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1.
Biophys J ; 120(15): 2984-2997, 2021 08 03.
Article in English | MEDLINE | ID: mdl-34214524

ABSTRACT

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.


Subject(s)
Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Actin Cytoskeleton , Actins/genetics , Cytokinesis , Cytoskeletal Proteins , Formins , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics
2.
Mol Biol Cell ; 22(20): 3826-39, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21865598

ABSTRACT

Fission yeast expresses three formins required for distinct actin cytoskeletal processes: Cdc12 (cytokinesis), For3 (polarization), and Fus1 (mating). We propose that in addition to differential regulation, key actin-assembly properties tailor formins for a particular role. In direct comparison to the well-studied Cdc12, we report the first in vitro characterization of the actin-assembly properties of For3 and Fus1. All three share fundamental formin activities; however, particular reaction rates vary significantly. Cdc12 is an efficient nucleator (one filament per approximately 3 Cdc12 dimers) that processively elongates profilin-actin at a moderate rate of 10 subunits s(-1) µM(-1), but lacks filament-bundling activity. Fus1 is also an efficient nucleator, yet processively elongates profilin-actin at one-half the rate of and dissociates 10-fold more rapidly than Cdc12; it also bundles filaments. For3 nucleates filaments 100-fold less well than Fus1, but like Cdc12, processively elongates profilin-actin at a moderate rate and lacks filament-bundling activity. Additionally, both the formin homology FH1 and FH2 domains contribute to the overall rate of profilin-actin elongation. We also confirmed the physiological importance of the actin-assembly activity of the fission yeast formins. Point mutants that disrupt their ability to stimulate actin assembly in vitro do not function properly in vivo.


Subject(s)
Actins/metabolism , Cell Cycle Proteins/metabolism , Cytoskeletal Proteins/metabolism , Cytoskeleton/metabolism , Gene Expression Regulation, Fungal , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Signal Transduction/genetics , Actins/genetics , Cell Cycle Proteins/genetics , Cytoskeletal Proteins/genetics , Cytoskeleton/genetics , Formins , Microscopy, Fluorescence , Mutation , Plasmids , Profilins/genetics , Profilins/metabolism , Protein Binding , Protein Structure, Tertiary , Schizosaccharomyces/cytology , Schizosaccharomyces/genetics , Schizosaccharomyces/growth & development , Schizosaccharomyces pombe Proteins/genetics , Transduction, Genetic
3.
Chem Biol ; 16(11): 1158-68, 2009 Nov 25.
Article in English | MEDLINE | ID: mdl-19942139

ABSTRACT

Formins stimulate actin filament assembly for fundamental cellular processes including division, adhesion, establishing polarity, and motility. A formin inhibitor would be useful because most cells express multiple formins whose functions are not known and because metastatic tumor formation depends on the deregulation of formin-dependent processes. We identified a general small molecule inhibitor of formin homology 2 domains (SMIFH2) by screening compounds for the ability to prevent formin-mediated actin assembly in vitro. SMIFH2 targets formins from evolutionarily diverse organisms including yeast, nematode worm, and mice, with a half-maximal inhibitor concentration of approximately 5 to 15 microM. SMIFH2 prevents both formin nucleation and processive barbed end elongation and decreases formin's affinity for the barbed end. Furthermore, low micromolar concentrations of SMIFH2 disrupt formin-dependent, but not Arp2/3 complex-dependent, actin cytoskeletal structures in fission yeast and mammalian NIH 3T3 fibroblasts.


Subject(s)
Actin Cytoskeleton/drug effects , Actins/metabolism , Carrier Proteins/antagonists & inhibitors , Microfilament Proteins/antagonists & inhibitors , Thiones/pharmacology , Uracil/analogs & derivatives , Actin Cytoskeleton/metabolism , Actin-Related Protein 2-3 Complex/metabolism , Actins/chemistry , Animals , Cell Line, Tumor , Cytoskeleton/drug effects , Formins , Humans , Mice , Microscopy, Fluorescence , NIH 3T3 Cells , Protein Structure, Tertiary , Pyrimidinones/chemistry , Pyrimidinones/pharmacology , Schizosaccharomyces/drug effects , Small Molecule Libraries , Structure-Activity Relationship , Thiones/chemistry , Thiones/toxicity , Uracil/chemistry , Uracil/pharmacology , Uracil/toxicity
4.
Mol Biol Cell ; 20(8): 2160-73, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19244341

