Modular complexes that regulate actin assembly in budding yeast

The actin cytoskeleton of budding yeast contains an extensive set of actin-associated proteins with conserved mammalian counterparts. For more than 20 years, yeast has been used as a model organism to dissect the in vivo functions of these factors, revealing an intricate web of genetic interactions in the cell. Now, a surge of biochemical reports is defining the physical interactions and activities of these proteins and providing mechanistic insights into their cellular roles. The emerging view is that most actin-associated proteins do not act alone but, rather, associate to form modular protein complexes that regulate actin assembly and organization.     

Mapping actin surfaces required for functional interactions in vivo

An in vivo strategy to identify amino acids of actin required for functional interactions with actin-binding proteins was developed. This approach is based on the assumption that an actin mutation that specifically impairs the interaction with an actin-binding protein will cause a phenotype similar to a null mutation in the gene that encodes the actin-binding protein. 21 actin mutations were analyzed in budding yeast, and specific regions of actin subdomain 1 were implicated in the interaction with fimbrin, an actin filament-bundling protein. Mutations in this actin subdomain were shown to be, like a null allele of the yeast fimbrin gene (SAC6), lethal in combination with null mutations in the ABP1 and SLA2 genes, and viable in combination with a null mutation in the SLA1 gene. Biochemical experiments with act1-120 actin (E99A, E100A) verified a defect in the fimbrin-actin interaction. Genetic interactions between mutant alleles of the yeast actin gene and null alleles of the SAC6, ABP1, SLA1, and SLA2 genes also demonstrated that the effects of the 21 actin mutations are diverse and allowed four out of seven pseudo-wild-type actin alleles to be distinguished from the wild-type gene for the first time, providing evidence for functional redundancy between different surfaces of actin.     

Scd5p and clathrin function are important for cortical actin organization,endocytosis, and localization of sla2p in yeast

SCD5 was identified as a multicopy suppressor of clathrin HC-deficient yeast. SCD5 is essential, but an scd5-Delta338 mutant, expressing Scd5p with a C-terminal truncation of 338 amino acids, is temperature sensitive for growth. Further studies here demonstrate that scd5-Delta338 affects receptor-mediated and fluid-phase endocytosis and normal actin organization. The scd5-Delta338 mutant contains larger and depolarized cortical actin patches and a prevalence of G-actin bars. scd5-Delta338 also displays synthetic negative genetic interactions with mutations in several other proteins important for cortical actin organization and endocytosis. Moreover, Scd5p colocalizes with cortical actin. Analysis has revealed that clathrin-deficient yeast also have a major defect in cortical actin organization and accumulate G-actin. Overexpression of SCD5 partially suppresses the actin defect of clathrin mutants, whereas combining scd5-Delta338 with a clathrin mutation exacerbates the actin and endocytic phenotypes. Both Scd5p and yeast clathrin physically associate with Sla2p, a homologue of the mammalian huntingtin interacting protein HIP1 and the related HIP1R. Furthermore, Sla2p localization at the cell cortex is dependent on Scd5p and clathrin function. Therefore, Scd5p and clathrin are important for actin organization and endocytosis, and Sla2p may provide a critical link between clathrin and the actin cytoskeleton in yeast, similar to HIP1(R) in animal cells.     

Mutational analysis of arginine 177 in the nucleotide binding site of beta-actin.

Actin ADP-ribosylated at arginine 177 is unable to hydrolyze ATP, and the R177 side chain is in a position similar to that of the catalytically essential lysine 71 in heat shock cognate protein Hsc70, another member of the actin-fold family of proteins. Therefore, actin residue R177 has been implicated in the mechanism of ATP hydrolysis. This paper compares wild-type beta-actin with a mutant in which R177 has been replaced by aspartic acid. The mutant beta-actin was expressed in Saccharomyces cerevisiae and purified by DNase I-affinity chromatography. The mutant protein exhibited a reduced thermal stability and an increased nucleotide exchange rate, suggesting a weakened interdomain connection. The ATPase activity of G-actin and the ATPase activity expressed during polymerization were unaffected by the R177D replacement, showing that this residue is not involved in catalysis. In the presence of polymerizing salts, ATP hydrolysis by both wild-type Mg-beta-actin and the mutant protein preceded filament formation. With the mutant actin, the initial rate of ATP hydrolysis was as high as with wild-type actin, but polymer formation was slower, reached lower steady-state levels, and the polymers formed exhibited much lower viscosity. The critical concentration of polymerization (Acc) of the mutant actin was increased 10-fold as compared to wild-type actin. Filaments formed from the R177D mutant beta-actin bound phalloidin.     

