scholarly journals Purification, characterization, and immunofluorescence localization of Saccharomyces cerevisiae capping protein

1992 ◽  
Vol 117 (5) ◽  
pp. 1067-1076 ◽  
Author(s):  
JF Amatruda ◽  
JA Cooper
Keyword(s):  
Sequence Similarity ◽  
Critical Concentration ◽  
Beta Subunits ◽  
Cortical Actin ◽  
Gene Encoding ◽  
Capping Protein ◽  
Bud Emergence ◽  
End Capping ◽  
Actin Cables

Capping protein binds the barbed ends of actin filaments and nucleates actin filament assembly in vitro. We purified capping protein from Saccharomyces cervisiae. One of the two subunits is the product of the CAP2 gene, which we previously identified as the gene encoding the beta subunit of capping protein based on its sequence similarity to capping protein beta subunits in chicken and Dictyostelium (Amatruda, J. F., J. F. Cannon, K. Tatchell, C. Hug, and J. A. Cooper. 1990. Nature (Lond.) 344:352-354). Yeast capping protein has activity in critical concentration and low-shear viscometry assays consistent with barbed-end capping activity. Like chicken capping protein, yeast capping protein is inhibited by PIP2. By immunofluorescence microscopy yeast capping protein colocalizes with cortical actin spots at the site of bud emergence and at the tips of growing buds and shmoos. In contrast, capping protein does not colocalize with actin cables or with actin rings at the site of cytokinesis.

scholarly journals Interactions with PIP2, ADP-actin monomers, and capping protein regulate the activity and localization of yeast twinfilin

2001 ◽  
Vol 155 (2) ◽  
pp. 251-260 ◽  
Author(s):  
Sandra Palmgren ◽  
Pauli J. Ojala ◽  
Martin A. Wear ◽  
John A. Cooper ◽  
Pekka Lappalainen
Keyword(s):  
Actin Filament ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Actin Monomer ◽  
Cortical Actin ◽  
Gene Encoding ◽  
Capping Protein ◽  
Filament Dynamics ◽  
Filament Assembly

Twinfilin is a ubiquitous actin monomer–binding protein that regulates actin filament turnover in yeast and mammalian cells. To elucidate the mechanism by which twinfilin contributes to actin filament dynamics, we carried out an analysis of yeast twinfilin, and we show here that twinfilin is an abundant protein that localizes to cortical actin patches in wild-type yeast cells. Native gel assays demonstrate that twinfilin binds ADP-actin monomers with higher affinity than ATP-actin monomers. A mutant twinfilin that does not interact with actin monomers in vitro no longer localizes to cortical actin patches when expressed in yeast, suggesting that the ability to interact with actin monomers may be essential for the localization of twinfilin. The localization of twinfilin to the cortical actin cytoskeleton is also disrupted in yeast strains where either the CAP1 or CAP2 gene, encoding for the α and β subunits of capping protein, is deleted. Purified twinfilin and capping protein form a complex on native gels. Twinfilin also interacts with phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2), and its actin monomer–sequestering activity is inhibited by PI(4,5)P2. Based on these results, we propose a model for the biological role of twinfilin as a protein that localizes actin monomers to the sites of rapid filament assembly in cells.

Mutations that enhance the cap2 null mutant phenotype in Saccharomyces cerevisiae affect the actin cytoskeleton, morphogenesis and pattern of growth.

Genetics ◽  
1993 ◽  
Vol 135 (3) ◽  
pp. 693-709 ◽  
Author(s):  
T S Karpova ◽  
M M Lepetit ◽  
J A Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Actin Cytoskeleton ◽  
Synthetic Lethality ◽  
Actin Binding ◽  
Cortical Actin ◽  
Gene Encoding ◽  
Synthetic Lethal ◽  
Capping Protein ◽  
Colony Color ◽  
Actin Cables

