Bil2 Is a Novel Inhibitor of the Yeast Formin Bnr1 Required for Proper Actin Cable Organization and Polarized Secretion

Cell growth in budding yeast depends on rapid and on-going assembly and turnover of polarized actin cables, which direct intracellular transport of post-Golgi vesicles to the bud tip. Saccharomyces cerevisiae actin cables are polymerized by two formins, Bni1 and Bnr1. Bni1 assembles cables in the bud, while Bnr1 is anchored to the bud neck and assembles cables that specifically extend filling the mother cell. Here, we report a formin regulatory role for YGL015c, a previously uncharacterized open reading frame, which we have named Bud6 Interacting Ligand 2 (BIL2). bil2Δ cells display defects in actin cable architecture and partially-impaired secretory vesicle transport. Bil2 inhibits Bnr1-mediated actin filament nucleation in vitro, yet has no effect on the rate of Bnr1-mediated filament elongation. This activity profile for Bil2 resembles that of another yeast formin regulator, the F-BAR protein Hof1, and we find that bil2Δ with hof1Δ are synthetic lethal. Unlike Hof1, which localizes exclusively to the bud neck, GFP-Bil2 localizes to the cytosol, secretory vesicles, and sites of polarized cell growth. Further, we provide evidence that Hof1 and Bil2 inhibitory effects on Bnr1 are overcome by distinct mechanisms. Together, our results suggest that Bil2 and Hof1 perform distinct yet genetically complementary roles in inhibiting the actin nucleation activity of Bnr1 to control actin cable assembly and polarized secretion.

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

10.1091/mbc.e14-03-0850 ◽

2014 ◽

Vol 25 (11) ◽

pp. 1730-1743 ◽

Cited By ~ 23

Author(s):

Brian R. Graziano ◽

Hoi-Ying E. Yu ◽

Salvatore L. Alioto ◽

Julian A. Eskin ◽

Casey A. Ydenberg ◽

...

Keyword(s):

Secretory Vesicle ◽

Polarized Growth ◽

Vesicle Traffic ◽

Src Homology 3 ◽

Polarized Secretion ◽

Cytoskeleton Remodeling ◽

Src Homology ◽

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.

Aip1 and Cofilin Promote Rapid Turnover of Yeast Actin Patches and Cables: A Coordinated Mechanism for Severing and Capping Filaments

10.1091/mbc.e06-02-0135 ◽

2006 ◽

Vol 17 (7) ◽

pp. 2855-2868 ◽

Cited By ~ 80

Author(s):

Kyoko Okada ◽

Harini Ravi ◽

Ellen M. Smith ◽

Bruce L. Goode

Keyword(s):

Close Correlation ◽

Actin Binding ◽

Temperature Sensitive ◽

Interacting Protein ◽

Rapid Turnover ◽

Actin Cable ◽

Actin Turnover ◽

Rapid turnover of actin structures is required for dynamic remodeling of the cytoskeleton and cell morphogenesis, but the mechanisms driving actin disassembly are poorly defined. Cofilin plays a central role in promoting actin turnover by severing/depolymerizing filaments. Here, we analyze the in vivo function of a ubiquitous actin-interacting protein, Aip1, suggested to work with cofilin. We provide the first demonstration that Aip1 promotes actin turnover in living cells. Further, we reveal an unanticipated role for Aip1 and cofilin in promoting rapid turnover of yeast actin cables, dynamic structures that are decorated and stabilized by tropomyosin. Through systematic mutagenesis of Aip1 surfaces, we identify two well-separated F-actin–binding sites, one of which contributes to actin filament binding and disassembly specifically in the presence of cofilin. We also observe a close correlation between mutations disrupting capping of severed filaments in vitro and reducing rates of actin turnover in vivo. We propose a model for balanced regulation of actin cable turnover, in which Aip1 and cofilin function together to “prune” tropomyosin-decorated cables along their lengths. Consistent with this model, deletion of AIP1 rescues the temperature-sensitive growth and loss of actin cable defects of tpm1Δ mutants.

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

10.1091/mbc.e11-05-0404 ◽

2011 ◽

Vol 22 (21) ◽

pp. 4016-4028 ◽

Cited By ~ 45

Author(s):

Brian R. Graziano ◽

Amy Grace DuPage ◽

Alphee Michelot ◽

Dennis Breitsprecher ◽

James B. Moseley ◽

...

Keyword(s):

Actin Assembly ◽

Actin Cable ◽

Polarized Cell Growth ◽

Cable Assembly ◽

Nucleation Promoting Factor ◽

Actin Cables ◽

Elongation Phase

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.

The rho-GAP encoded by BEM2 regulates cytoskeletal structure in budding yeast.

10.1091/mbc.6.8.1011 ◽

1995 ◽

Vol 6 (8) ◽

pp. 1011-1024 ◽

Cited By ~ 51

Author(s):

T Wang ◽

A Bretscher

Keyword(s):

Functional Organization ◽

Synthetic Lethality ◽

Gtpase Activating Protein ◽

Functional Link ◽

Synthetic Lethal ◽

Cytoskeletal Structure ◽

Polarized Secretion ◽

Actin Cables ◽

Microtubule Function ◽

Six Genes

Microfilaments are required for polarized growth and morphogenesis in Saccharomyces cerevisiae. To accomplish this, actin cables and patches are redistributed during the cell cycle to direct secretory components to appropriate sites for cell growth. A major component of actin cables is tropomyosin I, encoded by TPM1, that determines or stabilizes these structures. Disruption of TPM1 is not lethal but results in the loss of actin cables and confers a partial defect in polarized secretion. Using a synthetic lethal screen, we have identified seven mutations residing in six genes whose products are required in the absence of Tpm1p. Each mutant exhibited a morphological defect, suggesting a functional link to the actin cytoskeleton. Complementation cloning of one mutation revealed that it lies in BEM2, which encodes a GTPase-activating protein for the RHO1 product. bem2 mutations also show synthetic lethality with rho1 and mutations in certain other cytoskeletal genes (ACT1, MYO1, MYO2, and SAC6) but not with mutations in several noncytoskeletal genes. These data therefore provide a genetic link between the GAP encoded by BEM2 and the functional organization of microfilaments. In addition, we show that bem2 mutations confer benomyl sensitivity and have abnormal microtubule arrays, suggesting that the BEM2 product may also be involved directly or indirectly in regulating microtubule function.

Phosphoregulation of tropomyosin is crucial for actin cable turnover and division site placement

The Journal of Cell Biology ◽

10.1083/jcb.201809089 ◽

2019 ◽

Vol 218 (11) ◽

pp. 3548-3559 ◽

Cited By ~ 7

Author(s):

Saravanan Palani ◽

Darius V. Köster ◽

Tomoyuki Hatano ◽

Anton Kamnev ◽

Taishi Kanamaru ◽

...

Keyword(s):

Fission Yeast ◽

Actin Filaments ◽

Coiled Coil ◽

Actin Binding ◽

Division Site ◽

Actin Cable ◽

Nonmuscle Cells ◽

The Stability ◽

Tropomyosin is a coiled-coil actin binding protein key to the stability of actin filaments. In muscle cells, tropomyosin is subject to calcium regulation, but its regulation in nonmuscle cells is not understood. Here, we provide evidence that the fission yeast tropomyosin, Cdc8, is regulated by phosphorylation of a serine residue. Failure of phosphorylation leads to an increased number and stability of actin cables and causes misplacement of the division site in certain genetic backgrounds. Phosphorylation of Cdc8 weakens its interaction with actin filaments. Furthermore, we show through in vitro reconstitution that phosphorylation-mediated release of Cdc8 from actin filaments facilitates access of the actin-severing protein Adf1 and subsequent filament disassembly. These studies establish that phosphorylation may be a key mode of regulation of nonmuscle tropomyosins, which in fission yeast controls actin filament stability and division site placement.

Identification of a novel modulator of the actin cytoskeleton, mitochondria, nutrient metabolism and lifespan in yeast

10.1101/2021.10.07.463535 ◽

2021 ◽

Author(s):

Cierra N Sing ◽

Enrique J Garcia ◽

Thomas Lipkin ◽

Thomas Huckaba ◽

Catherine A Tsang ◽

...

Keyword(s):

Nutrient Metabolism ◽

Mitochondrial Quality Control ◽

Reading Frame ◽

Daughter Cells ◽

Actin Cable ◽

Branched Chain Amino Acid ◽

Genome Wide ◽

Branched Chain ◽

Actin Cables ◽

And Function

In yeast, actin cables are F-actin bundles that are essential for cell division through their function as tracks for cargo movement from mother to daughter cell. Actin cables also affect yeast lifespan by promoting transport and inheritance of higher-functioning mitochondria to daughter cells. Here, we report that actin cable stability declines with age. Our genome-wide screen for genes that affect actin cable stability identified the open reading frame YKL075C. Deletion of YKL075C results in increases in actin cable stability and abundance, mitochondrial fitness, and replicative lifespan. Transcriptome analysis revealed a role for YKL075C in regulating branched-chain amino acid (BCAA) metabolism. Consistent with this, modulation of BCAA metabolism or decreasing leucine levels promotes actin cable stability and function in mitochondrial quality control. Our studies support a role for actin stability in yeast lifespan, and demonstrate that this process is controlled by BCAA and a previously uncharacterized ORF YKL075C, which we refer to as actin, aging and nutrient modulator protein 1 (AAN1).

Polarisome scaffolder Spa2-mediated macromolecular condensation of Aip5 for actin polymerization

Nature Communications ◽

10.1038/s41467-019-13125-1 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 5

Author(s):

Ying Xie ◽

Jialin Sun ◽

Xiao Han ◽

Alma Turšić-Wunder ◽

Joel D. W. Toh ◽

...

