snail we studied the impact of 16- (Foton M-2) and 12-day (Foton M-3) exposure to microgravity in unmanned orbital missions on: (i) the whole animal behavior (Helix lucorum L.), (ii) the statoreceptor responses to tilt in an isolated neural preparation (Helix lucorum L.), and (iii) the differential expression of the Helix pedal peptide (HPep) and the tetrapeptide FMRFamide genes in neural structures (Helix aspersa L.). Experiments were performed 13–42 hours after return to Earth. Latency of body re-orientation to sudden 90° head-down pitch was significantly reduced in postflight snails indicating an enhanced negative gravitaxis response. Statoreceptor responses to tilt in postflight snails were independent of motion direction, in contrast to a directional preference observed in control animals. Positive relation between tilt velocity and firing rate was observed in both control and postflight snails, but the response magnitude was significantly larger in postflight snails indicating an enhanced sensitivity to acceleration. A significant increase in mRNA expression of the gene encoding HPep, a peptide linked to ciliary beating, in statoreceptors was observed in postflight snails; no differential expression of the gene encoding FMRFamide, a possible neurotransmission modulator, was observed.

Conclusions/Significance

Upregulation of statocyst function in snails following microgravity exposure parallels that observed in vertebrates suggesting fundamental principles underlie gravi-sensing and the organism''s ability to adapt to gravity changes. This simple animal model offers the possibility to describe general subcellular mechanisms of nervous system''s response to conditions on Earth and in space.     

A Highlights from MBoC Selection: Calcium signaling mediates a biphasic mechanoadaptive response of endothelial cells to cyclic mechanical stretch

The vascular system is precisely regulated to adjust blood flow to organismal demand, thereby guaranteeing adequate perfusion under varying physiological conditions. Mechanical forces, such as cyclic circumferential stretch, are among the critical stimuli that dynamically adjust vessel distribution and diameter, but the precise mechanisms of adaptation to changing forces are unclear. We find that endothelial monolayers respond to cyclic stretch by transient remodeling of the vascular endothelial cadherin–based adherens junctions and the associated actomyosin cytoskeleton. Time-resolved proteomic profiling reveals that this remodeling is driven by calcium influx through the mechanosensitive Piezo1 channel, triggering Rho activation to increase actomyosin contraction. As the mechanical stimulus persists, calcium signaling is attenuated through transient down-regulation of Piezo1 protein. At the same time, filamins are phosphorylated to increase monolayer stiffness, allowing mechanoadaptation to restore junctional integrity despite continuing exposure to stretch. Collectively, this study identifies a biphasic response to cyclic stretch, consisting of an initial calcium-driven junctional mechanoresponse, followed by mechanoadaptation facilitated by monolayer stiffening.     

Frustule morphogenesis of raphid pennate diatom <Emphasis Type="Italic">Encyonema ventricosum</Emphasis> (Agardh) Grunow

Diatoms stand out among other microalgae due to the high diversity of species-specific silica frustules whose components (valves and girdle bands) are formed within the cell in special organelles called silica deposition vesicles (SDVs). Research on cell structure and morphogenesis of frustule elements in diatoms of different taxonomic groups has been carried out since the 1950s but is still relevant today. Here, cytological features and valve morphogenesis in the freshwater raphid pennate diatom Encyonema ventricosum (Agardh) Grunow have been studied using light and transmission electron microscopy of cleaned frustules and ultrathin sections of cells, and scanning electron and atomic force microscopy of the frustule surface. Data have been obtained on chloroplast structure: the pyrenoid is spherical, penetrated by a lamella (a stack of two thylakoids); the girdle lamella consists of several short lamellae. The basic stages of frustule morphogenesis characteristic of raphid pennate diatoms have been traced, with the presence of cytoskeletal elements near SDVs being observed throughout this process. Degradation of the plasmalemma and silicalemma is shown to take place when the newly formed valve is released into the space between sister cells. The role of vesicular transport and exocytosis in the gliding of pennate diatoms is discussed.     

Further studies on using multiple-cross mapping (MCM) to map quantitative trait loci

We have completed whole-genome scans for quantitative trait loci (QTLs) associated with acute ethanol-induced activation in the six F₂ intercrosses that can be formed from the C57BL/6J (B6), DBA/2J (D2) , BALB/cJ (C), and LP/J (LP) inbred strains. The goal was to test the hypothesis that given the relatively simple structure of the laboratory mouse genome, the same QTLs will be detected in multiple crosses which in turn will provide support for the strategy of multiple-cross mapping (MCM). QTLs with LOD scores greater than 4 were detected on Chrs 1, 2, 3, 8, 9, 13, 14, and 16. Only for the QTL on distal Chr 1 was there convincing evidence that the same or at least a very similar QTL was detected in multiple crosses. We also mapped the Chr 2 QTL directly in heterogeneous stock (HS) animals derived from the four inbred strains. At G₁₉ the QTL was mapped to an approximately 3-Mbp interval and this interval was associated with a haplotype block with a largely biallelic structure: B6-L:C-D2. We conclude that mapping in HS animals not only provides significantly greater QTL resolution, at least in some cases it provides significantly more information about the QTL haplotype structure.     

