Interaction of the Gonococcal Porin P.IB with G- and F-Actin

Wen, Kuo‐Kuang; Giardina, Peter C.; Blake, M. S.; Edwards, Jennifer; Apicella, Michael A.; Rubenstein, Peter A.

doi:10.1021/bi000241j

Cited by 33 publications

(22 citation statements)

References 49 publications

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“…Uninfected, control cell monolayers were simultaneously processed with challenged cell monolayers. Infected and uninfected (control) cell monolayers were subsequently processed for LSCM, SEM or TEM as described previously , or the cells were harvested for immunoprecipitation (Wen et al, 2000) assays before fixation.…”

Section: Bacteria and Infection Studiesmentioning

confidence: 99%

The role of complement receptor 3 (CR3) inNeisseria gonorrhoeaeinfection of human cervical epithelia

Edwards¹,

Brown²,

Ault³

et al. 2001

Cellular Microbiology

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Section: Bacteria and Infection Studiesmentioning

confidence: 99%

The role of complement receptor 3 (CR3) inNeisseria gonorrhoeaeinfection of human cervical epithelia

Edwards¹,

Brown²,

Ault³

et al. 2001

Cellular Microbiology

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“…Protein Preparation-WT yeast actin and S265C yeast actin were prepared from frozen cells as described previously (32). Modification of the actins with N-(1-pyrenyl) maleimide (Sigma) on C374 of WT actin and C265C and C374 of S265C actin was carried out according to Feng et al (33).…”

Section: Methodsmentioning

confidence: 99%

Differential Regulation of Actin Polymerization and Structure by Yeast Formin Isoforms

Wen

Rubenstein

2009

Journal of Biological Chemistry

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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 isoformspecific. 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 pyrenelabeled wild type and S265C Mg-F actins enhanced the pyreneand 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 c...

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“…Supporting evidence lies in the fact that modification of the actin C-terminus by proteolysis, antibody binding and by mutagenesis can significantly impact actin polymerization properties [Johannes and Gallwitz, 1991;Crosbie et al, 1994;Orlova and Egelman, 1995;Phan et al 1997;Hegyi et al, 1998;Kim et al, 1998]. Attachment of fluorometric probes to different parts of the actin showed that polymerization per se and the binding of specific proteins to the actin filament at a distance from the probe site could affect probe behavior [Feng et al, 1997;Moens and dos Remedios, 1997;Wen et al, 2000;Bobkov et al, 2002Bobkov et al, , 2004Borovikov et al, 2004]. EM studies revealed that decoration of filaments by cofilin changed the filament twist [McGough et al, 1997;McGough and Chiu, 1999;Galkin et al, 2003Galkin et al, , 2011.…”

Section: Budding Yeast As An Expression System For Mutant Actinsmentioning

confidence: 99%

“…Rubenstein and Wen CYTOSKELETON specific proteins to the actin filament at a distance from the probe site could affect probe behavior [Feng et al, 1997;Moens and dos Remedios, 1997;Wen et al, 2000;Bobkov et al, 2002Bobkov et al, , 2004 Borovikov et al, 2004]. EM studies revealed that decoration of filaments by cofilin changed the filament twist [McGough et al, 1997;McGough and Chiu, 1999;Galkin et al, 2003Galkin et al, , 2011.…”

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confidence: 99%

Insights into the effects of disease‐causing mutations in human actins

Rubenstein

Wen

2014

Cytoskeleton

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Mutations in all six actins in humans have now been shown to cause diseases. However, a number of factors have made it difficult to gain insight into how the changes in actin functions brought about by these pathogenic mutations result in the disease phenotype. These include the presence of multiple actins in the same cell, limited accessibility to pure mutant material, and complexities associated with the structures and their component cells that manifest the diseases. To try to circumvent these difficulties, investigators have turned to the use of model systems. This review describes these various approaches, the initial results obtained using them, and the insight they have provided into allosteric mechanisms that govern actin function. Although results so far have not explained a particular disease phenotype at the molecular level, they have provided valuable insight into actin function at the mechanistic level which can be utilized in the future to delineate the molecular bases of these different actinopathies. Key Words: actin; allostery; disease; mutation; isoforms Introduction A ctin mutations can cause human disease. However, little is known concerning the mechanisms by which these mutations lead to the disease phenotypes they produce. The goal of this review is to describe the general properties of actin, the diseases associated with mutations in actin, methodologies that might be employed to achieve a mechanistic understanding of actin based disease and efforts that have been made toward this goal. Properties of G-and F-ActinA discussion of the effects of pathogenic mutations on actin function requires a basic understanding of actin structure and its solution properties. Actin is an abundant 42 kD monomeric protein, found in virtually all eukaryotic cells, that can cycle between either a monomeric (G-actin) or polymeric (F-actin) state. It plays both contractile and structural roles in the cytoplasm, and recent studies have documented that actin in the nucleus acts as a promoter of transcription or as a member of a number of chromatin remodeling complexes Philimonenko et al., 2004;Miyamoto and Gurdon, 2011; Miyamoto et al., 2011a,b;Weston et al., 2012].G-actin is a clam-shaped protein with two domains, the inner and outer domains ( Fig. 1), connected by a hinge region, and separated by a deep cleft. Outer (subdomains 1 and 2) and inner (subdomains 3 and 4) refer to their position in the actin filament. The N and C-termini reside on opposite faces of the protein in subdomain 1. An adenine nucleotide binds deep within the inter-domain cleft, held in place by a divalent cation, and imparts stability to the protein and the filament. This latter role depends on its ability to cycle between the ATP and ADP state by virtue of a polymerization-dependent ATPase activity [Carlier et al., 1987;Korn et al., 1987]. F-actin is a polar structure with pointed (2) and barbed (1) ends referring to the arrowhead pattern formed when myosin S1 binds to F-actin in the absence of ATP [Craig et al., 1985]. The polarity of th...

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Interaction of the Gonococcal Porin P.IB with G- and F-Actin

Cited by 33 publications

References 49 publications

The role of complement receptor 3 (CR3) inNeisseria gonorrhoeaeinfection of human cervical epithelia

The role of complement receptor 3 (CR3) inNeisseria gonorrhoeaeinfection of human cervical epithelia

Differential Regulation of Actin Polymerization and Structure by Yeast Formin Isoforms

Insights into the effects of disease‐causing mutations in human actins

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