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David C Amberg

Publications and source records attributed to David C Amberg.

10 recordsLinked to original sources
PubMed2006

Modeling complex genetic interactions in a simple eukaryotic genome: actin displays a rich spectrum of complex haploinsufficiencies.

Multigenic influences are major contributors to human genetic disorders. Since humans are highly polymorphic, there are a high number of possible detrimental, multiallelic gene pairs. The actin cytoskeleton of yeast was used to determine the potential for deleterious bigenic interactions; approximately 4800 complex hemizygote strains were constructed between an actin-null allele and the nonessential gene deletion collection. We found 208 genes that have deleterious complex haploinsufficient (CHI) interactions with actin. This set is enriched for genes with gene ontology terms shared with actin, including several actin-binding protein genes, and nearly half of the CHI genes have defects in actin organization when deleted. Interactions were frequently seen with genes for multiple components of a complex or with genes involved in the same function. For example, many of the genes for the large ribosomal subunit (RPLs) were CHI with act1Delta and had actin organization defects when deleted. This was generally true of only one RPL paralog of apparently duplicate genes, suggesting functional specialization between ribosomal genes. In many cases, CHI interactions could be attributed to localized defects on the actin protein. Spatial congruence in these data suggest that the loss of binding to specific actin-binding proteins causes subsets of CHI interactions.

Actins↗
PubMed2006

A genetic dissection of Aip1p's interactions leads to a model for Aip1p-cofilin cooperative activities.

Actin interacting protein 1 (Aip1p) and cofilin cooperate to disassemble actin filaments in vitro and are thought to promote rapid turnover of actin networks in vivo. The precise method by which Aip1p participates in these activities has not been defined, although severing and barbed-end capping of actin filaments have been proposed. To better describe the mechanisms and biological consequences of Aip1p activities, we undertook an extensive mutagenesis of AIP1 aimed at disrupting and mapping Aip1p interactions. Site-directed mutagenesis suggested that Aip1p has two actin binding sites, the primary actin binding site lies on the edge of its N-terminal beta-propeller and a secondary actin binding site lies in a comparable location on its C-terminal beta-propeller. Random mutagenesis followed by screening for separation of function mutants led to the identification of several mutants specifically defective for interacting with cofilin but still able to interact with actin. These mutants suggested that cofilin binds across the cleft between the two propeller domains, leaving the actin binding sites exposed and flanking the cofilin binding site. Biochemical, genetic, and cell biological analyses confirmed that the actin binding- and cofilin binding-specific mutants are functionally defective, whereas the genetic analyses further suggested a role for Aip1p in an early, internalization step of endocytosis. A complementary, unbiased molecular modeling approach was used to derive putative structures for the Aip1p-cofilin complex, the most stable of which is completely consistent with the mutagenesis data. We theorize that Aip1p-severing activity may involve simultaneous binding to two actin subunits with cofilin wedged between the two actin binding sites of the N- and C-terminal propeller domains.

Actin Cytoskeleton↗
PubMed2005

Eliminating gene conversion improves high-throughput genetics in Saccharomyces cerevisiae.

Synthetic genetic analysis was improved by eliminating leaky expression of the HIS3 reporter and gene conversion between the HIS3 reporter and his3Delta1. Leaky expression was eliminated using 3-aminotriazole and gene conversion was eliminated by using the Schizosaccharomyces pombe his5+ gene, resulting in a 5- to 10-fold improvement in the efficiency of SGA.

Amitrole↗
PubMed2004

Old yellow enzyme protects the actin cytoskeleton from oxidative stress.

Old Yellow Enzyme (OYE) has long served as a paradigm for the study of flavin-containing NADPH oxido-reductases and yet its physiological role has remained a mystery. A two-hybrid interaction between Oye2p and actin led us to investigate a possible function in the actin cytoskeleton. We found that oye deletion strains have an overly elaborate actin cytoskeleton that cannot be attributed to changes in actin concentration but likely reflect stabilization of actin filaments, resulting in excessive actin assembly. Cells expressing the actin mutant act1-123p, which has a weakened interaction with Oye2p, show comparable defects in actin organization to the oye deletion strain that can be suppressed by overexpression of Oye2p. Similarly, mutation of either conserved cysteine of the potential disulfide pair Cys285-Cys374 in actin completely suppresses the actin organization defect of the oyeDelta phenotype. Strains lacking Oye function are also sensitive to oxidative stress as induced by H2O2, menadione, and diamide treatment. Mutation of either Cys285 or Cys374 of actin suppresses the sensitivity of oyeDelta strains to oxidative stress and in fact confers super-resistance to oxidative stress in otherwise wild-type strains. These results suggest that oxidative damage to actin, like that which has been observed in irreversibly sickled red blood cells, may be a general phenomenon and that OYE functions to control the redox state of actin thereby maintaining the proper plasticity of the actin cytoskeleton. In addition to uncovering a long sought biological function for Old Yellow Enzyme, these results establish that cellular sensitivity to oxidative stress can in part be directly attributed to a specific form (C285-C374 disulfide bond formation) of oxidative damage to actin.