The class V myosin motor protein, Myo2, plays a major role in mitochondrial motility in Saccharomyces cerevisiae

The actin cytoskeleton is essential for polarized, bud-directed movement of cellular membranes in Saccharomyces cerevisiae and thus ensures accurate inheritance of organelles during cell division. Also, mitochondrial distribution and inheritance depend on the actin cytoskeleton, though the precise molecular mechanisms are unknown. Here, we establish the class V myosin motor protein, Myo2, as an important mediator of mitochondrial motility in budding yeast. We found that mutants with abnormal expression levels of Myo2 or its associated light chain, Mlc1, exhibit aberrant mitochondrial morphology and loss of mitochondrial DNA. Specific mutations in the globular tail of Myo2 lead to aggregation of mitochondria in the mother cell. Isolated mitochondria lacking functional Myo2 are severely impaired in their capacity to bind to actin filaments in vitro. Time-resolved fluorescence microscopy revealed a block of bud-directed anterograde mitochondrial movement in cargo binding-defective myo2 mutant cells. We conclude that Myo2 plays an important and direct role for mitochondrial motility and inheritance in budding yeast.     

The EH-domain-containing protein Pan1 is required for normal organization of the actin cytoskeleton in Saccharomyces cerevisiae.

Normal cell growth and division in the yeast Saccharomyces cerevisiae involve dramatic and frequent changes in the organization of the actin cytoskeleton. Previous studies have suggested that the reorganization of the actin cytoskeleton in accordance with cell cycle progression is controlled, directly or indirectly, by the cyclin-dependent kinase Cdc28. Here we report that by isolating rapid-death mutants in the background of the Start-deficient cdc28-4 mutation, the essential yeast gene PAN1, previously thought to encode the yeast poly(A) nuclease, is identified as a new factor required for normal organization of the actin cytoskeleton. We show that at restrictive temperature, the pan1 mutant exhibited abnormal bud growth, failed to maintain a proper distribution of the actin cytoskeleton, was unable to reorganize actin the cytoskeleton during cell cycle, and was defective in cytokinesis. The mutant also displayed a random pattern of budding even at permissive temperature. Ectopic expression of PAN1 by the GAL promoter caused abnormal distribution of the actin cytoskeleton when a single-copy vector was used. Immunofluorescence staining revealed that the Pan1 protein colocalized with the cortical actin patches, suggesting that it may be a filamentous actin-binding protein. The Pan1 protein contains an EF-hand calcium-binding domain, a putative Src homology 3 (SH3)-binding domain, a region similar to the actin cytoskeleton assembly control protein Sla1, and two repeats of a newly identified protein motif known as the EH domain. These findings suggest that Pan1, recently recognized as not responsible for the poly(A) nuclease activity (A. B. Sachs and J. A. Deardorff, erratum, Cell 83:1059, 1995; R. Boeck, S. Tarun, Jr., M. Rieger, J. A. Deardorff, S. Muller-Auer, and A. B. Sachs, J. Biol. Chem. 271:432-438, 1996), plays an important role in the organization of the actin cytoskeleton in S. cerevisiae.     

Thoracic Aortic Aneurysm (TAAD)-causing Mutation in Actin Affects Formin Regulation of Polymerization

More than 30 mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause autosomal dominant thoracic aortic aneurysm and dissection. The mutation R256H is of particular interest because it also causes patent ductus arteriosus and moyamoya disease. R256H is one of the more prevalent mutations and, based on its molecular location near the strand-strand interface in the actin filament, may affect F-actin stability. To understand the molecular ramifications of the R256H mutation, we generated Saccharomyces cerevisiae yeast cells expressing only R256H yeast actin as a model system. These cells displayed abnormal cytoskeletal morphology and increased sensitivity to latrunculin A. After cable disassembly induced by transient exposure to latrunculin A, mutant cells were delayed in reestablishing the actin cytoskeleton. In vitro, mutant actin exhibited a higher than normal critical concentration and a delayed nucleation. Consequently, we investigated regulation of mutant actin by formin, a potent facilitator of nucleation and a protein needed for normal vascular smooth muscle cell development. Mutant actin polymerization was inhibited by the FH1-FH2 fragment of the yeast formin, Bni1. This fragment strongly capped the filament rather than facilitating polymerization. Interestingly, phalloidin or the presence of wild type actin reversed the strong capping behavior of Bni1. Together, the data suggest that the R256H actin mutation alters filament conformation resulting in filament instability and misregulation by formin. These biochemical effects may contribute to abnormal histology identified in diseased arterial samples from affected patients.     

