NPF motifs in the vaccinia virus protein A36 recruit intersectin-1 to promote Cdc42:N-WASP-mediated viral release from infected cells.

During its egress, vaccinia virus transiently recruits AP-2 and clathrin after fusion with the plasma membrane. This recruitment polarizes the viral protein A36 beneath the virus, enhancing actin polymerization and the spread of infection. We now demonstrate that three NPF motifs in the C-terminus of A36 recruit AP-2 and clathrin by interacting directly with the Epsin15 homology domains of Eps15 and intersectin-1. A36 is the first identified viral NPF motif containing protein shown to interact with endocytic machinery. Vaccinia still induces actin tails in the absence of the A36 NPF motifs. Their loss, however, reduces the cell-to-cell spread of vaccinia. This is due to a significant reduction in virus release from infected cells, as the lack of intersectin-1 recruitment leads to a loss of Cdc42 activation, impairing N-WASP-driven Arp2/3-mediated actin polymerization. Our results suggest that initial A36-mediated virus release plays a more important role than A36-driven super-repulsion in promoting the cell-to-cell spread of vaccinia.

A human interactome in three quantitative dimensions organized by stoichiometries and abundances.

The organization of a cell emerges from the interactions in protein networks. The interactome is critically dependent on the strengths of interactions and the cellular abundances of the connected proteins, both of which span orders of magnitude. However, these aspects have not yet been analyzed globally. Here, we have generated a library of HeLa cell lines expressing 1,125 GFP-tagged proteins under near-endogenous control, which we used as input for a next-generation interaction survey. Using quantitative proteomics, we detect specific interactions, estimate interaction stoichiometries, and measure cellular abundances of interacting proteins. These three quantitative dimensions reveal that the protein network is dominated by weak, substoichiometric interactions that play a pivotal role in defining network topology. The minority of stable complexes can be identified by their unique stoichiometry signature. This study provides a rich interaction dataset connecting thousands of proteins and introduces a framework for quantitative network analysis.

Targeting Human Long Noncoding Transcripts by Endoribonuclease-Prepared siRNAs.

Broad sequencing enterprises such as the FANTOM or ENCODE projects have substantially extended our knowledge of the human transcriptome. They have revealed that a large portion of genomic DNA is actively transcribed and have identified a plethora of novel transcripts. Many newly identified transcripts belong to the class of long noncoding RNAs (lncRNAs), which range from a few hundred bases to multiple kilobases in length and harbor no protein-coding potential. Although the biological activity of some lncRNAs is understood, the functions of most lncRNAs remain elusive. Tools that allow rapid and cost-effective access to functional data of lncRNAs are therefore essential. Here, we describe the construction and validation of an endoribonuclease-prepared siRNA (esiRNA) library designed to target 1779 individual human lncRNAs by RNA interference. We present a compendium of lncRNA expression data for 11 human cancer cell lines. Furthermore, we show that the resource is suitable for combined knockdown and localization analysis. We discuss challenges in sequence annotation of lncRNAs with respect to their often low and cell type-specific expression and specify esiRNAs that are suitable for targeting lncRNAs in commonly used human cell lines.

WIP provides an essential link between Nck and N-WASP during Arp2/3-dependent actin polymerization.

Nck links phosphotyrosine-based signaling to Arp2/3-dependent actin polymerization during many different cellular processes as well as actin-based motility of enteropathogenic Escherichia coli (EPEC), vaccinia, and other vertebrate poxviruses by interacting with N-WASP/WASP. Nck also binds WASP-interacting protein (WIP), which inhibits the ability of N-WASP to activate the Arp2/3 complex until it receives an appropriate signaling input. Using mouse embryonic fibroblasts (MEFs) lacking Nck, WIP, or N-WASP, we have investigated whether an interaction of Nck with both WIP and N-WASP is required for their recruitment to vaccinia during Arp2/3-dependent actin assembly. We find that WIP or its homolog WIRE is required for N-WASP recruitment and actin-based motility of the virus. WIP contains two Nck-binding sites and is recruited to the virus, bound to N-WASP, by interacting with the second SH3 domain of Nck. N-WASP also contains two Nck-binding sites, but its recruitment is dependent on its interaction with WIP rather than Nck. The first and third SH3 domains of Nck are not required to recruit the WIP:N-WASP complex but are essential to stimulate actin assembly. We have established that WIP acts as an essential link between Nck and N-WASP. Our observations provide important insights into the hierarchy and connections in one of the major cellular signaling networks stimulating Arp2/3 complex-dependent actin polymerization.

The rate of N-WASP exchange limits the extent of ARP2/3-complex-dependent actin-based motility.

