Publications (7)128.83 Total impact
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Article: Dynamic actin remodeling during epithelial-mesenchymal transition depends on increased moesin expression.
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ABSTRACT: Remodeling of actin filaments is necessary for epithelial-mesenchymal transition (EMT); however, understanding of how this is regulated in real time is limited. We used an actin filament reporter and high-resolution live-cell imaging to analyze the regulated dynamics of actin filaments during transforming growth factor-β-induced EMT of mammary epithelial cells. Progressive changes in cell morphology were accompanied by reorganization of actin filaments from thin cortical bundles in epithelial cells to thick, parallel, contractile bundles that disassembled more slowly but remained dynamic in transdifferentiated cells. We show that efficient actin filament remodeling during EMT depends on increased expression of the ezrin/radixin/moesin (ERM) protein moesin. Cells suppressed for moesin expression by short hairpin RNA had fewer, thinner, and less stable actin bundles, incomplete morphological transition, and decreased invasive capacity. These cells also had less α-smooth muscle actin and phosphorylated myosin light chain in cortical patches, decreased abundance of the adhesion receptor CD44 at membrane protrusions, and attenuated autophosphorylation of focal adhesion kinase. Our findings suggest that increased moesin expression promotes EMT by regulating adhesion and contractile elements for changes in actin filament organization. We propose that the transciptional program driving EMT controls progressive remodeling of actin filament architectures.Molecular biology of the cell 12/2011; 22(24):4750-64. · 5.98 Impact Factor -
Article: The biologically relevant targets and binding affinity requirements for the function of the yeast actin-binding protein 1 Src-homology 3 domain vary with genetic context.
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ABSTRACT: Many protein-protein interaction domains bind to multiple targets. However, little is known about how the interactions of a single domain with many proteins are controlled and modulated under varying cellular conditions. In this study, we investigated the in vivo effects of Abp1p SH3 domain mutants that incrementally reduce target-binding affinity in four different yeast mutant backgrounds in which Abp1p activity is essential for growth. Although the severity of the phenotypic defects observed generally increased as binding affinity was reduced, some genetic backgrounds (prk1 Delta and sla1 Delta) tolerated large affinity reductions while others (sac6 Delta and sla2 Delta) were much more sensitive to these reductions. To elucidate the mechanisms behind these observations, we determined that Ark1p is the most important Abp1p SH3 domain interactor in prk1 Delta cells, but that interactions with multiple targets, including Ark1p and Scp1p, are required in the sac6 Delta background. We establish that the Abp1p SH3 domain makes different, functionally important interactions under different genetic conditions, and these changes in function are reflected by changes in the binding affinity requirement of the domain. These data provide the first evidence of biological relevance for any Abp1p SH3 domain-mediated interaction. We also find that considerable reductions in binding affinity are tolerated by the cell with little effect on growth rate, even when the actin cytoskeletal morphology is significantly perturbed.Genetics 06/2007; 176(1):193-208. · 4.01 Impact Factor -
Article: The synthetic genetic interaction spectrum of essential genes.
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ABSTRACT: The nature of synthetic genetic interactions involving essential genes (those required for viability) has not been previously examined in a broad and unbiased manner. We crossed yeast strains carrying promoter-replacement alleles for more than half of all essential yeast genes to a panel of 30 different mutants with defects in diverse cellular processes. The resulting genetic network is biased toward interactions between functionally related genes, enabling identification of a previously uncharacterized essential gene (PGA1) required for specific functions of the endoplasmic reticulum. But there are also many interactions between genes with dissimilar functions, suggesting that individual essential genes are required for buffering many cellular processes. The most notable feature of the essential synthetic genetic network is that it has an interaction density five times that of nonessential synthetic genetic networks, indicating that most yeast genetic interactions involve at least one essential gene.Nature Genetics 11/2005; 37(10):1147-52. · 35.53 Impact Factor -
Article: Exploration of essential gene functions via titratable promoter alleles.