Identification of novel mutations in ACT1 and SLA2 that suppress the actin-cable-overproducing phenotype caused by overexpression of a dominant active form of Bni1p in Saccharomyces cerevisiae

A formin Bni1p nucleates actin to assemble actin cables, which guide the polarized transport of secretory vesicles in budding yeast. We identified mutations that suppressed both the lethality and the excessive actin cable formation caused by overexpression of a truncated Bni1p (BNI1DeltaN). Two recessive mutations, act1-301 in the actin gene and sla2-82 in a gene involved in cortical actin patch assembly, were identified. The isolation of sla2-82 was unexpected, because cortical actin patches are required for the internalization step of endocytosis. Both act1-301 and sla2-82 exhibited synthetic growth defects with bni1Delta. act1-301, which resulted in an E117K substitution, interacted genetically with mutations in profilin (PFY1) and BUD6, suggesting that Act1-301p was not fully functional in formin-mediated polymerization. sla2-82 also interacted genetically with genes involved in actin cable assembly. Some experiments, however, suggested that the effects of sla2-82 were caused by depletion of actin monomers, because the temperature-sensitive growth phenotype of the bni1Delta sla2-82 mutant was suppressed by increased expression of ACT1. The isolation of suppressors of the BNI1DeltaN phenotype may provide a useful system for identification of actin amino-acid residues that are important for formin-mediated actin polymerization and mutations that affect the availability of actin monomers.     

Interactions of mitochondria with the actin cytoskeleton

Interactions between mitochondria and the cytoskeleton are essential for normal mitochondrial morphology, motility and distribution. While microtubules and their motors have been established as important factors for mitochondrial transport, emerging evidence indicates that mitochondria interact with the actin cytoskeleton in many cell types. In certain fungi, such as the budding yeast and Aspergillus, or in plant cells mitochondrial motility is largely actin-based. Even in systems such as neurons, where microtubules are the primary means of long-distance mitochondrial transport, the actin cytoskeleton is required for short-distance mitochondrial movements and for immobilization of the organelle at the cell cortex. The actin cytoskeleton is also involved in the immobilization of mitochondria at the cortex in cultured tobacco cells and in budding yeast. While the exact nature of these immobilizations is not known, they may be important for retaining mitochondria at sites of high ATP utilization or at other cellular locations where they are needed. Recent findings also indicate that mutations in actin or actin-binding proteins can influence mitochondrial pathways leading to cell death. Thus, mitochondria-actin interactions contribute to apoptosis.     

Locking the hydrophobic loop 262-274 to G-actin surface by a disulfide bridge prevents filament formation

Models of F-actin structure predict the importance of hydrophobic loop 262-274 at the interface of subdomains 3 and 4 to interstrand interactions in filaments. If this premise is correct, prevention of the loop conformational change--its swinging motion--should abort filament formation. To test this hypothesis, we used site-directed mutagenesis to create yeast actin triple mutant (LC)2CA (L180C/L269C/C374A). This mutation places two cysteine residues in positions potentially enabling the locking of loop 262-274 to the monomer surface via disulfide formation. Exposure of the purified mutant to oxidation catalysts resulted in an increased electrophoretic mobility of actin on SDS PAGE and a loss of two cysteines by DTNB titrations, consistent with disulfide formation. The polymerization of un-cross-linked mutant actin by MgCl2 was inhibited strongly but could be restored to wild type actin levels by phalloidin and improved greatly through copolymerization with the wild-type actin. Light scattering measurements revealed nonspecific aggregation of the cross-linked actin under the same conditions. Electron microscopy confirmed the absence of filaments and the presence of amorphous aggregates in the cross-linked actin samples. Reduction of the disulfide bond by DTT restored normal actin polymerization in the presence of MgCl2 and phalloidin. These observations provide strong experimental support for a critical role of the hydrophobic loop 262-274 in the polymerization of actin into filaments.     