ABSTRACT

Like animal cells, fission yeast divides by assembling actin filaments into a contractile ring. In addition to formin Cdc12p and profilin, the single tropomyosin isoform SpTm is required for contractile ring assembly. Cdc12p nucleates actin filaments and remains processively associated with the elongating barbed end while driving the addition of profilin-actin. SpTm is thought to stabilize mature filaments, but it is not known how SpTm localizes to the contractile ring and whether SpTm plays a direct role in Cdc12p-mediated actin polymerization. Using "bulk" and single actin filament assays, we discovered that Cdc12p can recruit SpTm to actin filaments and that SpTm has diverse effects on Cdc12p-mediated actin assembly. On its own, SpTm inhibits actin filament elongation and depolymerization. However, Cdc12p completely overcomes the combined inhibition of actin nucleation and barbed end elongation by profilin and SpTm. Furthermore, SpTm increases the length of Cdc12p-nucleated actin filaments by enhancing the elongation rate twofold and by allowing them to anneal end to end. In contrast, SpTm ultimately turns off Cdc12p-mediated elongation by "trapping" Cdc12p within annealed filaments or by dissociating Cdc12p from the barbed end. Therefore, SpTm makes multiple contributions to contractile ring assembly during and after actin polymerization.


Subject(s)
Actin Cytoskeleton/metabolism , Cell Cycle Proteins/metabolism , Cytoskeletal Proteins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Tropomyosin/metabolism , Actins/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Microbial Viability , Microscopy, Fluorescence , Models, Biological , Protein Binding , Schizosaccharomyces/cytology
5.
J Biol Chem ; 284(1): 673-684, 2009 Jan 02.
Article in English | MEDLINE | ID: mdl-18978356

ABSTRACT

Cells contain multiple formin isoforms that drive the assembly of profilin-actin for diverse processes. Given that many organisms also contain several profilin isoforms, specific formin/profilin pairs might be matched to optimally stimulate actin polymerization. We utilized a combination of bulk actin polymerization and single filament total internal reflection fluorescence microscopy assays to measure the effect of different profilin isoforms on the actin assembly properties of the cytokinesis formins from fission yeast (Cdc12p) and the nematode worm (CYK-1). We discovered that Cdc12p only effectively utilizes the single fission yeast profilin isoform SpPRF. Conversely, CYK-1 prefers the essential worm cytokinesis profilin CePFN-1 to the two non-essential worm profilin isoforms (SpPRF = CePFN-1 > CePFN-2 > CePFN-3). Chimeras containing the profilin-binding formin homology 1 (FH1) domain from one formin and the barbed-end associated FH2 domain from the other formin, revealed that both the FH1 and FH2 domains help confer profilin isoform specialization. Although the Cdc12p and CYK-1 FH1 domains cannot differentiate between profilin isoforms in the absence of actin, formin FH1 domains appear to preferentially select specific isoforms of profilin-actin. Surprisingly, analysis of profilin point mutants revealed that differences in highly conserved residues in both the poly-L-proline and actin binding regions of profilin do not explain their differential utilization by formin. Therefore, rapid formin-mediated elongation of profilin-actin depends upon favorable interactions of profilin-actin with the FH1 domain as well as the barbed-end associated FH2 domain. Specific formin FH1FH2 domains are tailored to optimally utilize actin bound to particular profilin isoforms.


Subject(s)
Actin Cytoskeleton/chemistry , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans/chemistry , Microfilament Proteins/chemistry , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces/chemistry , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Point Mutation , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary/physiology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Sequence Homology, Amino Acid
6.
J Biol Chem ; 283(35): 23872-83, 2008 Aug 29.
Article in English | MEDLINE | ID: mdl-18577519

ABSTRACT

Formins drive actin filament assembly for diverse cellular processes including motility, establishing polarity, and cell division. To investigate the mechanism of contractile ring assembly in animal cells, we directly compared the actin assembly properties of formins required for cytokinesis in the nematode worm early embryo (CYK-1) and fission yeast (Cdc12p). Like Cdc12p and most other formins, CYK-1 nucleates actin filament assembly and remains processively associated with the elongating barbed end while facilitating the addition of profilin-actin above the theoretical diffusion-limited rate. However, specific properties differ significantly between Cdc12p and CYK-1. Cdc12p efficiently nucleates filaments that in the presence of profilin elongate at approximately the same rate as control filaments without formin (approximately 10.0 subunits/s). CYK-1 is an inefficient nucleator but allows filaments to elongate profilin-actin 6-fold faster than Cdc12p (approximately 60 subunits/s). Both Cdc12p and CYK-1 bind to pre-assembled actin filaments with low nanomolar affinity, but CYK-1 dissociates 2 orders of magnitude more quickly. However, CYK-1 rapidly re-associates with free barbed ends. Cdc12p allows barbed ends to elongate in the presence of excess capping protein, whereas capping protein inhibits CYK-1-mediated actin assembly. Therefore, these evolutionarily diverse formins can drive contractile ring assembly by a generally similar mechanism, but cells with unique dimensions and physical parameters might require proteins with carefully tuned actin assembly properties.


Subject(s)
Actin Cytoskeleton/chemistry , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans/chemistry , Cytoskeletal Proteins/chemistry , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces/chemistry , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cytokinesis/physiology , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Mice , Profilins/chemistry , Profilins/genetics , Profilins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Species Specificity
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