Coordination of actin plus-end dynamics by IQGAP1, formin, and capping protein.

Cell processes require precise regulation of actin polymerization that is mediated by plus-end regulatory proteins. Detailed mechanisms that explain plus-end dynamics involve regulators with opposing roles, including factors that enhance assembly, e.g., the formin mDia1, and others that stop growth (Capping Protein, CPz). We explore IQGAP1's roles regulating actin filament plus-ends and the consequences of perturbing its activity in cells. We confirm that IQGAP1 pauses elongation and interacts with plus ends through two residues (C756 and C781). We directly visualize the dynamic interplay between IQGAP1 and mDia1, revealing that IQGAP1 displaces the formin to influence actin assembly. Using four-color TIRF we show that IQGAP1's displacement activity extends to formin-CPz 'decision complexes', promoting end-binding protein turnover at plus-ends. Loss of IQGAP1 or its plus-end activities disrupts morphology and migration, emphasizing its essential role. These results reveal a new role for IQGAP1 in promoting protein turnover on filament ends and provide new insights into how plus-end actin assembly is regulated in cells.

Purification of Human Cytoplasmic Actins From Saccharomyces cerevisiae.

Eukaryotic cells rely on actin to support cellular structure, motility, transport, and a wide variety of other cytoplasmic functions and nuclear activities. Humans and other mammals express six closely related isoforms of actin, four of which are found primarily in skeletal, cardiac, and smooth muscle tissues. The final two isoforms, ß and γ, are found in non-muscle cells. Due to the ease of purification, many biochemical studies surveying the functions of actin and its regulators have been carried out with protein purified from skeletal muscle. However, it has become increasingly clear that some activities are isoform specific, necessitating more accessible sources of non-muscle actin isoforms. Recent innovations permit the purification of non-muscle actins from human cell culture and heterologous systems, such as insect cell culture and the yeast Pichia pastoris. However, these systems generate mixtures of actin types or require additional steps to remove purification-related tags. We have developed strains of Saccharomyces cerevisiae (budding yeast) that express single untagged isoforms of either human non-muscle actin (ß or γ) as their sole actin, allowing the purification of individual homogeneous actin isoforms by conventional purification techniques. Key features • Easy growth of yeast as a source of human cytoplasmic actin isoforms. Uses well-established actin purification methods. • The tag-free system requires no post-purification processing.

Purification of human ß- and γ-actin from budding yeast.

Biochemical studies of human actin and its binding partners rely heavily on abundant and easily purified α-actin from skeletal muscle. Therefore, muscle actin has been used to evaluate and determine the activities of most actin regulatory proteins but there is an underlying concern that these proteins perform differently from actin present in non-muscle cells. To provide easily accessible and relatively abundant sources of human ß- or γ-actin (i.e. cytoplasmic actins), we developed Saccharomyces cerevisiae strains that express each as their sole source of actin. Both ß- or γ-actin purified in this system polymerize and interact with various binding partners, including profilin, mDia1 (formin), fascin and thymosin-ß4 (Tß4). Notably, Tß4 and profilin bind to ß- or γ-actin with higher affinity than to α-actin, emphasizing the value of testing actin ligands with specific actin isoforms. These reagents will make specific isoforms of actin more accessible for future studies on actin regulation.

Biochemical characterization of actin assembly mechanisms with ALS-associated profilin variants.

Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for many neuronal cell processes through its regulation of lipids, nuclear signals, and cytoskeletal dynamics, including actin filament assembly. Direct interactions between profilin and actin monomers inhibit actin filament polymerization. In contrast, profilin can also stimulate polymerization by simultaneously binding actin monomers and proline-rich tracts found in other proteins. Whether the ALS-associated mutations in profilin compromise these actin assembly functions is unclear. We performed a quantitative biochemical comparison of the direct and formin mediated impact for the eight ALS-associated profilin variants on actin assembly using classic protein-binding and single-filament microscopy assays. We determined that the binding constant of each profilin for actin monomers generally correlates with the actin nucleation strength associated with each ALS-related profilin. In the presence of formin, the A20T, R136W, Q139L, and C71G variants failed to activate the elongation phase of actin assembly. This diverse range of formin-activities is not fully explained through profilin-poly-L-proline (PLP) interactions, as all ALS-associated variants bind a formin-derived PLP peptide with similar affinities. However, chemical denaturation experiments suggest that the folding stability of these profilins impact some of these effects on actin assembly. Thus, changes in profilin protein stability and alterations in actin filament polymerization may both contribute to the profilin-mediated actin disruptions in ALS.