Pan1p, End3p, and S1a1p, three yeast proteins required for normal cortical actin cytoskeleton organization, associate with each other and play essential roles in cell wall morphogenesis

The EH domain proteins Pan1p and End3p of budding yeast have been known to form a complex in vivo and play important roles in organization of the actin cytoskeleton and endocytosis. In this report, we describe new findings concerning the function of the Pan1p-End3p complex. First, we found that the Pan1p-End3p complex associates with Sla1p, another protein known to be required for the assembly of cortical actin structures. Sla1p interacts with the first long repeat region of Pan1p and the N-terminal EH domain of End3p, thus leaving the Pan1p-End3p interaction, which requires the second long repeat of Pan1p and the C-terminal repeat region of End3p, undisturbed. Second, Pan1p, End3p, and Sla1p are also required for normal cell wall morphogenesis. Each of the Pan1-4, sla1Delta, and end3Delta mutants displays the abnormal cell wall morphology previously reported for the act1-1 mutant. These cell wall defects are also exhibited by wild-type cells overproducing the C-terminal region of Sla1p that is responsible for interactions with Pan1p and End3p. These results indicate that the functions of Pan1p, End3p, and Sla1p in cell wall morphogenesis may depend on the formation of a heterotrimeric complex. Interestingly, the cell wall abnormalities exhibited by these cells are independent of the actin cytoskeleton organization on the cell cortex, as they manifest despite the presence of apparently normal cortical actin cytoskeleton. Examination of several act1 mutants also supports this conclusion. These observations suggest that the Pan1p-End3p-Sla1p complex is required not only for normal actin cytoskeleton organization but also for normal cell wall morphogenesis in yeast.     

A Highlights from MBoC Selection: Initial Polarized Bud Growth by Endocytic Recycling in the Absence of Actin Cable–dependent Vesicle Transport in Yeast

The assembly of filamentous actin is essential for polarized bud growth in budding yeast. Actin cables, which are assembled by the formins Bni1p and Bnr1p, are thought to be the only actin structures that are essential for budding. However, we found that formin or tropomyosin mutants, which lack actin cables, are still able to form a small bud. Additional mutations in components for cortical actin patches, which are assembled by the Arp2/3 complex to play a pivotal role in endocytic vesicle formation, inhibited this budding. Genes involved in endocytic recycling were also required for small-bud formation in actin cable-less mutants. These results suggest that budding yeast possesses a mechanism that promotes polarized growth by local recycling of endocytic vesicles. Interestingly, the type V myosin Myo2p, which was thought to use only actin cables to track, also contributed to budding in the absence of actin cables. These results suggest that some actin network may serve as the track for Myo2p-driven vesicle transport in the absence of actin cables or that Myo2p can function independent of actin filaments. Our results also show that polarity regulators including Cdc42p were still polarized in mutants defective in both actin cables and cortical actin patches, suggesting that the actin cytoskeleton does not play a major role in cortical assembly of polarity regulators in budding yeast.     

Differential Support of Aspergillus fumigatus Morphogenesis by Yeast and Human Actins

The actin cytoskeleton is highly conserved among eukaryotes and is essential for cellular processes regulating growth and differentiation. In fungi, filamentous actin (F-actin) orchestrates hyphal tip structure and extension via organization of exocytic and endocytic processes at the hyphal tip. Although highly conserved, there are key differences among actins of fungal species as well as between mammalian and fungal actins. For example, the F-actin stabilizing molecules, phalloidin and jasplakinolide, bind to actin structures in yeast and human cells, whereas phalloidin does not bind actin structures of Aspergillus. These discrepancies suggest structural differences between Aspergillus actin filaments and those of human and yeast cells. Additionally, fungal actin kinetics are much faster than those of humans, displaying 5-fold faster nucleation and 40-fold faster nucleotide exchange rates. Limited published studies suggest that these faster actin kinetics are required for normal growth and morphogenesis of yeast cells. In the current work, we show that replacement of Aspergillus actin with yeast actin generates a morphologically normal strain, suggesting that Aspergillus actin kinetics are similar to those of yeast. In contrast to wild type A. fumigatus, F-actin in this strain binds phalloidin, and pharmacological stabilization of these actin structures with jasplakinolide inhibits germination and alters morphogenesis in a dose-dependent manner. We also show that human β-actin cannot support Aspergillus viability, even though the amino acid sequences of human and Aspergillus actins are 89.3% identical. Our findings show that minor differences in actin protein sequence account for loss of phalloidin and jasplakinolide sensitivity in Aspergillus species.     

