Differential Regulation of Actin Polymerization and Structure by Yeast Formin Isoforms

The budding yeast formins, Bnr1 and Bni1, behave very differently with respect to their interactions with muscle actin. However, the mechanisms underlying these differences are unclear, and these formins do not interact with muscle actin in vivo. We use yeast wild type and mutant actins to further assess these differences between Bnr1 and Bni1. Low ionic strength G-buffer does not promote actin polymerization. However, Bnr1, but not Bni1, causes the polymerization of pyrene-labeled Mg-G-actin in G-buffer into single filaments based on fluorometric and EM observations. Polymerization by Bnr1 does not occur with Ca-G-actin. By cosedimentation, maximum filament formation occurs at a Bnr1:actin ratio of 1:2. The interaction of Bnr1 with pyrene-labeled S265C Mg-actin yields a pyrene excimer peak, from the cross-strand interaction of pyrene probes, which only occurs in the context of F-actin. In F-buffer, Bnr1 promotes much faster yeast actin polymerization than Bni1. It also bundles the F-actin in contrast to the low ionic strength situation where only single filaments form. Thus, the differences previously observed with muscle actin are not actin isoform-specific. The binding of both formins to F-actin saturate at an equimolar ratio, but only about 30% of each formin cosediments with F-actin. Finally, addition of Bnr1 but not Bni1 to pyrene-labeled wild type and S265C Mg-F actins enhanced the pyrene- and pyrene-excimer fluorescence, respectively, suggesting Bnr1 also alters F-actin structure. These differences may facilitate the ability of Bnr1 to form the actin cables needed for polarized delivery of nutrients and organelles to the growing yeast bud.Bni1 and Bnr1 are the two formin isoforms expressed in Saccharomyces cerevisiae (1, 2). These proteins, as other isoforms in the formin family, are large multidomain proteins (3, 4). Several regulatory domains, including one for binding the G-protein rho, are located at the N-terminal half of the protein (4–7). FH1, FH2, and Bud6 binding domains are located in the C-terminal half of the protein (8). The formin homology 1 (FH1)² domain contains several sequential poly-l-proline motifs, and it interacts with the profilin/actin complex to recruit actin monomers and regulate the insertion of actin monomers at the barbed end of actin (9–11). The fomin homology domain 2 (FH2) forms a donut-shaped homodimer, which wraps around actin dimers at the barbed end of actin filaments (12, 13). One important function of formin is to facilitate actin polymerization by stabilizing actin dimers or trimers under polymerization conditions and then to processively associate with the barbed end of the elongating filament to control actin filament elongation kinetics (13–18).A major unsolved protein in the study of formins is the elucidation of the individual functions of different isoforms and their regulation. In vivo, these two budding yeast formins have distinct cellular locations and dynamics (1, 2, 19, 20). Bni1 concentrates at the budding site before the daughter cell buds from the mother cell, moves along with the tip of the daughter cell, and then travels back to the neck between daughter and mother cells at the end of segregation. Bnr1 localizes only at the neck of the budding cell in a very short period of time after bud emergence. Although a key cellular function of these two formins in yeast is to promote actin cable formation (8, 18), the roles of the individual formins in different cellular process is unclear because deleting either individual formin gene has limited impact on cell growth and deleting both genes together is lethal (21).Although each of the two formins can nucleate actin filament formation in vitro, the manner in which they affect polymerization is distinctly isoform-specific. Most of this mechanistic work in vitro has used formin fragments containing the FH1 and FH2 domains. Bni1 alone processively caps the barbed end of actin filaments partially inhibiting polymerization at this end (14, 16, 18). The profilin-actin complex, recruited to the actin barbed end through its binding to Bni1 FH1 domain, possibly raises the local actin concentration and appears to allow this inhibition to be overcome, thereby, accelerating barbed end polymerization. It has also been shown that this complex modifies the kinetics of actin dynamics at the barbed end (9, 11, 18, 22). Moreover, Bni1 participation leads only to the formation of single filaments (8). In comparison, the Bnr1 FH1-FH2 domain facilitates actin polymerization much more efficiently than does Bni1. Moseley and Goode (8) showed Bnr1 accelerates actin polymerization up to 10 times better than does Bni and produces actin filament bundles when the Bnr1/actin molar ratio is above 1:2. Finally, the regulation of Bni1 and Bnr1 by formin binding is different. For example, Bud 6/Aip3, a yeast cell polarity factor, binds to Bni1, but not Bnr1, and also stimulates its activity in vitro.For their studies, Moseley and Goode (8) utilized mammalian skeletal muscle actin instead of the S. cerevisiae actin with which the yeast formins are designed to function. It is entirely possible that the differences observed with the two formins are influenced quantitatively or qualitatively by the nature of the actin used in the study. This possibility must be seriously considered because although yeast and muscle actins are 87% identical in sequence, they display marked differences in their polymerization behavior (23). Yeast actin nucleates filaments better than muscle actin (24, 25). It appears to form shorter and more flexible filaments than does muscle actin (26, 27). Finally, the disposition of the P_i released during the hydrolysis of ATP that occurs during polymerization is different. Yeast actin releases its P_i concomitant with hydrolysis of the bound ATP whereas muscle actin retains the P_i for a significant amount of time following nucleotide hydrolysis (28, 29). This difference is significant because ADP-P_i F-actin has been shown to be more stable than ADP F-actin (30). Another example of this isoform dependence is the interaction of yeast Arp2/3 with yeast versus muscle actins (31). Yeast Arp2/3 complex accelerates polymerization of muscle actin only in the presence of a nucleation protein factor such as WASP. However, with yeast actin, no such auxiliary protein is required. In light of these actin behavioral differences, to better understand the functional differences of these two formins in vivo, we have studied the behavior of Bni 1 and Bnr 1 with WT and mutant yeast actins, and we have also explored the molecular basis underlying the Bnr 1-induced formation of actin nuclei from G-actin.     

