Structure and control of the actin regulatory WAVE complex.

Members of the Wiskott-Aldrich syndrome protein (WASP) family control cytoskeletal dynamics by promoting actin filament nucleation with the Arp2/3 complex. The WASP relative WAVE regulates lamellipodia formation within a 400-kilodalton, hetero-pentameric WAVE regulatory complex (WRC). The WRC is inactive towards the Arp2/3 complex, but can be stimulated by the Rac GTPase, kinases and phosphatidylinositols. Here we report the 2.3-ångstrom crystal structure of the WRC and complementary mechanistic analyses. The structure shows that the activity-bearing VCA motif of WAVE is sequestered by a combination of intramolecular and intermolecular contacts within the WRC. Rac and kinases appear to destabilize a WRC element that is necessary for VCA sequestration, suggesting the way in which these signals stimulate WRC activity towards the Arp2/3 complex. The spatial proximity of the Rac binding site and the large basic surface of the WRC suggests how the GTPase and phospholipids could cooperatively recruit the complex to membranes.

Hierarchical regulation of WASP/WAVE proteins.

Members of the Wiskott-Aldrich syndrome protein (WASP) family control actin dynamics in eukaryotic cells by stimulating the actin nucleating activity of the Arp2/3 complex. The prevailing paradigm for WASP regulation invokes allosteric relief of autoinhibition by diverse upstream activators. Here we demonstrate an additional level of regulation that is superimposed upon allostery: dimerization increases the affinity of active WASP species for Arp2/3 complex by up to 180-fold, greatly enhancing actin assembly by this system. This finding explains a large and apparently disparate set of observations under a common mechanistic framework. These include WASP activation by the bacterial effector EspFu and a large number of SH3 domain proteins, the effects on WASP of membrane localization/clustering and assembly into large complexes, and cooperativity between different family members. Allostery and dimerization act in hierarchical fashion, enabling WASP/WAVE proteins to integrate different classes of inputs to produce a wide range of cellular actin responses.

The VieB auxiliary protein negatively regulates the VieSA signal transduction system in Vibrio cholerae.

BACKGROUND: Vibrio cholerae is a facultative pathogen that lives in the aquatic environment and the human host. The ability of V. cholerae to monitor environmental changes as it transitions between these diverse environments is vital to its pathogenic lifestyle. One way V. cholerae senses changing external stimuli is through the three-component signal transduction system, VieSAB, which is encoded by the vieSAB operon. The VieSAB system plays a role in the inverse regulation of biofilm and virulence genes by controlling the concentration of the secondary messenger, cyclic-di-GMP. While the sensor kinase, VieS, and the response regulator, VieA, behave similar to typical two-component phosphorelay systems, the role of the auxiliary protein, VieB, is unclear. RESULTS: Here we show that VieB binds to VieS and inhibits its autophosphorylation and phosphotransfer activity thus preventing phosphorylation of VieA. Additionally, we show that phosphorylation of the highly conserved Asp residue in the receiver domain of VieB regulates the inhibitory activity of VieB. CONCLUSION: Taken together, these data point to an inhibitory role of VieB on the VieSA phosphorelay, allowing for additional control over the signal output. Insight into the function and regulatory mechanism of the VieSAB system improves our understanding of how V. cholerae controls gene expression as it transitions between the aquatic environment and human host.

A genome-wide approach to discovery of small RNAs involved in regulation of virulence in Vibrio cholerae.