Cofilin (ADF) affects lateral contacts in F-actin

The effect of yeast cofilin on lateral contacts between protomers of yeast and skeletal muscle actin filaments was examined in solution. These contacts are presumably stabilized by the interactions of loop 262-274 of one protomer with two other protomers on the opposite strand in F-actin. Cofilin inhibited several-fold the rate of interstrand disulfide cross-linking between Cys265 and Cys374 in yeast S265C mutant F-actin, but enhanced excimer formation between pyrene probes attached to these cysteine residues. The possibility that these effects are due to a translocation of the C terminus of actin by cofilin was ruled out by measurements of fluorescence resonance energy transfer (FRET) from tryptophan residues and ATP to acceptor probes at Cys374. Such measurements did not reveal cofilin-induced changes in FRET efficiency, suggesting that changes in Cys265-Cys374 cross-linking and excimer formation stem from the perturbation of loop 262-274 by cofilin. Changes in lateral interactions in F-actin were indicated also by the cofilin-induced partial release of rhodamine phalloidin. Disulfide cross-linking of S265C yeast F-actin inhibited strongly and reversibly the release of rhodamine phalloidin by cofilin. Overall, this study provides solution evidence for the weakening of lateral interactions in F-actin by cofilin.     

A role for GEA1 and GEA2 in the organization of the actin cytoskeleton in Saccharomyces cerevisiae

Profilin is an actin monomer-binding protein implicated in the polymerization of actin filaments. In the budding yeast Saccharomyces cerevisiae, the pfy1-111 rho2delta double mutant has severe growth and actin cytoskeletal defects. The GEA1 and GEA2 genes, which code for paralog guanosine exchange factors for Arf proteins, were identified as multicopy suppressors of the mutant phenotype. These two genes restored the polarized distribution of actin cortical patches and produced visible actin cables in both the pfy1-111 rho2delta and pfy1delta cells. Thus, overexpression of GEA1 or GEA2 bypassed the requirement for profilin in actin cable formation. In addition, gea1 gea2 double mutants showed defects in budding and in actin cytoskeleton organization, while overexpression of GEA1 or GEA2 led to the formation of supernumerary actin cable-like structures in a Bni1p/Bnr1p-dependent manner. The ADP-ribosylation factor Arf3p may be a target of Gea1p/Gea2p, since overexpression of ARF3 partially suppressed the profilin-deficient phenotype and a deletion of ARF3 exacerbated the phenotype of a pfy1-111 mutant. Gea1p, Gea2p, Arf1p, and Arf2p but not Arf3p are known to function in vesicular transport between the endoplasmic reticulum and the Golgi. In this work, we demonstrate a role for Gea1p, Gea2p, and Arf3p in the organization of the actin cytoskeleton.     

Septin ring assembly requires concerted action of polarisome components, a PAK kinase Cla4p, and the actin cytoskeleton in Saccharomyces cerevisiae

Septins are filament-forming proteins that function in cytokinesis in a wide variety of organisms. In budding yeast, the small GTPase Cdc42p triggers the recruitment of septins to the incipient budding site and the assembly of septins into a ring. We herein report that Bni1p and Cla4p, effectors of Cdc42p, are required for the assembly of the septin ring during the initiation of budding but not for its maintenance after the ring converts to a septin collar. In bni1Delta cla4-75-td mutant, septins were recruited to the incipient budding site. However, the septin ring was not assembled, and septins remained at the polarized growing sites. Bni1p, a formin family protein, is a member of the polarisome complex with Spa2p, Bud6p, and Pea2p. All spa2Delta cla4-75-td, bud6Delta cla4-75-td, and pea2Delta cla4-75-td mutants showed defects in septin ring assembly. Bni1p stimulates actin polymerization for the formation of actin cables. Point mutants of BNI1 that are specifically defective in actin cable formation also exhibited septin ring assembly defects in the absence of Cla4p. Consistently, treatment of cla4Delta mutant with the actin inhibitor latrunculin A inhibited septin ring assembly. Our results suggest that polarisome components and Cla4p are required for the initial assembly of the septin ring and that the actin cytoskeleton is involved in this process.     