Fission Yeast Sec3 Bridges the Exocyst Complex to the Actin Cytoskeleton

The exocyst complex tethers post‐Golgi secretory vesicles to the plasma membrane prior to docking and fusion. In this study, we identify Sec3, the missing component of the Schizosaccharomyces pombe exocyst complex (SpSec3). SpSec3 shares many properties with its orthologs, and its mutants are rescued by human Sec3/EXOC1. Although involved in exocytosis, SpSec3 does not appear to mark the site of exocyst complex assembly at the plasma membrane. It does, however, mark the sites of actin cytoskeleton recruitment and controls the organization of all three yeast actin structures: the actin cables, endocytic actin patches and actomyosin ring. Specifically, SpSec3 physically interacts with For3 and sec3 mutants have no actin cables as a result of a failure to polarize this nucleating formin. SpSec3 also interacts with actin patch components and sec3 mutants have depolarized actin patches of reduced endocytic capacity. Finally, the constriction and disassembly of the cytokinetic actomyosin ring is compromised in these sec3 mutant cells. We propose that a role of SpSec3 is to spatially couple actin machineries and their independently polarized regulators. As a consequence of its dual role in secretion and actin organization, Sec3 appears as a major co‐ordinator of cell morphology in fission yeast .     

Model of For3p-Mediated Actin Cable Assembly in Fission Yeast

Formin For3p nucleates actin cables at the tips of fission yeast cells for polarized cell growth. The results of prior experiments have suggested a possible mechanism for actin cable assembly that involves association of For3p near cell tips, For3p-mediated actin polymerization, retrograde flow of actin cables toward the cell center, For3p dissociation from cell tips, and cable disassembly. We used analytical and computational modeling to test the validity and implications of the proposed coupled For3p/actin mechanism. We compared the model to prior experiments quantitatively and generated predictions for the expected behavior of the actin cable system upon changes of parameter values. We found that the model generates stable steady states with realistic values of rate constants and actin and For3p concentrations. Comparison of our results to previous experiments monitoring the FRAP of For3p-3GFP and the response of actin cables to treatments with actin depolymerizing drugs provided further support for the model. We identified the set of parameter values that produces results in agreement with experimental observations. We discuss future experiments that will help test the model''s predictions and eliminate other possible mechanisms. The results of the model suggest that flow of actin cables may establish actin and For3p concentration gradients in the cytoplasm that could be important in global cell patterning.     

An InCytes from MBC Selection: Pob1 Participates in the Cdc42 Regulation of Fission Yeast Actin Cytoskeleton