Small RNAs (sRNAs) are becoming increasingly recognized as important regulators in bacteria. To investigate the contribution of sRNA mediated regulation to virulence in Vibrio cholerae, we performed high throughput sequencing of cDNA generated from sRNA transcripts isolated from a strain ectopically expressing ToxT, the major transcriptional regulator within the virulence gene regulon. We compared this data set with ToxT binding sites determined by pulldown and deep sequencing to identify sRNA promoters directly controlled by ToxT. Analysis of the resulting transcripts with ToxT binding sites in cis revealed two sRNAs within the Vibrio Pathogenicity Island. When deletions of these sRNAs were made and the resulting strains were competed against the parental strain in the infant mouse model of V. cholerae colonization, one, TarB, displayed a variable colonization phenotype dependent on its physiological state at the time of inoculation. We identified a target of TarB as the mRNA for the secreted colonization factor, TcpF. We verified negative regulation of TcpF expression by TarB and, using point mutations that disrupted interaction between TarB and tpcF mRNA, showed that loss of this negative regulation was primarily responsible for the colonization phenotype observed in the TarB deletion mutant.

PhoB regulates both environmental and virulence gene expression in Vibrio cholerae.

Vibrio cholerae is a facultative pathogen that thrives in two nutritionally disparate environments, aquatic and human small intestine. Phosphate (P(i) ) is an essential nutrient that is limited in aquatic ecosystems and of unknown availability in the small intestine. Here, we show that the P(i) (Pho) regulon, which is controlled by the P(i)-specific transporter (Pst) and two-component system PhoBR, is required for V. cholerae survival in both environments, though for differing reasons. While induction of P(i) acquisition systems including Pst is critical for survival in the aquatic environment, regulation of virulence genes by PhoB and not P(i) transport per se is required for colonization of the small intestine. We show that PhoB regulates virulence genes by directly controlling expression of a key upstream transcriptional regulator, tcpPH. Thus, the Pho regulon includes virulence genes and represents a diverse gene set essential to pathogenic V. cholerae throughout its life cycle.

Predictors of stimulation-induced seizures during perirolandic glioma resection using intraoperative mapping techniques.

BACKGROUND: Intraoperative mapping techniques maximize safety and efficacy during perirolandic glioma resection but may induce seizures and limit the procedure. We aim to report the incidence and predictors of stimulation-induced seizures during mapping either patient is awake or under general anesthesia (GA). METHODS: Retrospective analysis of 64 patients (40 awake and 24 GA) with perirolandic glioma underwent resection using intraoperative mapping techniques between 2014 and 2019. Preoperative data, operative details, postoperative neurological status, and extent of resection (EOR) were analyzed. Predictors of intraoperative seizures were assessed. RESULTS: The mean cortical and subcortical stimulation intensities needed to evoke motor responses were significantly lower in awake cases than in GA patients (4.9 ± 0.42 vs. 8.9 ± 1.2 mA) and (8.3 ± 0.62 vs. 12.1 ± 1.1 mA), respectively (P = 0.01). Incidence of intraoperative seizures was lower but statistically non-significant in awake cases (10% vs. 12.5%) (P = 0.76). Preoperative multiple antiepileptic drugs (AEDs) (P = 0.03) and low-grade glioma (P = 0.04) were statistically significant predictors for intraoperative seizures. Mean EOR in awake cases was 92.03% and 90.05% in GA cases (P = 0.23). Postoperative deficits were permanent after 3 months only in 5% of awake patients versus 8.3% of GA group (P = 0.59). CONCLUSION: Awake craniotomy with intraoperative mapping can be done safely for perirolandic gliomas with lower but statistically nonsignificant incidence of intraoperative seizures and this could be attributed to statistically significant lower stimulation intensities required for mapping. Preoperative multiple AEDs and low-grade glioma are significant predictors for intraoperative seizures.

The WAVE regulatory complex is inhibited.

The WAVE regulatory complex (WRC) transmits information from the Rac GTPase to the actin nucleator Arp2/3 complex. We have reconstituted recombinant human and Drosophila WRC in several forms and shown that they are inactive toward Arp2/3 complex but can be activated by Rac in a nucleotide-dependent fashion. Our observations identify core components needed for WAVE inhibition, reconcile contradictory existing mechanisms and reveal common regulatory principles for the WAVE/WASP family of proteins.