Abstract Mutations conferring synthetic lethality in combination with null mutations in CAP2, the gene encoding the beta subunit of capping protein of Saccharomyces cerevisiae, were obtained in a colony color assay. Monogenic inheritance was found for four mutations, which were attributed to three genetic loci. One mutation, sac6-69, is in the gene encoding fimbrin, another actin-binding protein, which was expected because null mutations in SAC6 and CAP2 are known to be synthetic-lethal. The other two loci were designated slc for synthetic lethality with cap2. These loci include the mutations slc1-66, slc1-87 and slc2-107. The slc mutations are semi-dominant, as shown by incomplete complementation in slc/SLC cap2/cap2 heterozygotes. The slc mutations and sac6-69 interact with each other, as shown by enhanced phenotypes in diheterozygotes. Moreover, the haploid slc2-107 sac6-69 double mutant is inviable. In a CAP2 background, the slc mutations lead to temperature and osmotic sensitivity. They alter the distribution of the actin cytoskeleton, including deficits in the presence of actin cables and the polarization of cortical actin patches. The slc mutations also lead to a pseudomycelial growth pattern. Together these results suggest that slc1 and slc2 encode components of the actin cytoskeleton in yeast and that the actin cytoskeleton can regulate the patterns of growth.

scholarly journals Signaling through Adenylyl Cyclase Is Essential for Hyphal Growth and Virulence in the Pathogenic FungusCandida albicans

2001 ◽  
Vol 12 (11) ◽  
pp. 3631-3643 ◽  
Author(s):  
Cintia R. C. Rocha ◽  
Klaus Schröppel ◽  
Doreen Harcus ◽  
Anne Marcil ◽  
Daniel Dignard ◽  
...  
Keyword(s):  
Mouse Model ◽  
Adenylyl Cyclase ◽  
Sequence Similarity ◽  
Hyphal Growth ◽  
Antifungal Drugs ◽  
Growth Defect ◽  
Adenylyl Cyclases ◽  
Gene Encoding ◽  
Mutant Cells

The human fungal pathogen Candida albicans switches from a budding yeast form to a polarized hyphal form in response to various external signals. This morphogenetic switching has been implicated in the development of pathogenicity. We have cloned theCaCDC35 gene encoding C. albicansadenylyl cyclase by functional complementation of the conditional growth defect of Saccharomyces cerevisiae cells with mutations in Ras1p and Ras2p. It has previously been shown that these Ras homologues regulate adenylyl cyclase in yeast. The C. albicans adenylyl cyclase is highly homologous to other fungal adenylyl cyclases but has less sequence similarity with the mammalian enzymes. C. albicans cells deleted for both alleles ofCaCDC35 had no detectable cAMP levels, suggesting that this gene encodes the only adenylyl cyclase in C. albicans. The homozygous mutant cells were viable but grew more slowly than wild-type cells and were unable to switch from the yeast to the hyphal form under all environmental conditions that we analyzed in vitro. Moreover, this morphogenetic switch was completely blocked in mutant cells undergoing phagocytosis by macrophages. However, morphogenetic switching was restored by exogenous cAMP. On the basis of epistasis experiments, we propose that CaCdc35p acts downstream of the Ras homologue CaRas1p. These epistasis experiments also suggest that the putative transcription factor Efg1p and components of the hyphal-inducing MAP kinase pathway depend on the function of CaCdc35p in their ability to induce morphogenetic switching. Homozygouscacdc35Δ cells were unable to establish vaginal infection in a mucosal membrane mouse model and were avirulent in a mouse model for systemic infections. These findings suggest that fungal adenylyl cyclases and other regulators of the cAMP signaling pathway may be useful targets for antifungal drugs.

scholarly journals Visualization and Molecular Analysis of Actin Assembly in Living Cells

1998 ◽  
Vol 143 (7) ◽  
pp. 1919-1930 ◽  
Author(s):  
Dorothy A. Schafer ◽  
Matthew D. Welch ◽  
Laura M. Machesky ◽  
Paul C. Bridgman ◽  
Shelley M. Meyer ◽  
...  
Keyword(s):  
Actin Filament ◽  
Eukaryotic Cell ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Lim Kinase ◽  
Permeabilized Cells ◽  
Capping Protein ◽  
Filament Assembly

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

scholarly journals Dia- and Rok-dependent enrichment of capping proteins in a cortical region

2021 ◽  
Author(s):  
Anja Schmidt ◽  
Long Li ◽  
Zhiyi Lv ◽  
Shuling Yan ◽  
Jörg Großhans
Keyword(s):  
Myosin Ii ◽  
Rho Kinase ◽  
Cortical Region ◽  
Protein Enrichment ◽  
Cortical Actin ◽  
Capping Protein ◽  
Capping Proteins ◽  
Muscle Myosin ◽  
Drosophila Embryos

Rho signaling with its major targets the formin Dia, Rho kinase (Rok) and non-muscle myosin II control turnover, amount and contractility of actomyosin. Much less investigated has been a potential function for the distribution of F-actin plus and minus ends. In syncytial Drosophila embryos Rho1 signaling is high between actin caps, i. e. the cortical intercap region. Capping protein binds to free plus ends of F-actin to prevent elongation of the filament. Capping protein has served as a marker to visualize the distribution of F-actin plus ends in cells and in vitro. Here, we probed the distribution of plus ends with capping protein in syncytial Drosophila embryos. We found that Capping proteins are specifically enriched in the intercap region similar to Dia and MyoII but distinct from overall F-actin. The intercap enrichment of Capping protein was impaired in dia mutants and embryos, in which Rok and MyoII activation was inhibited. Our observations reveal that Dia and Rok/MyoII control Capping protein enrichment and support a model that Dia and Rok/MyoII control the organization of cortical actin cytoskeleton downstream of Rho1 signaling.

scholarly journals Tropomodulin caps the pointed ends of actin filaments.

1994 ◽  
Vol 127 (6) ◽  
pp. 1627-1635 ◽  
Author(s):  
A Weber ◽  
C R Pennise ◽  
G G Babcock ◽  
V M Fowler
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Actin Filaments ◽  
Striated Muscle ◽  
Critical Concentration ◽  
Thin Filaments ◽  
Capping Protein ◽  
End Capping ◽  
Pointed End ◽  
A Site

Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411-420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd < or = 1 nM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 microM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate of extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells.

scholarly journals The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin

2003 ◽  
Vol 161 (5) ◽  
pp. 875-887 ◽  
Author(s):  
David R. Kovar ◽  
Jeffrey R. Kuhn ◽  
Andrea L. Tichy ◽  
Thomas D. Pollard
Keyword(s):  
Fission Yeast ◽  
Actin Filaments ◽  
Maximum Yield ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Contractile Ring ◽  
Capping Protein ◽  
Ring Assembly ◽  
End Capping ◽  
Actin Cables

Cytokinesis in most eukaryotes requires the assembly and contraction of a ring of actin filaments and myosin II. The fission yeast Schizosaccharomyces pombe requires the formin Cdc12p and profilin (Cdc3p) early in the assembly of the contractile ring. The proline-rich formin homology (FH) 1 domain binds profilin, and the FH2 domain binds actin. Expression of a construct consisting of the Cdc12 FH1 and FH2 domains complements a conditional mutant of Cdc12 at the restrictive temperature, but arrests cells at the permissive temperature. Cells overexpressing Cdc12(FH1FH2)p stop growing with excessive actin cables but no contractile rings. Like capping protein, purified Cdc12(FH1FH2)p caps the barbed end of actin filaments, preventing subunit addition and dissociation, inhibits end to end annealing of filaments, and nucleates filaments that grow exclusively from their pointed ends. The maximum yield is one filament pointed end per six formin polypeptides. Profilins that bind both actin and poly-l-proline inhibit nucleation by Cdc12(FH1FH2)p, but polymerization of monomeric actin is faster, because the filaments grow from their barbed ends at the same rate as uncapped filaments. On the other hand, Cdc12(FH1FH2)p blocks annealing even in the presence of profilin. Thus, formins are profilin-gated barbed end capping proteins with the ability to initiate actin filaments from actin monomers bound to profilin. These properties explain why contractile ring assembly requires both formin and profilin and why viability depends on the ability of profilin to bind both actin and poly-l-proline.