Understanding cell motility will require detailed knowledge not only of the localization of signalling networks regulating actin polymerization, but also of their dynamics. Unfortunately, many signalling networks are not amenable to such analysis, as they are frequently transient and dispersed. By contrast, the signalling pathways used by pathogens undergoing actin-based motility are highly localized and operate in a constitutive fashion. Taking advantage of this, we have analysed the dynamics of neuronal Wiskott-Aldrich syndrome protein (N-WASP), WASP-interacting protein (WIP), GRB2 and NCK, which are required to stimulate actin-related protein (ARP)2/3-complex-dependent actin-based motility of vaccinia virus, using fluorescence recovery after photobleaching. Here we show that all four proteins are rapidly exchanging, albeit at different rates, and that the turnover of N-WASP depends on its ability to stimulate ARP2/3-complex-mediated actin polymerization. Conversely, disruption of the interaction of N-WASP with GRB2 and/or the barbed ends of actin filaments increases its exchange rate and results in a faster rate of virus movement. We suggest that the exchange rate of N-WASP controls the rate of ARP2/3-complex-dependent actin-based motility by regulating the extent of actin polymerization by antagonizing filament capping.

Abl collaborates with Src family kinases to stimulate actin-based motility of vaccinia virus.

Local activation of Src at the plasma membrane by extracellular vaccinia virus results in a signalling cascade that acts to stimulate actin polymerization beneath the virus to enhance its cell-to-cell spread. Initiation of this signalling cascade involves Src-mediated phosphorylation of tyrosine 112 and 132 of the viral membrane protein A36R. Here we show that recruitment of Src is dependent on its myristoylation and an interaction with A36R upstream of tyrosine 112 and 132. We further show that Src, Fyn and Yes have unique specificities towards these tyrosine residues. Using cell lines deficient in Src, Fyn and Yes, we demonstrate that multiple Src family members can stimulate vaccinia-induced actin polymerization and also uncover a role for Abl family kinases. Additionally, Abl and Arg are able to phosphorylate A36R in vitro and are recruited to vaccinia-induced actin tails. The ability of multiple families of tyrosine kinases to directly phosphorylate A36R ensures robust cell-to-cell spread of vaccinia virus will occur under a variety of cellular conditions.

The leading edge is a lipid diffusion barrier.

Actin polymerization drives many cellular events, including endocytosis, pathogen rocketing, and cell spreading. Force generation and polymerization regulation are intimately linked where an actin meshwork attaches to, and pushes against, an interface. We reasoned that interaction with actin filament plus-ends might stabilize the position of components within the plasma membrane at the leading edge, thereby slowing the diffusion of lipids within the bilayer where filament growth occurs. To test this hypothesis we focally labeled the outer membrane leaflet of migrating keratocytes and compared the initial diffusion of carbocyanine dyes in the dorsal and ventral lamellipodium membranes using sequential TIRF and epi-fluorescent imaging. Global diffusion analysis shows that lateral mobility of lipids in the outer membrane leaflet is blocked at the leading edge during protrusion. Cytochalasin treatment abolished this diffusion barrier, but we found no evidence to support the involvement of membrane microdomains. Our results demonstrate the immobilization of membrane components at the leading edge, and suggest that interaction between actin filaments and the plasma membrane is mediated by densely packed molecular complexes. We propose that actin polymerization traps regulatory proteins at the leading edge in a positive-feedback loop.

Stimulation of alkaline phosphatase activity in Ishikawa cells induced by various phytoestrogens and synthetic estrogens.

Xenoestrogens, phytoestrogens and synthetic estrogens, are able to bind to estrogen receptors, and to mimic estrogenic activities in a cell and tissue specific manner. For the characterization of environmental estrogens mainly mammary derived and yeast based models have been used. The aim of this study was therefore to assess selected natural and synthetic compounds in an endometrial derived model. We measured the relative estrogenic potency of phytoestrogens (genistein, daidzein, coumestrol, some naringenins), synthetic estrogens (bisphenol A, octylphenol, nonylphenol, o,p'-DDT), mycoestrogen (zearalanone) as well as extracts of Cimicifuga racemosa on alkaline phosphatase (AlkP) activity in the endometrial derived adenocarcinoma cell line Ishikawa. We used a modified multiwell plate in vitro bioassay based on the estrogen-specific and dose-dependent enhancement of AlkP activity in this cell line. Estradiol, which induced AlkP at levels as low as 10(-8)M, was used as positive control. Most of the compounds studied showed a clear dose-dependent estrogenic effect. Compared to the vehicle control (ethanol) all phyto- and mycoestrogens, stimulated the AlkP activity 2-4-fold at a concentration of 10(-6)M. The synthetic chemicals bisphenol A and nonylphenol showed an effect at 10(-6)M, octylphenol at 10(-5)M. Effects of o,p'-DTT could not be measured. ICI 182,780, a pure estrogen receptor antagonist, significantly inhibited these effects. The latter result demonstrated the estrogen receptor dependency of this process. In summary, most of the phytoestrogens and industrial chemicals tested, behaved as estrogen receptor agonists in terms of the stimulation of AlkP activity.