A simple mathematical model of signaling resulting from the binding of lipopolysaccharide with Toll-like receptor 4 demonstrates inherent preconditioning behavior

The complex biology of Gram-negative bacterial lipopolysaccharide (LPS) is central to the acute inflammatory response in sepsis and related diseases. Repeated treatment with LPS can lead to desensitization or enhancement of subsequent responses both in vitro and in vivo (a phenomenon known as preconditioning). Previous computational studies have demonstrated a role for anti-inflammatory influences in this process (J. Day, J. Rubin, Y. Vodovotz, C.C. Chow, A. Reynolds, G. Clermont, A reduced mathematical model of the acute inflammatory response: II. Capturing scenarios of repeated endotoxin administration. J. Theor. Biol. 242 (2006) 237). Since LPS signals via Toll-like receptor 4 (TLR4), we created a simple mathematical model in order to address the role of this receptor in both the normal and preconditioned response to LPS. We created a non-linear system of ordinary differential equations, consisting of free LPS, free TLR4, bound complex LPS-TLR4, and an intracellular signaling cascade (lumped into a single variable). We simulate the effects of preconditioning by small and large repeated doses of LPS on the system, varying the timing of the doses as well as the rate of expression of TLR4. Our simulations suggest that a simplified model of LPS/TLR4 signaling can account for complex preconditioning phenomena without invoking a specific signaling inhibition mechanism, but rather based on the dynamics of the signaling response itself, as well as the timing and magnitude of the LPS stimuli.     

Differentiation inducing factor-1 (DIF-1) induces gene and protein expression of the Dictyostelium nuclear calmodulin-binding protein nucleomorphin

The nucleomorphin gene numA1 from Dictyostelium codes for a multi-domain, calmodulin binding protein that regulates nuclear number. To gain insight into the regulation of numA, we assessed the effects of the stalk cell differentiation inducing factor-1 (DIF-1), an extracellular signalling molecule, on the expression of numA1 RNA and protein. For comparison, the extracellular signalling molecules cAMP (mediates chemotaxis, prestalk and prespore differentiation) and ammonia (NH₃/NH₄⁺; antagonizes DIF) were also studied. Starvation, which is a signal for multicellular development, results in a greater than 80% decrease in numA1 mRNA expression within 4 h. Treatment with ammonium chloride led to a greater than 90% inhibition of numA1 RNA expression within 2 h. In contrast, the addition of DIF-1 completely blocked the decrease in numA1 gene expression caused by starvation. Treatment of vegetative cells with cAMP led to decreases in numA1 RNA expression that were equivalent to those seen with starvation. Western blotting after various morphogen treatments showed that the maintenance of vegetative levels of numA1 RNA by DIF-1 in starved cells was reflected in significantly increased numA1 protein levels. Treatment with cAMP and/or ammonia led to decreased protein expression and each of these morphogens suppressed the stimulatory effects of DIF-1. Protein expression levels of CBP4a, a calcium-dependent binding partner of numA1, were regulated in the same manner as numA1 suggesting this potential co-regulation may be related to their functional relationship. NumA1 is the first calmodulin binding protein shown to be regulated by developmental morphogens in Dictyostelium being upregulated by DIF-1 and down-regulated by cAMP and ammonia.     

The Nup107-160 nucleoporin complex is required for correct bipolar spindle assembly

The Nup107-160 complex is a critical subunit of the nuclear pore. This complex localizes to kinetochores in mitotic mammalian cells, where its function is unknown. To examine Nup107-160 complex recruitment to kinetochores, we stained human cells with antisera to four complex components. Each antibody stained not only kinetochores but also prometaphase spindle poles and proximal spindle fibers, mirroring the dual prometaphase localization of the spindle checkpoint proteins Mad1, Mad2, Bub3, and Cdc20. Indeed, expanded crescents of the Nup107-160 complex encircled unattached kinetochores, similar to the hyperaccumulation observed of dynamic outer kinetochore checkpoint proteins and motors at unattached kinetochores. In mitotic Xenopus egg extracts, the Nup107-160 complex localized throughout reconstituted spindles. When the Nup107-160 complex was depleted from extracts, the spindle checkpoint remained intact, but spindle assembly was rendered strikingly defective. Microtubule nucleation around sperm centrosomes seemed normal, but the microtubules quickly disassembled, leaving largely unattached sperm chromatin. Notably, Ran-GTP caused normal assembly of microtubule asters in depleted extracts, indicating that this defect was upstream of Ran or independent of it. We conclude that the Nup107-160 complex is dynamic in mitosis and that it promotes spindle assembly in a manner that is distinct from its functions at interphase nuclear pores.