Replication Stress Induces Global Chromosome Breakage in the Fragile X Genome.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1 gene and deficiency of a functional FMRP protein. FMRP is known as a translation repressor whose nuclear function is not understood. We investigated the global impact on genome stability due to FMRP loss. Using Break-seq, we map spontaneous and replication stress-induced DNA double-strand breaks (DSBs) in an FXS patient-derived cell line. We report that the genomes of FXS cells are inherently unstable and accumulate twice as many DSBs as those from an unaffected control. We demonstrate that replication stress-induced DSBs in FXS cells colocalize with R-loop forming sequences. Exogenously expressed FMRP in FXS fibroblasts ameliorates DSB formation. FMRP, not the I304N mutant, abates R-loop-induced DSBs during programmed replication-transcription conflict. These results suggest that FMRP is a genome maintenance protein that prevents R-loop accumulation. Our study provides insights into the etiological basis for FXS.

Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict between DNA replication and transcription.

We have previously demonstrated that in Saccharomyces cerevisiae replication, checkpoint inactivation via a mec1 mutation leads to chromosome breakage at replication forks initiated from virtually all origins after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Here we sought to determine whether all replication forks containing single-stranded DNA gaps have equal probability of producing double-strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-seq, that combines our previously described DSB labeling with next generation sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed a greater distance in MEC1 cells than in mec1 cells during recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of those transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. We further propose that replication inhibitors can induce unscheduled encounters between replication and transcription and give rise to distinct patterns of chromosome fragile sites.

Using two-hybrid interactions to identify separation-of-function mutations.

Protein functions within cells frequently require they interact physically with a number of partner proteins to coordinate the appropriate biochemical processes. Mutational analysis has been quite useful for analyzing how the loss of a gene or protein impacts cell function or more specifically particular pathways. However, the genetics approach to studying gene function can be limited by not having the right mutations; for example because the mutant allele ablates all function, as is the case for deletion (null) alleles and most temperature-sensitive alleles. To dissect the relative contributions of a protein's interactions, the researcher needs mutations that specifically affect one but not all of the protein's interactions. In genetics parlance such mutations are called separation-of-function mutations. The yeast two-hybrid system has been exploited for two decades to identify protein-binding partners. Here we describe a fairly simple protocol, within reach of laboratories with molecular biology experience, for using the two-hybrid system to identify separation-of-function mutations.

Coordination of the filament stabilizing versus destabilizing activities of cofilin through its secondary binding site on actin.

Cofilin is a ubiquitous modulator of actin cytoskeleton dynamics that can both stabilize and destabilize actin filaments depending on its concentration and/or the presence of regulatory co-factors. Three charge-reversal mutants of yeast cofilin, located in cofilin's filament-specific secondary binding site, were characterized in order to understand why disruption of this site leads to enhanced filament disassembly. Crystal structures of the mutants showed that the mutations specifically affect the secondary actin-binding interface, leaving the primary binding site unaltered. The mutant cofilins show enhanced activity compared to wild-type cofilin in severing and disassembling actin filaments. Electron microscopy and image analysis revealed long actin filaments in the presence of wild-type cofilin, while the mutants induced many short filaments, consistent with enhanced severing. Real-time fluorescence microscopy of labeled actin filaments confirmed that the mutants, unlike wild-type cofilin, were functioning as constitutively active severing proteins. In cells, the mutant cofilins delayed endocytosis, which depends on rapid actin turnover. We conclude that mutating cofilin's secondary actin-binding site increases cofilin's ability to sever and de-polymerize actin filaments. We hypothesize that activators of cofilin severing, like Aip1p, may act by disrupting the interface between cofilin's secondary actin-binding site and the actin filament.

Linking genetics to structural biology: complex heterozygosity screening with actin alanine scan alleles identifies functionally related surfaces on yeast actin.

Previous genome-level genetic interaction screens with the single essential actin gene of yeast identified 238 nonessential genes that upon deletion result in deleterious, digenic complex haploinsufficiences with an actin null allele. Deletion alleles of these 238 genes were tested for complex heterozygous interactions with 32 actin alanine scan alleles, which target clusters of residues on the surface of actin. A total of 891 deleterious digenic combinations were identified with 203 of the 238 genes. Two-dimensional hierarchical cluster analysis of the interactions identified nine distinct groups, and the alleles within clusters tended to affect localized regions on the surface of actin. The mutants in one cluster all affect electrostatic interactions between stacked subunits in the long pitch helix of the actin filament. A second cluster that contains the most highly interactive alleles may disrupt the tropomyosin/myosin system, as one of the mutants in that cluster cannot support Type V myosin-dependent movement of secretory vesicles in haploids and causes processivity defects in heterozygous diploids. These examples suggest the clusters represent mutations with shared protein-protein interaction defects. These results show that complex heterozygous interaction screens have benefit for detecting actin-related genes and suggest that having actin filaments of mixed composition, containing both mutant and wild-type subunits, presents unique challenges to the cell.