Keyword(s):

Phase Separation ◽

Cell Growth ◽

Actin Polymerization ◽

Stress Conditions ◽

Scaffolding Protein ◽

Actin Assembly ◽

Intrinsically Disordered ◽

Abstract A multiprotein complex polarisome nucleates actin cables for polarized cell growth in budding yeast and filamentous fungi. However, the dynamic regulations of polarisome proteins in polymerizing actin under physiological and stress conditions remains unknown. We identify a previously functionally unknown polarisome member, actin-interacting-protein 5 (Aip5), which promotes actin assembly synergistically with formin Bni1. Aip5-C terminus is responsible for its activities by interacting with G-actin and Bni1. Through N-terminal intrinsically disordered region, Aip5 forms high-order oligomers and generate cytoplasmic condensates under the stresses conditions. The molecular dynamics and reversibility of Aip5 condensates are regulated by scaffolding protein Spa2 via liquid-liquid phase separation both in vitro and in vivo. In the absence of Spa2, Aip5 condensates hamper cell growth and actin cable structures under stress treatment. The present study reveals the mechanisms of actin assembly for polarity establishment and the adaptation in stress conditions to protect actin assembly by protein phase separation.

Regulation of the Formin for3p by cdc42p and bud6p

10.1091/mbc.e07-02-0094 ◽

2007 ◽

Vol 18 (10) ◽

pp. 4155-4167 ◽

Cited By ~ 104

Author(s):

Sophie G. Martin ◽

Sergio A. Rincón ◽

Roshni Basu ◽

Pilar Pérez ◽

Fred Chang

Keyword(s):

Cell Growth ◽

Fission Yeast ◽

Rho Gtpase ◽

Intramolecular Interaction ◽

Actin Cable ◽

Polarized Cell Growth ◽

N Terminus ◽

Cable Assembly ◽

Formins are conserved actin nucleators responsible for the assembly of diverse actin structures. Many formins are controlled through an autoinhibitory mechanism involving the interaction of a C-terminal DAD sequence with an N-terminal DID sequence. Here, we show that the fission yeast formin for3p, which mediates actin cable assembly and polarized cell growth, is regulated by a similar autoinhibitory mechanism in vivo. Multiple sites govern for3p localization to cell tips. The localization and activity of for3p are inhibited by an intramolecular interaction of divergent DAD and DID-like sequences. A for3p DAD mutant expressed at endogenous levels produces more robust actin cables, which appear to have normal organization and dynamics. We identify cdc42p as the primary Rho GTPase involved in actin cable assembly and for3p regulation. Both cdc42p, which binds at the N terminus of for3p, and bud6p, which binds near the C-terminal DAD-like sequence, are needed for for3p localization and full activity, but a mutation in the for3p DAD restores for3p localization and other phenotypes of cdc42 and bud6 mutants. In particular, the for3p DAD mutation suppresses the bipolar growth (NETO) defect of bud6Δ cells. These findings suggest that cdc42p and bud6p activate for3p by relieving autoinhibition.

Stable and Dynamic Axes of Polarity Use Distinct Formin Isoforms in Budding Yeast

10.1091/mbc.e04-04-0296 ◽

2004 ◽

Vol 15 (11) ◽

pp. 4971-4989 ◽

Cited By ~ 116

Author(s):

David Pruyne ◽

Lina Gao ◽

Erfei Bi ◽

Anthony Bretscher

Keyword(s):

Mother Cell ◽

Feedback Signal ◽

Secretory Vesicles ◽

Rho Proteins ◽

Polarized Secretion ◽

Bud Neck ◽

Morphological Defects ◽

Bud Growth ◽

Actin Cables ◽

Cytoskeletal Elements

Bud growth in yeast is guided by myosin-driven delivery of secretory vesicles from the mother cell to the bud. We find transport occurs along two sets of actin cables assembled by two formin isoforms. The Bnr1p formin assembles cables that radiate from the bud neck into the mother, providing a stable mother-bud axis. These cables also depend on septins at the neck and are required for efficient transport from the mother to the bud. The Bni1p formin assembles cables that line the bud cortex and target vesicles to varying locations in the bud. Loss of these cables results in morphological defects as vesicles accumulate at the neck. Assembly of these cables depends on continued polarized secretion, suggesting vesicular transport provides a positive feedback signal for Bni1p activation, possibly by rho-proteins. By coupling different formin isoforms to unique cortical landmarks, yeast uses common cytoskeletal elements to maintain stable and dynamic axes in the same cell.

Yeast Formins Bni1 and Bnr1 Utilize Different Modes of Cortical Interaction during the Assembly of Actin Cables

10.1091/mbc.e06-09-0820 ◽

2007 ◽

Vol 18 (5) ◽

pp. 1826-1838 ◽

Cited By ~ 84

Author(s):

Shawnna M. Buttery ◽

Satoshi Yoshida ◽

David Pellman

Keyword(s):

Mother Cell ◽

Fluorescence Recovery After Photobleaching ◽

Actin Assembly ◽

Actin Cable ◽

Bud Neck ◽

Cable Assembly ◽

Different Populations ◽

Actin Cables ◽

Assembly Activity

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.