ABSTRACT
We have developed a new benign means of reversibly breaking emulsions and latexes by using "switchable water", an aqueous solution of switchable ionic strength. The conventional surfactant sodium dodecyl sulfate (SDS) is not normally stimuli-responsive when CO2 is used as the stimulus but becomes CO2 -responsive or "switchable" in the presence of a switchable water additive. In particular, changes in the air/water surface tension and oil/water interfacial tension can be triggered by addition and removal of CO2 . A switchable water additive, N,N-dimethylethanolamine (DMEA), was found to be an effective and efficient additive for the reversible reduction of interfacial tension and can lower the tension of the dodecane/water interface in the presence of SDS surfactant to ultra-low values at very low additive concentrations. Switchable water was successfully used to reversibly break an emulsion containing SDS as surfactant, and dodecane as organic liquid. Also, the addition of CO2 and switchable water can result in aggregation of polystyrene (PS) latexes; the later removal of CO2 neutralizes the DMEA and decreases the ionic strength allowing for the aggregated PS latex to be redispersed and recovered in its original state.
ABSTRACT
Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.
Subject(s)
4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/metabolism , Actins/metabolism , Cytoskeletal Proteins/metabolism , Actin-Related Protein 2 , Actin-Related Protein 3 , Actins/chemistry , Alleles , Cell Nucleus/metabolism , Centrifugation, Density Gradient , Gene Deletion , Gene Expression Regulation, Fungal , Genetic Complementation Test , Immunoblotting , Kinetics , Microscopy, Fluorescence , Mutation , Plasmids/metabolism , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Saccharomyces cerevisiae/metabolism , Sucrose/pharmacology , Temperature , Time Factors , Two-Hybrid System TechniquesABSTRACT
We have used comprehensive synthetic lethal screens and biochemical assays to examine the biological role of the yeast amphiphysin homologues Rvs161p and Rvs167p, two proteins that play a role in regulation of the actin cytoskeleton, endocytosis, and sporulation. We found that unlike some forms of amphiphysin, Rvs161p-Rvs167p acts as an obligate heterodimer during vegetative growth and neither Rvs161p nor Rvs167p forms a homodimer in vivo. RVS161 and RVS167 have an identical set of 49 synthetic lethal interactions, revealing functions for the Rvs proteins in cell polarity, cell wall synthesis, and vesicle trafficking as well as a shared role in mating. Consistent with these roles, we show that the Rvs167p-Rvs161p heterodimer, like its amphiphysin homologues, can bind to phospholipid membranes in vitro, suggesting a role in vesicle formation and/or fusion. Our genetic screens also reveal that the interaction between Abp1p and the Rvs167p Src homology 3 (SH3) domain may be important under certain conditions, providing the first genetic evidence for a role for the SH3 domain of Rvs167p. Our studies implicate heterodimerization of amphiphysin family proteins in various functions related to cell polarity, cell integrity, and vesicle trafficking during vegetative growth and the mating response.
Subject(s)
Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Actins/metabolism , Animals , Carrier Proteins/metabolism , Cattle , Cell Line , Cell Membrane/metabolism , Chitin/metabolism , Dimerization , Diploidy , Gene Deletion , Genes, Mating Type, Fungal , Genetic Complementation Test , Insecta , Microfilament Proteins , Pheromones/pharmacology , Protein Binding , Protein Structure, Quaternary , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/drug effects , Thermodynamics , src Homology DomainsABSTRACT
A genetic interaction network containing approximately 1000 genes and approximately 4000 interactions was mapped by crossing mutations in 132 different query genes into a set of approximately 4700 viable gene yeast deletion mutants and scoring the double mutant progeny for fitness defects. Network connectivity was predictive of function because interactions often occurred among functionally related genes, and similar patterns of interactions tended to identify components of the same pathway. The genetic network exhibited dense local neighborhoods; therefore, the position of a gene on a partially mapped network is predictive of other genetic interactions. Because digenic interactions are common in yeast, similar networks may underlie the complex genetics associated with inherited phenotypes in other organisms.