Chemical suppression of defects in mitotic spindle assembly, redox control, and sterol biosynthesis by hydroxyurea.

We describe the results of a systematic search for a class of hitherto-overlooked chemical-genetic interactions in the Saccharomyces cerevisiae genome, which exists between a detrimental genetic mutation and a chemical/drug that can ameliorate, rather than exacerbate, that detriment. We refer to this type of interaction as "chemical suppression." Our work was driven by the hypothesis that genome instability in a certain class of mutants could be alleviated by mild replication inhibition using chemicals/drugs. We queried a collection of conditionally lethal, i.e., temperature-sensitive, alleles representing 40% of the yeast essential genes for those mutants whose growth defect can be suppressed by hydroxyurea (HU), known as a potent DNA replication inhibitor, at the restrictive temperature. Unexpectedly, we identified a number of mutants defective in diverse cellular pathways other than DNA replication. Here we report that HU suppresses selected mutants defective in the kinetochore-microtubule attachment pathway during mitotic chromosome segregation. HU also suppresses an ero1-1 mutant defective for a thiol oxidase of the endoplasmic reticulum by providing oxidation equivalents. Finally, we report that HU suppresses an erg26-1 mutant defective for a C-3 sterol dehydrogenase through regulating iron homeostasis and in turn impacting ergosterol biosynthesis. We further demonstrate that cells carrying the erg26-1 mutation show an increased rate of mitochondrial DNA loss and delayed G1 to S phase transition. We conclude that systematic gathering of a compendium of "chemical suppression" of yeast mutants by genotoxic drugs will not only enable the identification of novel functions of both chemicals and genes, but also have profound implications in cautionary measures of anticancer intervention in humans.

Actin dosage lethality screening in yeast mediated by selective ploidy ablation reveals links to urmylation/wobble codon recognition and chromosome stability.

The actin cytoskeleton exists in a dynamic equilibrium with monomeric and filamentous states of its subunit protein actin. The spatial and temporal regulation of actin dynamics is critical to the many functions of actin. Actin levels are remarkably constant, suggesting that cells have evolved to function within a narrow range of actin concentrations. Here we report the results of screens in which we have increased actin levels in strains deleted for the ~4800 nonessential yeast genes using a technical advance called selective ploidy ablation. We detected 83 synthetic dosage interactions with actin, 78 resulted in reduced growth, whereas in 5 cases overexpression of actin suppressed the growth defects caused by the deleted genes. The genes were highly enriched in several classes, including transfer RNA wobble uridine modification, chromosome stability and segregation, cell growth, and cell division. We show that actin overexpression sequesters a limited pool of eEF1A, a bifunctional protein involved in aminoacyl-transfer RNA recruitment to the ribosome and actin filament cross-linking. Surprisingly, the largest class of genes is involved in chromosome stability and segregation. We show that actin mutants have chromosome segregation defects, suggesting a possible role in chromosome structure and function. Monomeric actin is a core component of the INO80 and SWR chromatin remodeling complexes and the NuA4 histone modification complex, and our results suggest these complexes may be sensitive to actin stoichiometry. We propose that the resulting effects on chromatin structure can lead to synergistic effects on chromosome stability in strains lacking genes important for chromosome maintenance.

F1-ATPase of Escherichia coli: the ε- inhibited state forms after ATP hydrolysis, is distinct from the ADP-inhibited state, and responds dynamically to catalytic site ligands.

F1-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit ε, which partially inserts into the enzyme's central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F1·Îµ binding and dissociation, we show that formation of the extended, inhibitory conformation of ε (εX) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the εX state, and post-hydrolysis conditions stabilize it. We also show that ε inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of ε is responsible for initial binding to F1 and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the ε N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F1-bound ε suggest dynamic conformational and rotational mobility in F1 that is paused near the catalytic dwell position.

Novel interactions between actin and the proteasome revealed by complex haploinsufficiency.