Rho GTPases regulate the actin cytoskeleton in all eukaryotes. Fission yeast Cdc42 is involved in actin cable assembly and formin For3 regulation. We isolated cdc42-879 as a thermosensitive strain with actin cable and For3 localization defects. In a multicopy suppressor screening, we identified pob1⁺ as suppressor of cdc42-879 thermosensitivity. Pob1 overexpression also partially restores actin cables and localization of For3 in the mutant strain. Pob1 interacts with Cdc42 and this GTPase regulates Pob1 localization and/or stability. The C-terminal pleckstrin homology (PH) domain of Pob1 is required for Cdc42 binding. Pob1 also binds to For3 through its N-terminal sterile alpha motif (SAM) domain and contributes to the formin localization at the cell tips. The previously described pob1-664 mutant strain (Mol. Biol. Cell. 10, 2745–2757, 1999), which carries a mutation in the PH domain, as well as pob1 mutant strains in which Pob1 lacks the N-terminal region (pob1ΔN) or the SAM domain (pob1ΔSAM), have cytoskeletal defects similar to that of cdc42-879 cells. Expression of constitutively active For3DAD* partially restores actin organization in cdc42-879, pob1-664, pob1ΔN, and pob1ΔSAM. Therefore, we propose that Pob1 is required for For3 localization to the tips and facilitates Cdc42-mediated relief of For3 autoinhibition to stimulate actin cable formation.     

Mapping of Unconventional Myosins in Mouse and Human

Myosins are molecular motors that move along filamentous actin. Seven classes of myosin are expressed in vertebrates: conventional myosin, or myosin-II, as well as the 6 unconventional myosin classes -I, -V, -VI, -VII, -IX, and -X. We have mapped in mouse 22 probes encompassing all known unconventional myosins and, as a result, have identified 16 potential unconventional myosin genes. These genes include 7 myosins-I, 2 myosins-V, 1 myosin-VI, 3 myosins-VII, 2 myosins-IX, and 1 myosin-X. The map location of 5 of these genes was identified in human chromosomes by fluorescencein situhybridization.     

Insight into Actin Organization and Function in Cytokinesis from Analysis of Fission Yeast Mutants

Actin is a key cytoskeletal protein with multiple roles in cellular processes such as polarized growth, cytokinesis, endocytosis, and cell migration. Actin is present in all eukaryotes as highly dynamic filamentous structures, such as linear cables and branched filaments. Detailed investigation of the molecular role of actin in various processes has been hampered due to the multifunctionality of the protein and the lack of alleles defective in specific processes. The actin cytoskeleton of the fission yeast, Schizosaccharomyces pombe, has been extensively characterized and contains structures analogous to those in other cell types. In this study, primarily with the view to uncover actin function in cytokinesis, we generated a large bank of fission yeast actin mutants that affect the organization of distinct actin structures and/or discrete physiological functions of actin. Our screen identified 17 mutants with specific defects in cytokinesis. Some of these cytokinesis mutants helped in dissecting the function of specific actin structures during ring assembly. Further genetic analysis of some of these actin mutants revealed multiple genetic interactions with mutants previously known to affect the actomyosin ring assembly. We also characterize a mutant allele of actin that is suppressed upon overexpression of Cdc8p-tropomyosin, underscoring the utility of this mutant bank. Another 22 mutant alleles, defective in polarized growth and/or other functions of actin obtained from this screen, are also described in this article. This mutant bank should be a valuable resource to study the physiological and biochemical functions of actin.     

Regulation of the Cortical Actin Cytoskeleton in Budding Yeast by Twinfilin,a Ubiquitous Actin Monomer-sequestering Protein

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)–twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.     

Mathematical Model for Growth Regulation of Fission Yeast Schizosaccharomyces pombe

Regulation of polarised cell growth is essential for many cellular processes including spatial coordination of cell morphology changes during the division cycle. We present a mathematical model of the core mechanism responsible for the regulation of polarised growth dynamics during the fission yeast cell cycle. The model is based on the competition of growth zones localised at the cell tips for a common substrate distributed uniformly in the cytosol. We analyse the bifurcations in this model as the cell length increases, and show that the growth activation dynamics provides an explanation for the new-end take-off (NETO) as a saddle-node bifurcation at which the cell sharply switches from monopolar to bipolar growth. We study the parameter sensitivity of the bifurcation diagram and relate qualitative changes of the growth pattern, e.g. delayed or absent NETO, to previously observed mutant phenotypes. We investigate the effects of imperfect asymmetric cell division, and show that this leads to distinct growth patterns that provide experimentally testable predictions for validating the presented competitive growth zone activation model. Finally we discuss extension of the model for describing mutant cells with more than two growth zones.     

D-loop of Actin Differently Regulates the Motor Function of Myosins II and V

To gain more information on the manner of actin-myosin interaction, we examined how the motile properties of myosins II and V are affected by the modifications of the DNase I binding loop (D-loop) of actin, performed in two different ways, namely, the proteolytic digestion with subtilisin and the M47A point mutation. In an in vitro motility assay, both modifications significantly decreased the gliding velocity on myosin II-heavy meromyosin due to a weaker generated force but increased it on myosin V. On the other hand, single molecules of myosin V “walked” with the same velocity on both the wild-type and modified actins; however, the run lengths decreased sharply, correlating with a lower affinity of myosin for actin due to the D-loop modifications. The difference between the single-molecule and the ensemble measurements with myosin V indicates that in an in vitro motility assay the non-coordinated multiple myosin V molecules impose internal friction on each other via binding to the same actin filament, which is reduced by the weaker binding to the modified actins. These results show that the D-loop strongly modulates the force generation by myosin II and the processivity of myosin V, presumably affecting actin-myosin interaction in the actomyosin-ADP·P_i state of both myosins.     

Multiple Regulation of Rad51-Mediated Homologous Recombination by Fission Yeast Fbh1

Fbh1, an F-box helicase related to bacterial UvrD, has been proposed to modulate homologous recombination in fission yeast. We provide several lines of evidence for such modulation. Fbh1, but not the related helicases Srs2 and Rqh1, suppressed the formation of crossover recombinants from single HO-induced DNA double-strand breaks. Purified Fbh1 in complex with Skp1 (Fbh1-Skp1 complex) inhibited Rad51-driven DNA strand exchange by disrupting Rad51 nucleoprotein filaments in an ATP-dependent manner; this disruption was alleviated by the Swi5-Sfr1 complex, an auxiliary activator of Rad51. In addition, the reconstituted SCF^Fbh1 complex, composed of purified Fbh1-Skp1 and Pcu1-Rbx1, displayed ubiquitin-ligase E3 activity toward Rad51. Furthermore, Fbh1 reduced the protein level of Rad51 in stationary phase in an F-box-dependent, but not in a helicase domain-independent manner. These results suggest that Fbh1 negatively regulates Rad51-mediated homologous recombination via its two putative, unrelated activities, namely DNA unwinding/translocation and ubiquitin ligation. In addition to its anti-recombinase activity, we tentatively suggest that Fbh1 might also have a pro-recombination role in vivo, because the Fbh1-Skp1 complex stimulated Rad51-mediated strand exchange in vitro after strand exchange had been initiated.     

Suppressors of Yeast Actin Mutations

Suppressors of a temperature-sensitive mutation (act1-1) in the single actin gene of Saccharomyces cerevisiae were selected that had simultaneously acquired a cold-sensitive growth phenotype. Five genes, called SAC (suppressor of actin) were defined by complementation tests; both suppression and cold-sensitive phenotypes were recessive. Three of the genes (SAC1, SAC2 and SAC3) were subjected to extensive genetic and phenotypic analysis, including molecular cloning. Suppression was found to be allele-specific with respect to actin alleles. The sac mutants, even in ACT1+ genetic backgrounds, displayed phenotypes similar to those of actin mutants, notably aberrant organization of intracellular actin and deposition of chitin at the cell surface. These results are interpreted as being consistent with the idea that the SAC genes encode proteins that interact with actin, presumably as components or controllers of the assembly or stability of the yeast actin cytoskeleton. Two unexpected properties of the SAC1 gene were noted. Disruptions of the gene indicated that its function is essential only at temperatures below about 17 degrees and all sac1 alleles are inviable when combined with act1-2. These properties are interpreted in the context of the evolution of the actin cytoskeleton of yeast.     

裂殖酵母Papl应答H2O2氧化胁迫的机制

转录因子Papl是裂殖酵母(Schizosaccharomyces pombe)应答H2O2氧化胁迫反应中的关键调控因子.高浓度的H2O2激活蛋白激酶Styl途径清除过量的H2O2,使H2O2降至较低浓度再活化Papl;低浓度的则直接氧化活化Papl,导致Papl快速向细胞核内运输从而激活Papl相关基因的表达.本文综述了裂殖酵母中转录因子Papl在不同浓度H2O2胁迫下的激活途径,以及蛋白激酶Styl对Papl激活的重要作用.     

Feedback Regulation of SIN by Etd1 and Rho1 in Fission Yeast

In fission yeast, the septation initiation network (SIN) is thought to promote cytokinesis by downstream activation of Rho1, a conserved GTPase that controls cell growth and division. Here we show that Etd1 and PP2A-Pab1, antagonistic regulators of SIN, are Rho1 regulators. Our genetic and biochemical studies indicate that a C-terminal region of Etd1 may activate Rho1 by directly binding it, whereas an N-terminal domain confers its ability to localize at the growing tips and the division site where Rho1 functions. In opposition to Etd1, our results indicate that PP2A-Pab1 inhibits Rho1. The SIN cascade is upstream-regulated by the Spg1 GTPase. In the absence of Etd1, activity of Spg1 drops down prematurely, thereby inactivating SIN. Interestingly, we find that ectopic activation of Rho1 restores Spg1 activity in Etd1-depleted cells. By using a cytokinesis block strategy, we show that Rho1 is essential to feedback-activate Spg1 during actomyosin ring constriction. Therefore, activation of Spg1 by Rho1, which in turn is regulated by Etd1, uncovers a novel feedback loop mechanism that ensures SIN activity while cytokinesis is progressing.     