Suppressors of mdm20 in yeast identify new alleles of ACT1 and TPM1 predicted to enhance actin-tropomyosin interactions

The actin cytoskeleton is required for many aspects of cell division in yeast, including mitochondrial partitioning into growing buds (mitochondrial inheritance). Yeast cells lacking MDM20 function display defects in both mitochondrial inheritance and actin organization, specifically, a lack of visible actin cables and enhanced sensitivity to Latrunculin A. mdm20 mutants also exhibit a temperature-sensitive growth phenotype, which we exploited to isolate second-site suppressor mutations. Nine dominant suppressors selected in an mdm20/mdm20 background rescue temperature-sensitive growth defects and mitochondrial inheritance defects and partially restore actin cables in haploid and diploid mdm20 strains. The suppressor mutations define new alleles of ACT1 and TPM1, which encode actin and the major form of tropomyosin in yeast, respectively. The ACT1 mutations cluster in a region of the actin protein predicted to contact tropomyosin, suggesting that they stabilize actin cables by enhancing actin-tropomyosin interactions. The characteristics of the mutant ACT1 and TPM1 alleles and their potential effects on protein structure and binding are discussed.     

Role of intermonomer ionic bridges in the stabilization of the actin filament

Filament formation is required for most of the functions of actin. However, the intermonomer interactions that stabilize F-actin have not been elucidated because of a lack of an F-actin crystal structure. The Holmes muscle actin model suggests that an ionic interaction between Arg-39 of one monomer and Glu-167 of an adjacent monomer in the same strand contributes to this stabilization. Yeast actin has an Ala-167 instead. F-actin molecular dynamics modeling predicts another interaction between Arg-39 of one monomer and Asp-275 of an opposing strand monomer. In Toxoplasma gondii actin, which forms short stubby filaments, the Asp-275 equivalent is replaced by Arg leading to a potential filament-destabilizing charge-charge repulsion. Using yeast actin, we tested the effect of A167E as a potential stabilizer and A167R and D275R as potential filament disruptors. All mutations caused abnormal growth and mitochondrial malfunction. A167E and D275R actins polymerize normally and form relatively normal appearing filaments. A167R nucleates filaments more slowly and forms filament bundles. The R39D/A167R double mutant, which re-establishes an ionic bond in the opposite orientation, reverses this polymerization and bundling defect. Stoichiometric amounts of yeast cofilin have little effect on wild-type and A167E filaments. However, D275R and A167R actin depolymerization is profound with cofilin. Although our results suggest that disruption of an interaction between Arg-39 and Asp-275 is not sufficient to cause fragmentation, it suggests that it changes filament stability thereby disposing it for enhanced cofilin depolymerizing effects. Ala-167 results demonstrate the in vivo and in vitro importance of another potential Arg-39 ionic interaction.     

Purification and polymerization properties of two lethal yeast actin mutants

The budding yeast Saccharomyces cerevisiae contains a single actin gene and the gene product, actin, is essential for growth. Two mutants of yeast actin that do not support yeast growth were prepared from yeast by coexpressing the mutant and a 6-histidine-tagged wild-type actin followed by separation of the wild-type and mutant actin using Ni-NTA chromatography as described elsewhere [Buzan, J., Du, J., Karpova, T., and Frieden, C. (1999) Proc. Natl. Acad. Sci. USA 96, 2823-2827]. The mutations, in muscle actin numbering, were at positions 334 (Glu334Lys) and 168 (Gly168Arg) and were chosen based on phenotypic changes observed in the behavior of actin mutants of Caenorhabditis elegans. Glu334 is located on the surface of actin between subdomains 1 and 3 while Gly168 is located in a region near actin-actin contacts in the actin filament. The Glu334Lys mutant polymerized slightly faster than wild-type yeast actin, suggesting that loss of interactions with some actin binding protein, rather than loss of actin-actin contacts, was responsible for its inability to support yeast growth. The Gly168Arg mutant polymerized at a rate similar to wild-type but the extent was considerably less, kinetic characteristics suggesting a high critical concentration (ca. 4 microM) without a large change in the ability to form nuclei for the nucleation-elongation process.     

Actin hydrophobic loop 262-274 and filament nucleation and elongation

The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently with the use of the yeast mutant actin L180C/L269C/C374A, in which the hydrophobic loop could be locked in a “parked” conformation by a disulfide bond between C180 and C269. Such a cross-linked globular actin monomer does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin, to assist with actin nucleation, and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not individually, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin, the helical twist of filamentous actin (F-actin) changes by ∼ 5° per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin and a change of twist by ∼ 1° per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics in both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors.