Common formin-regulating sequences in Smy1 and Bud14 are required for the control of actin cable assembly in vivo

Formins comprise a large family of proteins with diverse roles in remodeling the actin cytoskeleton. However, the spatiotemporal mechanisms used by cells to control formin activities are only beginning to be understood. Here we dissected Smy1, which has dual roles in regulating formins and myosin. Using mutagenesis, we identified specific sequences in Smy1 critical for its in vitro inhibitory effects on the FH2 domain of the formin Bnr1. By integrating smy1 alleles targeting those sequences, we genetically uncoupled Smy1’s functions in regulating formins and myosin. Quantitative imaging analysis further demonstrated that the ability of Smy1 to directly control Bnr1 activity is crucial in vivo for proper actin cable length, shape, and velocity and, in turn, efficient secretory vesicle transport. A Smy1-like sequence motif was also identified in a different Bnr1 regulator, Bud14, and found to be essential for Bud14 functions in regulating actin cable architecture and function in vivo. Together these observations reveal unanticipated mechanistic ties between two distinct formin regulators. Further, they emphasize the importance of tightly controlling formin activities in vivo to generate specialized geometries and dynamics of actin structures tailored to their physiological roles.     

The F-BAR protein Hof1 tunes formin activity to sculpt actin cables during polarized growth

Asymmetric cell growth and division rely on polarized actin cytoskeleton remodeling events, the regulation of which is poorly understood. In budding yeast, formins stimulate the assembly of an organized network of actin cables that direct polarized secretion. Here we show that the Fer/Cip4 homology–Bin amphiphysin Rvs protein Hof1, which has known roles in cytokinesis, also functions during polarized growth by directly controlling the activities of the formin Bnr1. A mutant lacking the C-terminal half of Hof1 displays misoriented and architecturally altered cables, along with impaired secretory vesicle traffic. In vitro, Hof1 inhibits the actin nucleation and elongation activities of Bnr1 without displacing the formin from filament ends. These effects depend on the Src homology 3 domain of Hof1, the formin homology 1 (FH1) domain of Bnr1, and Hof1 dimerization, suggesting a mechanism by which Hof1 “restrains” the otherwise flexible FH1-FH2 apparatus. In vivo, loss of inhibition does not alter actin levels in cables but, instead, cable shape and functionality. Thus Hof1 tunes formins to sculpt the actin cable network.     

An InCytes from MBC Selection: Polarized Growth in Budding Yeast in the Absence of a Localized Formin

Polarity is achieved partly through the localized assembly of the cytoskeleton. During growth in budding yeast, the bud cortex and neck localized formins Bni1p and Bnr1p nucleate and assemble actin cables that extend along the bud-mother axis, providing tracks for secretory vesicle delivery. Localized formins are believed to determine the location and polarity of cables, hence growth. However, yeast expressing the nonlocalized actin nucleating/assembly formin homology (FH) 1-FH2 domains of Bnr1p or Bni1p as the sole formin grow well. Although cables are significantly disorganized, analysis of directed transport of secretory vesicles is still biased toward the bud, reflecting a bias in correctly oriented cables, thereby permitting polarized growth. Myosin II, localized at the bud neck, contributes to polarized growth as a mutant unable to interact with F-actin further compromises growth in cells with an unlocalized formin but not with a localized formin. Our results show that multiple mechanisms contribute to cable orientation and polarized growth, with localized formins and myosin II being two major contributors.     

Antenna Mechanism of Length Control of Actin Cables

Actin cables are linear cytoskeletal structures that serve as tracks for myosin-based intracellular transport of vesicles and organelles in both yeast and mammalian cells. In a yeast cell undergoing budding, cables are in constant dynamic turnover yet some cables grow from the bud neck toward the back of the mother cell until their length roughly equals the diameter of the mother cell. This raises the question: how is the length of these cables controlled? Here we describe a novel molecular mechanism for cable length control inspired by recent experimental observations in cells. This “antenna mechanism” involves three key proteins: formins, which polymerize actin, Smy1 proteins, which bind formins and inhibit actin polymerization, and myosin motors, which deliver Smy1 to formins, leading to a length-dependent actin polymerization rate. We compute the probability distribution of cable lengths as a function of several experimentally tuneable parameters such as the formin-binding affinity of Smy1 and the concentration of myosin motors delivering Smy1. These results provide testable predictions of the antenna mechanism of actin-cable length control.     

Formin-dependent actin assembly is regulated by distinct modes of Rho signaling in yeast

Formins are actin filament nucleators regulated by Rho-GTPases. In budding yeast, the formins Bni1p and Bnr1p direct the assembly of actin cables, which guide polarized secretion and growth. From the six yeast Rho proteins (Cdc42p and Rho1-5p), we have determined that four participate in the regulation of formin activity. We show that the essential function of Rho3p and Rho4p is to activate the formins Bni1p and Bnr1p, and that activated alleles of either formin are able to bypass the requirement for these Rho proteins. Through a separate signaling pathway, Rho1p is necessary for formin activation at elevated temperatures, acting through protein kinase C (Pkc1p), the major effector for Rho1p signaling to the actin cytoskeleton. Although Pkc1p also activates a MAPK pathway, this pathway does not function in formin activation. Formin-dependent cable assembly does not require Cdc42p, but in the absence of Cdc42p function, cable assembly is not properly organized during initiation of bud growth. These results show that formin function is under the control of three distinct, essential Rho signaling pathways.     

Mechanism and cellular function of Bud6 as an actin nucleation-promoting factor

Formins are a conserved family of actin assembly-promoting factors with diverse biological roles, but how their activities are regulated in vivo is not well understood. In Saccharomyces cerevisiae, the formins Bni1 and Bnr1 are required for the assembly of actin cables and polarized cell growth. Proper cable assembly further requires Bud6. Previously it was shown that Bud6 enhances Bni1-mediated actin assembly in vitro, but the biochemical mechanism and in vivo role of this activity were left unclear. Here we demonstrate that Bud6 specifically stimulates the nucleation rather than the elongation phase of Bni1-mediated actin assembly, defining Bud6 as a nucleation-promoting factor (NPF) and distinguishing its effects from those of profilin. We generated alleles of Bud6 that uncouple its interactions with Bni1 and G-actin and found that both interactions are critical for NPF activity. Our data indicate that Bud6 promotes filament nucleation by recruiting actin monomers to Bni1. Genetic analysis of the same alleles showed that Bud6 regulation of formin activity is critical for normal levels of actin cable assembly in vivo. Our results raise important mechanistic parallels between Bud6 and WASP, as well as between Bud6 and other NPFs that interact with formins such as Spire.     

A Highlights from MBoC Selection: Regulation of the formin Bnr1 by septins anda MARK/Par1-family septin-associated kinase

Formin-family proteins promote the assembly of linear actin filaments and are required to generate cellular actin structures, such as actin stress fibers and the cytokinetic actomyosin contractile ring. Many formin proteins are regulated by an autoinhibition mechanism involving intramolecular binding of a Diaphanous inhibitory domain and a Diaphanous autoregulatory domain. However, the activation mechanism for these Diaphanous-related formins (DRFs) is not completely understood. Although small GTPases play an important role in relieving autoinhibition, other factors likely contribute. Here we describe a requirement for the septin Shs1 and the septin-associated kinase Gin4 for the localization and in vivo activity of the budding yeast DRF Bnr1. In budding yeast strains in which the other formin, Bni1, is conditionally inactivated, the loss of Gin4 or Shs1 results in the loss of actin cables and cell death, similar to the loss of Bnr1. The defects in these strains can be suppressed by constitutive activation of Bnr1. Gin4 is involved in both the localization and activation of Bnr1, whereas the septin Shs1 is required for Bnr1 activation but not its localization. Gin4 promotes the activity of Bnr1 independently of the Gin4 kinase activity, and Gin4 lacking its kinase domain binds to the critical localization region of Bnr1. These data reveal novel regulatory links between the actin and septin cytoskeletons.     

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.     

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.     

An Arabidopsis Class II Formin,AtFH19, Nucleates Actin Assembly,Binds to the Barbed End of Actin Filaments and Antagonizes the Effect of AtFH1 on Actin Dynamics

Formin is a major protein responsible for regulating the nucleation of actin filaments, and as such, it permits the cell to control where and when to assemble actin arrays. It is encoded by a multigene family comprising 21 members in Arabidopsis thaliana. The Arabidopsis formins can be separated into two phylogenetically-distinct classes: there are 11 class I formins and 10 class II formins. Significant questions remain unanswered regarding the molecular mechanism of actin nucleation and elongation stimulated by each formin isovariant, and how the different isovariants coordinate to regulate actin dynamics in cells. Here, we characterize a class II formin, AtFH19, biochemically. We found that AtFH19 retains all general properties of the formin family, including nucleation and barbed end capping activity. It can also generate actin filaments from a pool of actin monomers bound to profilin. However, both the nucleation and barbed end capping activities of AtFH19 are less efficient compared to those of another well-characterized formin, AtFH1. Interestingly, AtFH19 FH1FH2 competes with AtFH1 FH1FH2 in binding actin filament barbed ends, and inhibits the effect of AtFH1 FH1FH2 on actin. We thus propose a mechanism in which two quantitatively different formins coordinate to regulate actin dynamics by competing for actin filament barbed ends.     

Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae

Myo2 protein (Myo2p), an unconventional myosin in the budding yeast Saccharomyces cerevisiae, has been implicated in polarized growth and secretion by studies of the temperature-sensitive myo2-66 mutant. Overexpression of Smy1p, which by sequence is a kinesin-related protein, can partially compensate for defects in the myo2 mutant (Lillie, S. H. and S. S. Brown, 1992. Nature (Lond.). 356:358-361). We have now immunolocalized Smy1p and Myo2p. Both are concentrated in regions of active growth, as caps at incipient bud sites and on small buds, at the mother-bud neck just before cell separation, and in mating cells as caps on shmoo tips and at the fusion bridge of zygotes. Double labeling of cells with either Myo2p or Smy1p antibody plus phalloidin was used to compare the localization of Smy1p and Myo2p to actin, and by extrapolation, to each other. These studies confirmed that Myo2p and Smy1p colocalize, and are concentrated in the same general regions of the cell as actin spots. However, neither colocalizes with actin. We noted a correlation in the behavior of Myo2p, Smy1p, and actin, but not microtubules, under a number of circumstances. In cdc4 and cdc11 mutants, which produce multiple buds, Myo2p and Smy1p caps were found only in the subset of buds that had accumulations of actin. Mutations in actin or secretory genes perturb actin, Smy1p and Myo2p localization. The rearrangements of Myo2p and Smy1p correlate temporally with those of actin spots during the cell cycle, and upon temperature and osmotic shift. In contrast, microtubules are not grossly affected by these perturbations. Although wild-type Myo2p localization does not require Smy1p, Myo2p staining is brighter when SMY1 is overexpressed. The myo2 mutant, when shifted to restrictive temperature, shows a permanent loss in Myo2p localization and actin polarization, both of which can be restored by SMY1 overexpression. However, the lethality of MYO2 deletion is not overcome by SMY1 overexpression. We noted that the myo2 mutant can recover from osmotic shift (unlike actin mutants; Novick, P., and D. Botstein. 1985. Cell. 40:405-416). We have also determined that the myo2-66 allele encodes a Lys instead of a Glu at position 511, which lies at an actin-binding face in the motor domain.     

Nucleation limits the lengths of actin filaments assembled by formin

Formins stimulate actin polymerization by promoting both filament nucleation and elongation. Because nucleation and elongation draw upon a common pool of actin monomers, the rate at which each reaction proceeds influences the other. This interdependent mechanism determines the number of filaments assembled over the course of a polymerization reaction, as well as their equilibrium lengths. In this study, we used kinetic modeling and in vitro polymerization reactions to dissect the contributions of filament nucleation and elongation to the process of formin-mediated actin assembly. We found that the rates of nucleation and elongation evolve over the course of a polymerization reaction. The period over which each process occurs is a key determinant of the total number of filaments that are assembled, as well as their average lengths at equilibrium. Inclusion of formin in polymerization reactions speeds filament nucleation, thus increasing the number and shortening the lengths of filaments that are assembled over the course of the reaction. Modulation of the elongation rate produces modest changes in the equilibrium lengths of formin-bound filaments. However, the dependence of filament length on the elongation rate is limited by the number of filament ends generated via formin’s nucleation activity. Sustained elongation of small numbers of formin-bound filaments, therefore, requires inhibition of nucleation via monomer sequestration and a low concentration of activated formin. Our results underscore the mechanistic advantage for keeping formin’s nucleation efficiency relatively low in cells, where unregulated actin assembly would produce deleterious effects on cytoskeletal dynamics. Under these conditions, differences in the elongation rates mediated by formin isoforms are most likely to impact the kinetics of actin assembly.     

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.     

Actin cable distribution and dynamics arising from cross-linking,motor pulling,and filament turnover

The growth of fission yeast relies on the polymerization of actin filaments nucleated by formin For3p, which localizes at tip cortical sites. These actin filaments bundle to form actin cables that span the cell and guide the movement of vesicles toward the cell tips. A big challenge is to develop a quantitative understanding of these cellular actin structures. We used computer simulations to study the spatial and dynamical properties of actin cables. We simulated individual actin filaments as semiflexible polymers in three dimensions composed of beads connected with springs. Polymerization out of For3p cortical sites, bundling by cross-linkers, pulling by type V myosin, and severing by cofilin are simulated as growth, cross-linking, pulling, and turnover of the semiflexible polymers. With the foregoing mechanisms, the model generates actin cable structures and dynamics similar to those observed in live-cell experiments. Our simulations reproduce the particular actin cable structures in myoVΔ cells and predict the effect of increased myosin V pulling. Increasing cross-linking parameters generates thicker actin cables. It also leads to antiparallel and parallel phases with straight or curved cables, consistent with observations of cells overexpressing α-actinin. Finally, the model predicts that clustering of formins at cell tips promotes actin cable formation.     

Assembly and mechanosensory function of focal adhesions: experiments and models

Initial integrin-mediated cell-matrix adhesions (focal complexes) appear underneath the lamellipodia, in the regions of the "fast" centripetal flow driven by actin polymerization. Once formed, these adhesions convert the flow behind them into a "slow", myosin II-driven mode. Some focal complexes then turn into elongated focal adhesions (FAs) associated with contractile actomyosin bundles (stress fibers). Myosin II inhibition does not suppress formation of focal complexes but blocks their conversion into mature FAs and further FA growth. Application of external pulling force promotes FA growth even under conditions when myosin II activity is blocked. Thus, individual FAs behave as mechanosensors responding to the application of force by directional assembly. We proposed a thermodynamic model for the mechanosensitivity of FAs, taking into account that an elastic molecular aggregate subject to pulling forces tends to grow in the direction of force application by incorporating additional subunits. This simple model can explain a variety of processes typical of FA behavior. Assembly of FAs is triggered by the small G-protein Rho via activation of two major targets, Rho-associated kinase (ROCK) and the formin homology protein, Dia1. ROCK controls creation of myosin II-driven forces, while Dia1 is involved in the response of FAs to these forces. Expression of the active form of Dia1, allows the external force-induced assembly of mature FAs, even in conditions when Rho is inhibited. Conversely, downregulation of Dia1 by siRNA prevents FA maturation even if Rho is activated. Dia1 and other formins cap barbed (fast growing) ends of actin filaments, allowing insertion of the new actin monomers. We suggested a novel mechanism of such "leaky" capping based on an assumption of elasticity of the formin/barbed end complex. Our model predicts that formin-mediated actin polymerization should be greatly enhanced by application of external pulling force. Thus, the formin-actin complex might represent an elementary mechanosensing device responding to force by enhancement of actin assembly. In addition to its role in actin polymerization, Dia1 seems to be involved in formation of links between actin filaments and microtubules affecting microtubule dynamics. Alpha-tubulin deacetylase HDAC6 cooperates with Dia1 in formation of such links. Since microtubules are known to promote FA disassembly, the Dia1-mediated effect on microtubule dynamics may possibly play a role in the negative feedback loop controlling size and turnover of FAs.     

Model of formin-associated actin filament elongation

Formin FH2 domains associate processively with actin-filament barbed ends and modify their rate of growth. We modeled how the elongation rate depends on the concentrations of profilin and actin for four different formins. We assume that (1) FH2 domains are in rapid equilibrium among conformations that block or allow actin addition and that (2) profilin-actin is transferred rapidly to the barbed end from multiple profilin binding sites in formin FH1 domains. In agreement with previous experiments discussed below, we find an optimal profilin concentration with a maximal elongation rate that can exceed the rate of actin alone. High profilin concentrations suppress elongation, largely because free profilin displaces profilin-actin from FH1. The model supports a common polymerization mechanism for the four formin FH1FH2 constructs with differences attributed to varying parameter values. The mechanism does not require ATP hydrolysis by polymerized actin, but we cannot exclude that formins accelerate hydrolysis.     

Yeast formins Bni1 and Bnr1 utilize different modes of cortical interaction during the assembly of actin cables

The budding yeast formins Bni1 and Bnr1 control the assembly of actin cables. These formins exhibit distinct patterns of localization and polymerize two different populations of cables: Bni1 in the bud and Bnr1 in the mother cell. We generated a functional Bni1-3GFP that improved the visualization of Bni1 in vivo at endogenous levels. Bni1 exists as speckles in the cytoplasm, some of which colocalize on actin cables. These Bni1 speckles display linear, retrograde-directed movements. Loss of polymerized actin or specifically actin cables abolished retrograde movement, and resulted in depletion of Bni1 speckles from the cytoplasm, with enhanced targeting of Bni1 to the bud tip. Mutations that impair the actin assembly activity of Bni1 abolished the movement of Bni1 speckles, even when actin cables were present. In contrast, Bnr1-GFP or 3GFP-Bnr1 did not detectably associate with actin cables and was not observed as cytoplasmic speckles. Finally, fluorescence recovery after photobleaching demonstrated that Bni1 was very dynamic, exchanging between polarized sites and the cytoplasm, whereas Bnr1 was confined to the bud neck and did not exchange with a cytoplasmic pool. In summary, our results indicate that formins can have distinct modes of cortical interaction during actin cable assembly.     