A Highlights from MBoC Selection: Mathematical Modeling of Endocytic Actin Patch Kinetics in Fission Yeast: Disassembly Requires Release of Actin Filament Fragments

We used the dendritic nucleation hypothesis to formulate a mathematical model of the assembly and disassembly of actin filaments at sites of clathrin-mediated endocytosis in fission yeast. We used the wave of active WASp recruitment at the site of the patch formation to drive assembly reactions after activation of Arp2/3 complex. Capping terminated actin filament elongation. Aging of the filaments by ATP hydrolysis and γ-phosphate dissociation allowed actin filament severing by cofilin. The model could simulate the assembly and disassembly of actin and other actin patch proteins using measured cytoplasmic concentrations of the proteins. However, to account quantitatively for the numbers of proteins measured over time in the accompanying article (Sirotkin et al., 2010 , MBoC 21: 2792–2802), two reactions must be faster in cells than in vitro. Conditions inside the cell allow capping protein to bind to the barbed ends of actin filaments and Arp2/3 complex to bind to the sides of filaments faster than the purified proteins in vitro. Simulations also show that depolymerization from pointed ends cannot account for rapid loss of actin filaments from patches in 10 s. An alternative mechanism consistent with the data is that severing produces short fragments that diffuse away from the patch.     

Linkers of Cell Polarity and Cell Cycle Regulation in the Fission Yeast Protein Interaction Network

The study of gene and protein interaction networks has improved our understanding of the multiple, systemic levels of regulation found in eukaryotic and prokaryotic organisms. Here we carry out a large-scale analysis of the protein-protein interaction (PPI) network of fission yeast (Schizosaccharomyces pombe) and establish a method to identify ‘linker’ proteins that bridge diverse cellular processes - integrating Gene Ontology and PPI data with network theory measures. We test the method on a highly characterized subset of the genome consisting of proteins controlling the cell cycle, cell polarity and cytokinesis and identify proteins likely to play a key role in controlling the temporal changes in the localization of the polarity machinery. Experimental inspection of one such factor, the polarity-regulating RNB protein Sts5, confirms the prediction that it has a cell cycle dependent regulation. Detailed bibliographic inspection of other predicted ‘linkers’ also confirms the predictive power of the method. As the method is robust to network perturbations and can successfully predict linker proteins, it provides a powerful tool to study the interplay between different cellular processes.     

G2/M Arrest Caused by Actin Disruption Is a Manifestation of the Cell Size Checkpoint in Fission Yeast

In budding yeast, actin disruption prevents nuclear division. This has been explained as activation of a morphogenesis checkpoint monitoring the integrity of the actin cytoskeleton. The checkpoint operates through inhibitory tyrosine phosphorylation of Cdc28, the budding yeast Cdc2 homolog. Wild-type Schizosaccharomyces pombe cells also arrest before mitosis after actin depolymerization. Oversized cells, however, enter mitosis uninhibited. We carried out a careful analysis of the kinetics of mitotic initiation after actin disruption in undersized and oversized cells. We show that an inability to reach the mitotic size threshold explains the arrest in smaller cells. Among the regulators that control the level of the inhibitory Cdc2-Tyr15 phosphorylation, the Cdc25 protein tyrosine phosphatase is required to link cell size monitoring to mitotic control. This represents a novel function of the Cdc25 phosphatase. Furthermore, we demonstrate that this cell size-monitoring system fulfills the formal criteria of a cell cycle checkpoint.     

裂殖酵母Pap1应答H2O2氧化胁迫的机制

转录因子Pap1是裂殖酵母(Schizosaccharomyces pombe)应答H2O2氧化胁迫反应中的关键调控因子.高浓度的H2O2激活蛋白激酶Sty1途径清除过量的H2O2,使H2O2降至较低浓度再活化Pap1;低浓度的则直接氧化活化Pap1,导致Pap1快速向细胞核内运输从而激活Pap1相关基因的表达.本文综述了裂殖酵母中转录因子Pap1在不同浓度H2O2胁迫下的激活途径,以及蛋白激酶Sty1对Pap1激活的重要作用     

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.