Saccharomyces cerevisiae has been a powerful model for uncovering the landscape of binary gene interactions through whole-genome screening. Complex heterozygous interactions are potentially important to human genetic disease as loss-of-function alleles are common in human genomes. We have been using complex haploinsufficiency (CHI) screening with the actin gene to identify genes related to actin function and as a model to determine the prevalence of CHI interactions in eukaryotic genomes. Previous CHI screening between actin and null alleles for non-essential genes uncovered ∼240 deleterious CHI interactions. In this report, we have extended CHI screening to null alleles for essential genes by mating a query strain to sporulations of heterozygous knock-out strains. Using an act1Δ query, knock-outs of 60 essential genes were found to be CHI with actin. Enriched in this collection were functional categories found in the previous screen against non-essential genes, including genes involved in cytoskeleton function and chaperone complexes that fold actin and tubulin. Novel to this screen was the identification of genes for components of the TFIID transcription complex and for the proteasome. We investigated a potential role for the proteasome in regulating the actin cytoskeleton and found that the proteasome physically associates with actin filaments in vitro and that some conditional mutations in proteasome genes have gross defects in actin organization. Whole-genome screening with actin as a query has confirmed that CHI interactions are important phenotypic drivers. Furthermore, CHI screening is another genetic tool to uncover novel functional connections. Here we report a previously unappreciated role for the proteasome in affecting actin organization and function.

Diverse protective roles of the actin cytoskeleton during oxidative stress.

Actin oxidation is known to result in changes in cytoskeleton organization and dynamics. Actin oxidation is clinically relevant since it occurs in the erythrocytes of sickle cell patients and may be the direct cause of the lack of morphological plasticity observed in irreversibly sickled red blood cells (ISCs). During episodes of crisis, ISCs accumulate C284-C373 intramolecularly disulfide bonded actin, which reduces actin filament dynamics. Actin cysteines 284 and 373 (285 and 374 in yeast) are conserved, suggesting that they play an important functional role. We have been investigating the physiological roles of these cysteines using the model eukaryote Saccharomyces cerevisiae in response to oxidative stress load. During acute oxidative stress, all of the F-actin in wild-type cells collapses into a few puncta that we call oxidation-induced actin bodies (OABs). In contrast, during acute oxidative stress the actin cytoskeleton in Cys-to-Ala actin mutants remains polarized longer, OABs are slower to form, and the cells recover more slowly than wild-type cells, suggesting that the OABs play a protective role. Live cell imaging revealed that OABs are large, immobile structures that contain actin-binding proteins and that can form by the fusion of actin cortical patches. We propose that actin's C285 and C374 may help to protect the cell from oxidative stress arising from normal oxidative metabolism and contribute to the cell's general adaptive response to oxidative stress.

Stable preanaphase spindle positioning requires Bud6p and an apparent interaction between the spindle pole bodies and the neck.

Faithful partitioning of genetic material during cell division requires accurate spatial and temporal positioning of nuclei within dividing cells. In Saccharomyces cerevisiae, nuclear positioning is regulated by an elegant interplay between components of the actin and microtubule cytoskeletons. Regulators of this process include Bud6p (also referred to as the actin-interacting protein Aip3p) and Kar9p, which function to promote contacts between cytoplasmic microtubule ends and actin-delimited cortical attachment points. Here, we present the previously undetected association of Bud6p with the cytoplasmic face of yeast spindle pole bodies, the functional equivalent of metazoan centrosomes. Cells lacking Bud6p show exaggerated movements of the nucleus between mother and daughter cells and display reduced amounts of time a given spindle pole body spends in close association with the neck region of budding cells. Furthermore, overexpression of BUD6 greatly enhances interactions between the spindle pole body and mother-bud neck in a spindle alignment-defective dynactin mutant. These results suggest that association of either spindle pole body with neck components, rather than simply entry of a spindle pole body into the daughter cell, provides a positive signal for the progression of mitosis. We propose that Bud6p, through its localization at both spindle pole bodies and at the mother-bud neck, supports this positive signal and provides a regulatory mechanism to prevent excessive oscillations of preanaphase nuclei, thus reducing the likelihood of mitotic delays and nuclear missegregation.

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.

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.

Role of a Cdc42p effector pathway in recruitment of the yeast septins to the presumptive bud site.

The septins are GTP-binding, filament-forming proteins that are involved in cytokinesis and other processes. In the yeast Saccharomyces cerevisiae, the septins are recruited to the presumptive bud site at the cell cortex, where they form a ring through which the bud emerges. We report here that in wild-type cells, the septins typically become detectable in the vicinity of the bud site several minutes before ring formation, but the ring itself is the first distinct structure that forms. Septin recruitment depends on activated Cdc42p but not on the normal pathway for bud-site selection. Recruitment occurs in the absence of F-actin, but ring formation is delayed. Mutant phenotypes and suppression data suggest that the Cdc42p effectors Gic1p and Gic2p, previously implicated in polarization of the actin cytoskeleton, also function in septin recruitment. Two-hybrid, in vitro protein binding, and coimmunoprecipitation data indicate that this role involves a direct interaction of the Gic proteins with the septin Cdc12p.