scholarly journals Expression of a Novel Leishmania Gene Encoding a Histone H1-Like Protein in Leishmania major Modulates Parasite Infectivity In Vitro

2002 ◽  
Vol 70 (12) ◽  
pp. 6976-6986 ◽  
Author(s):  
Fotini T. Papageorgiou ◽  
Ketty P. Soteriadou
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Leishmania Major ◽  
Sequence Similarity ◽  
Histone H1 ◽  
Nuclear Targeting ◽  
Developmentally Regulated ◽  
Gene Encoding

ABSTRACT We describe identification and characterization of a novel two-copy gene of the parasitic protozoan Leishmania that encodes a nuclear protein designated LNP18. This protein is highly conserved in the genus Leishmania, and it is developmentally regulated. It is an alanine- and lysine-rich protein with potential bipartite nuclear targeting sequence sites. LNP18 shows sequence similarity to H1 histones of trypanosomatids and of higher eukaryotes and in particular with histone H1 of Leishmania major. The nuclear localization of LNP18 was determined by indirect immunofluorescence and Western blot analysis of isolated nuclei by using antibodies raised against the recombinant protein as probes. The antibodies recognized predominantly a 18-kDa band or a 18-kDa-16-kDa doublet. Photochemical cross-linking of intact parasites followed by Western blot analysis provided evidence that LNP18 is indeed a DNA-binding protein. Generation of transfectants overexpressing LNP18 allowed us to determine the role of this protein in Leishmania infection of macrophages in vitro. These studies revealed that transfectants overexpressing LNP18 are significantly less infective than transfectants with the vector alone and suggested that the level of LNP18 expression modulates Leishmania infectivity, as assessed in vitro.

scholarly journals Capping Protein Terminates but Does Not Initiate Chemoattractant-induced Actin Assembly in Dictyostelium

1997 ◽  
Vol 139 (5) ◽  
pp. 1243-1253 ◽  
Author(s):  
R.J. Eddy ◽  
J. Han ◽  
J.S. Condeelis
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
De Novo ◽  
Leading Edge ◽  
Actin Assembly ◽  
Directed Movement ◽  
Capping Protein ◽  
End Capping

The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.

scholarly journals Yeast Aim21/Tda2 both regulates free actin by reducing barbed end assembly and forms a complex with Cap1/Cap2 to balance actin assembly between patches and cables

2018 ◽  
Vol 29 (8) ◽  
pp. 923-936 ◽  
Author(s):  
Myungjoo Shin ◽  
Jolanda van Leeuwen ◽  
Charles Boone ◽  
Anthony Bretscher
Keyword(s):  
Budding Yeast ◽  
Negative Regulator ◽  
Pivotal Role ◽  
Cortical Region ◽  
Actin Assembly ◽  
Actin Monomer ◽  
Filamentous Actin ◽  
Capping Protein ◽  
End Capping ◽  
Actin Cables

How cells balance the incorporation of actin into diverse structures is poorly understood. In budding yeast, a single actin monomer pool is used to build both actin cables involved in polarized growth and actin cortical patches involved in endocytosis. Here we report how Aim21/Tda2 is recruited to the cortical region of actin patches, where it negatively regulates actin assembly to elevate the available actin monomer pool. Aim21 has four polyproline regions and is recruited by two SH3-containing patch proteins, Bbc1 and Abp1. The C-terminal region, which is required for its function, binds Tda2. Cell biological and biochemical data reveal that Aim21/Tda2 is a negative regulator of barbed end filamentous actin (F-actin) assembly, and this activity is necessary for efficient endocytosis and plays a pivotal role in balancing the distribution of actin between cables and patches. Aim21/Tda2 also forms a complex with the F-actin barbed end capping protein Cap1/Cap2, revealing an interplay between regulators and showing the complexity of regulation of barbed end assembly.

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