Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin

Slingshot (SSH) phosphatases and LIM kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3zeta, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3zeta binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton.     

The impact of mutations in the quorum sensing systems of Aeromonas hydrophila, Vibrio anguillarum and Vibrio harveyi on their virulence towards gnotobiotically cultured Artemia franciscana

Disruption of quorum sensing, bacterial cell-to-cell communication by means of small signal molecules, has been suggested as a new anti-infective strategy for aquaculture. However, data about the impact of quorum sensing on the virulence of aquatic pathogens are scarce. In this study, a model system using gnotobiotically cultured Artemia franciscana was developed in order to determine the impact of mutations in the quorum sensing systems of Aeromonas hydrophila, Vibrio anguillarum and V. harveyi on their virulence. Mutations in the autoinducer 2 (AI-2) synthase gene luxS, the AI-2 receptor gene luxP or the response regulator gene luxO of the dual channel quorum sensing system of V. harveyi abolished virulence of the strain towards Artemia. Moreover, the addition of an exogenous source of AI-2 could restore the virulence of an AI-2 non-producing mutant. In contrast, none of the mutations in either the acylated homoserine lactone (AHL)-mediated component of the V. harveyi system or the quorum sensing systems of Ae. hydrophila and V. anguillarum had an impact on virulence of these bacteria towards Artemia. Our results indicate that disruption of quorum sensing could be a good alternative strategy to combat infections caused by V. harveyi.     

Rho-kinase controls cell shape changes during cytokinesis

BACKGROUND: Animal cell cytokinesis is characterized by a sequence of dramatic cortical rearrangements. How these are coordinated and coupled with mitosis is largely unknown. To explore the initiation of cytokinesis, we focused on the earliest cell shape change, cell elongation, which occurs during anaphase B and prior to cytokinetic furrowing. RESULTS: Using RNAi and live video microscopy in Drosophila S2 cells, we implicate Rho-kinase (Rok) and myosin II in anaphase cell elongation. rok RNAi decreased equatorial myosin II recruitment, prevented cell elongation, and caused a remarkable spindle defect where the spindle poles collided with an unyielding cell cortex and the interpolar microtubules buckled outward as they continued to extend. Disruption of the actin cytoskeleton with Latrunculin A, which abolishes cortical rigidity, suppressed the spindle defect. rok RNAi also affected furrowing, which was delayed and slowed, sometimes distorted, and in severe cases blocked altogether. Codepletion of the myosin binding subunit (Mbs) of myosin phosphatase, an antagonist of myosin II activation, only partially suppressed the cell-elongation defect and the furrowing delay, but prevented cytokinesis failures induced by prolonged rok RNAi. The marked sensitivity of cell elongation to Rok depletion was highlighted by RNAi to other genes in the Rho pathway, such as pebble, racGAP50C, and diaphanous, which had profound effects on furrowing but lesser effects on elongation. CONCLUSIONS: We show that cortical changes underlying cell elongation are more sensitive to depletion of Rok and myosin II, in comparison to other regulators of cytokinesis, and suggest that a distinct regulatory pathway promotes cell elongation.     

Killer whales are capable of vocal learning

The production learning of vocalizations by manipulation of the sound production organs to alter the physical structure of sound has been demonstrated in only a few mammals. In this natural experiment, we document the vocal behaviour of two juvenile killer whales, Orcinus orca, separated from their natal pods, which are the only cases of dispersal seen during the three decades of observation of their populations. We find mimicry of California sea lion (Zalophus californianus) barks, demonstrating the vocal production learning ability for one of the calves. We also find differences in call usage (compared to the natal pod) that may reflect the absence of a repertoire model from tutors or some unknown effect related to isolation or context.     