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ABSTRACT: Nearly 20% of yeast genes are required for viability, hindering genetic analysis with knockouts. We created promoter-shutoff strains for over two-thirds of all essential yeast genes and subjected them to morphological analysis, size profiling, drug sensitivity screening, and microarray expression profiling. We then used this compendium of data to ask which phenotypic features characterized different functional classes and used these to infer potential functions for uncharacterized genes. We identified genes involved in ribosome biogenesis (HAS1, URB1, and URB2), protein secretion (SEC39), mitochondrial import (MIM1), and tRNA charging (GSN1). In addition, apparent negative feedback transcriptional regulation of both ribosome biogenesis and the proteasome was observed. We furthermore show that these strains are compatible with automated genetic analysis. This study underscores the importance of analyzing mutant phenotypes and provides a resource to complement the yeast knockout collection.Cell 08/2004; 118(1):31-44. · 32.40 Impact Factor -
Article: Protein-protein interaction affinity plays a crucial role in controlling the Sho1p-mediated signal transduction pathway in yeast.
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ABSTRACT: Protein-protein interactions are required for most cellular functions, yet little is known about the relationship between protein-protein interaction affinity and biological activity. To investigate this issue, we engineered a series of mutants that incrementally reduced the affinity of the yeast Sho1p SH3 domain for its in vivo target, the MAP kinase kinase Pbs2p. We demonstrate a strong linear correlation between the binding energy of these mutants and quantitative in vivo outputs from the HOG high-osmolarity response pathway controlled by Sho1p. In addition, we find that reduction in binding affinity for the correct target within this pathway causes a proportional increase in misactivation of the related mating pheromone response pathway and that strong binding affinity alone does not guarantee efficient biological activity. Our experiments also indicate that a second binding surface on the Sho1p SH3 domain is required for its proper in vivo function.Molecular Cell 07/2004; 14(6):813-23. · 14.18 Impact Factor -
Article: Global Mapping of the Yeast Genetic Interaction Network
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ABSTRACT: A genetic interaction network containing�1000 genes and�4000 interactions was mapped by crossing mutations in 132 different query genes into a set of �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.Science 02/2004; 303:808. · 31.20 Impact Factor -
Article: Transcriptional Coregulation by the Cell Integrity Mitogen-Activated Protein Kinase Slt2 and the Cell Cycle Regulator Swi4
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ABSTRACT: In Saccharomyces cerevisiae, the heterodimeric transcription factor SBF (for SCB binding factor) is composed of Swi4 and Swi6 and activates gene expression at the G1/S-phase transition of the mitotic cell cycle. Cell cycle commitment is associated not only with major alterations in gene expression but also with highly polarized cell growth; the mitogen-activated protein kinase (MAPK) Slt2 is required to maintain cell wall integrity during periods of polarized growth and cell wall stress. We describe experiments aimed at defining the regulatory pathway involving the cell cycle transcription factor SBF and Slt2-MAPK. Gene expression assays and chromatin immunoprecipitation experiments revealed Slt2-dependent recruitment of SBF to the promoters of the G1 cyclins PCL1 and PCL2 after activation of the Slt2-MAPK pathway. We performed DNA microarray analysis and identified other genes whose expression was reduced in both SLT2 and SWI4 deletion strains. Genes that are sensitive to both Slt2 and Swi4 appear to be uniquely regulated and reveal a role for Swi4, the DNA-binding component of SBF, which is independent of the regulatory subunit Swi6. Some of the Swi4- and Slt2-dependent genes do not require Swi6 for either their expression or for Swi4 localization to their promoters. Consistent with these results, we found a direct interaction between Swi4 and Slt2. Our results establish a new Slt2-dependent mode of Swi4 regulation and suggest roles for Swi4 beyond its prominent role in controlling cell cycle transcription.Molecular and Cellular Biology 11/2001; · 5.53 Impact Factor
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- Genetics (1)
- Cell (1)
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- Science (1)
Institutions
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2011
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University of California, San Francisco
- Department of Cell and Tissue Biology
San Francisco, CA, USA
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2007
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University of Toronto
Toronto, Ontario, Canada
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