Actins↗
PubMed2003

Actin recovery and bud emergence in osmotically stressed cells requires the conserved actin interacting mitogen-activated protein kinase kinase kinase Ssk2p/MTK1 and the scaffold protein Spa2p.

Osmotic stress causes actin cytoskeleton disassembly, a cell cycle arrest, and activation of the high osmolarity growth mitogen-activated protein kinase pathway. A previous study showed that Ssk2p, a mitogen-activated protein kinase kinase kinase of the high osmolarity growth pathway, promotes actin cytoskeleton recovery to the neck of late cell cycle, osmotically stressed yeast cells. Data presented herein examined the role of Ssk2p in actin recovery early in the cell cycle. We found that actin recovery at all stages of the cell cycle is not controlled by Ssk1p, the known activator of Ssk2p, but required a polarized distribution of Ssk2p as well as its actin-interacting and kinase activity. Stress-induced localization of Ssk2p to the neck required the septin Shs1p, whereas localization to the bud cortex depended on the polarity scaffold protein Spa2p. spa2delta cells, like ssk2delta cells, were defective for actin recovery from osmotic stress. These spa2delta defects could be suppressed by overexpression of catalytically active Ssk2p. Furthermore, Spa2p could be precipitated by GST-Ssk2p from extracts of osmotically stressed cells. The Ssk2p mediated actin recovery pathway seems to be conserved; MTK1, a human mitogen-activated protein kinase kinase kinase of the p38 stress response pathway and Ssk2p homolog, was also able to localize at polarized growth sites, form a complex with actin and Spa2p, and complement actin recovery defects in osmotically stressed ssk2delta and spa2delta yeast cells. We hypothesize that osmotic stress-induced actin disassembly leads to the formation of an Ssk2p-actin complex and the polarized localization of Ssk2p. Polarized Ssk2p associates with the scaffold protein Spa2p in the bud and Shs1p in the neck, allowing Ssk2p to regulate substrates involved in polarized actin assembly.

Actins↗
PubMed2002

Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast.

Formins have been implicated in the regulation of cytoskeletal structure in animals and fungi. Here we show that the formins Bni1 and Bnr1 of budding yeast stimulate the assembly of actin filaments that function as precursors to tropomyosin-stabilized cables that direct polarized cell growth. With loss of formin function, cables disassemble, whereas increased formin activity causes the hyperaccumulation of cable-like filaments. Unlike the assembly of cortical actin patches, cable assembly requires profilin but not the Arp2/3 complex. Thus formins control a distinct pathway for assembling actin filaments that organize the overall polarity of the cell.

Actin-Related Protein 2↗
PubMed2002

Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast.

Formins have been implicated in the regulation of cytoskeletal structure in animals and fungi. Here we show that the formins Bni1 and Bnr1 of budding yeast stimulate the assembly of actin filaments that function as precursors to tropomyosin-stabilized cables that direct polarized cell growth. With loss of formin function, cables disassemble,whereas increased formin activity causes the hyperaccumulation of cable-like filaments. Unlike the assembly of cortical actin patches, cable assembly requires profilin but not the Arp2/3 complex. Thus formins control a distinct pathway for assembling actin filaments that organize the overall polarity of the cell.

Acetyltransferases↗
PubMed2002

The MEK kinase Ssk2p promotes actin cytoskeleton recovery after osmotic stress.

Saccharomyces cerevisiae adapts to osmotic stress through the activation of a conserved high-osmolarity growth (HOG) mitogen-activated protein (MAP) kinase pathway. Transmission through the HOG pathway is very well understood, yet other aspects of the cellular response to osmotic stress remain poorly understood, most notably regulation of actin organization. The actin cytoskeleton rapidly disassembles in response to osmotic insult and is induced to reassemble only after osmotic balance with the environment is reestablished. Here, we show that one of three MEK kinases of the HOG pathway, Ssk2p, is specialized to facilitate actin cytoskeleton reassembly after osmotic stress. Within minutes of cells' experiencing osmotic stress or catastrophic disassembly of the actin cytoskeleton through latrunculin A treatment, Ssk2p concentrates in the neck of budding yeast cells and concurrently forms a 1:1 complex with actin. These observations suggest that Ssk2p has a novel, previously undescribed function in sensing damage to the actin cytoskeleton. We also describe a second function for Ssk2p in facilitating reassembly of a polarized actin cytoskeleton at the end of the cell cycle, a prerequisite for efficient cell cycle completion. Loss of Ssk2p, its kinase activity, or its ability to localize and interact with actin led to delays in actin recovery and a resulting delay in cell cycle completion. These unique capabilities of Ssk2p are activated by a novel mechanism that does not involve known components of the HOG pathway.

Actins↗