Tropomyosin regulates elongation by formin at the fast-growing end of the actin filament

The balance between dynamic and stable actin filaments is essential for the regulation of cellular functions including the determination of cell shape and polarity, cell migration, and cytokinesis. Proteins that regulate polymerization at the filament ends and filament stability confer specificity to actin filament structure and cellular function. The dynamics of the barbed, fast-growing end of the filament are controlled in space and time by both positive and negative regulators of actin polymerization. Capping proteins inhibit the addition and loss of subunits, whereas other proteins, including formins, bind at the barbed end and allow filament growth. In this work, we show that tropomyosin regulates dynamics at the barbed end. Tropomyosin binds to constructs of FRL1 and mDia2 that contain the FH2 domain and modulates formin-dependent capping of the barbed end by relieving inhibition of elongation by FRL1-FH1FH2, mDia1-FH2, and mDia2-FH2 in an isoform-dependent fashion. In this role, tropomyosin functions as an activator of formin. Tropomyosin also inhibits the binding of FRL1-FH1FH2 to the sides of actin filaments independent of the isoform. In contrast, tropomyosin does not affect the ability of capping protein to block the barbed end. We suggest that tropomyosin and formin act together to ensure the formation of unbranched actin filaments, protected from severing, that could be capped in stable cellular structures. This role, in addition to its cooperative control of myosin function, establishes tropomyosin as a universal regulator of the multifaceted actin cytoskeleton.     

Differential activities and regulation of Saccharomyces cerevisiae formin proteins Bni1 and Bnr1 by Bud6

Formins are conserved proteins that nucleate actin assembly and tightly associate with the fast growing barbed ends of actin filaments to allow insertional growth. Most organisms express multiple formins, but it has been unclear whether they have similar or distinct activities and how they may be regulated differentially. We isolated and compared the activities of carboxyl-terminal fragments of the only two formins expressed in Saccharomyces cerevisiae, Bni1 and Bnr1. Bnr1 was an order of magnitude more potent than Bni1 in actin nucleation and processive capping, and unlike Bni1, Bnr1 bundled actin filaments. Profilin bound directly to Bni1 and Bnr1 and regulated their activities similarly. However, the cell polarity factor Bud6/Aip3 specifically bound to and stimulated Bni1, but not Bnr1. This was unexpected, since previous two-hybrid studies suggested Bud6 interacts with both formins. We mapped Bud6 binding activity to specific residues in the carboxyl terminus of Bni1 that are adjacent to its diaphanous autoregulatory domain (DAD). Fusion of the carboxyl terminus of Bni1 to Bnr1 conferred Bud6 stimulation to a Bnr1-Bni1 chimera. Thus, Bud6 differentially stimulates Bni1 and not Bnr1. We found that Bud6 is up-regulated during bud growth, when it is delivered to the bud tip on Bni1-nucleated actin cables. We propose that Bud6 stimulation of Bni1 promotes robust cable formation, which in turn delivers more Bud6 to the bud tip, reinforcing polarized cell growth through a positive feedback loop.     

Formin Cdc12’s specific actin assembly properties are tailored for cytokinesis in fission yeast

Formins generate unbranched actin filaments by a conserved, processive actin assembly mechanism. Most organisms express multiple formin isoforms that mediate distinct cellular processes and facilitate actin filament polymerization by significantly different rates, but how these actin assembly differences correlate to cellular activity is unclear. We used a computational model of fission yeast cytokinetic ring assembly to test the hypothesis that particular actin assembly properties help tailor formins for specific cellular roles. Simulations run in different actin filament nucleation and elongation conditions revealed that variations in formin’s nucleation efficiency critically impact both the probability and timing of contractile ring formation. To probe the physiological importance of nucleation efficiency, we engineered fission yeast formin chimera strains in which the FH1-FH2 actin assembly domains of full-length cytokinesis formin Cdc12 were replaced with the FH1-FH2 domains from functionally and evolutionarily diverse formins with significantly different actin assembly properties. Although Cdc12 chimeras generally support life in fission yeast, quantitative live-cell imaging revealed a range of cytokinesis defects from mild to severe. In agreement with the computational model, chimeras whose nucleation efficiencies are least similar to Cdc12 exhibit more severe cytokinesis defects, specifically in the rate of contractile ring assembly. Together, our computational and experimental results suggest that fission yeast cytokinesis is ideally mediated by a formin with properly tailored actin assembly parameters.