The N-terminal domain of hepatocyte growth factor inhibits the angiogenic behavior of endothelial cells independently from binding to the c-met receptor

Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an important role in complex biological processes such as embryogenesis, tissue regeneration, cancerogenesis, and angiogenesis. HGF promotes cell proliferation, survival, motility, and morphogenesis through binding to its receptor, a transmembrane tyrosine kinase encoded by the MET proto-oncogene (c-met). Structurally speaking, HGF is a polypeptide related to the enzymes of the blood coagulation cascade. Thus, it comprises kringle domains that in some other proteins have been shown to be responsible for the anti-angiogenic activity. To check whether the isolated kringles of HGF were able to inhibit angiogenesis, we produced them as recombinant proteins and compared their biological activity with that of the recombinant HGF N-terminal domain (N). We showed that (i) none of the isolated HGF kringle exhibits an anti-angiogenic activity; (ii) N is a new anti-angiogenic polypeptide; (iii) the inhibitory action of N is not specific toward HGF, because it antagonized the angiogenic activity of other growth factors, such as fibroblast growth factor-2 and vascular endothelial growth factor; and (iv) in contrast with full-length HGF, N does not bind to the c-met receptor in vitro, but fully retains its heparin-binding capacity. Our results suggest that N inhibits angiogenesis not by disrupting the HGF/c-met interaction but rather by interfering with the endothelial glycosaminoglycans, which are the secondary binding sites of HGF.     

Nucleus-encoded,plastid-targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) indicates a single origin for chromalveolate plastids

Plastids (the photosynthetic organelles of plants and algae) originated through endosymbiosis between a cyanobacterium and a eukaryote and subsequently spread to other eukaryotes by secondary endosymbioses between two eukaryotes. Mounting evidence favors a single origin for plastids of apicomplexans, cryptophytes, dinoflagellates, haptophytes, and heterokonts (together with their nonphotosynthetic relatives, termed chromalveolates), but so far, no single molecular marker has been described that supports this common origin. One piece of evidence comes from plastid-targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which originated by a gene duplication of the cytosolic form. However, no plastid GAPDH has been characterized from haptophytes, leaving an important piece of the puzzle missing. We have sequenced genes encoding cytosolic, mitochondrion-targeted, and plastid-targeted GAPDH proteins from a number of haptophytes and heterokonts and found haptophyte homologs that branch within a strongly supported clade of chromalveolate plastid-targeted genes, being more closely related to an apicomplexan homolog than was expected. The evolution of plastid-targeted GAPDH supports red algal ancestry of apicomplexan plastids and raises a number of questions about the importance of plastid loss and the possibility of cryptic plastids in nonphotosynthetic lineages such as ciliates.     

Gene expression patterns in ovarian carcinomas

We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers.     

New Insights into the Assembly of Bacterial Secretins: STRUCTURAL STUDIES OF THE PERIPLASMIC DOMAIN OF XcpQ FROM PSEUDOMONAS AERUGINOSA*

The type II secretion system is a multiprotein assembly spanning the inner and outer membranes in Gram-negative bacteria. It is found in almost all pathogenic bacteria where it contributes to virulence, host tissue colonization, and infection. The exoproteins are secreted across the outer membrane via a large translocation channel, the secretin, which typically adopts a dodecameric structure. These secretin channels have large periplasmic N-terminal domains that reach out into the periplasm for communication with the inner membrane platform and with a pseudopilus structure that spans the periplasm. Here we report the crystal structure of the N-terminal periplasmic domain of the secretin XcpQ from Pseudomonas aeruginosa, revealing a two-lobe dimeric assembly featuring parallel subunits engaging in well defined interactions at the tips of each lobe. We have employed structure-based engineering of disulfide bridges and native mass spectrometry to show that the periplasmic domain of XcpQ dimerizes in a concentration-dependent manner. Validation of these insights in the context of cellular full-length XcpQ and further evaluation of the functionality of disulfide-linked XcpQ establishes that the basic oligomerization unit of XcpQ is a dimer. This is consistent with the notion that the dodecameric secretin assembles as a hexamer of dimers to ensure correct projection of the N-terminal domains into the periplasm. Therefore, our studies provide a key conceptual advancement in understanding the assembly principles and dynamic function of type II secretion system secretins and challenge recent studies reporting monomers as the basic subunit of the secretin oligomer.