[1] Preparation and assay of cytoplasmic and secreted gelsolin

Hl, Yin

doi:10.1016/0076-6879(86)34069-2

Cited by 3 publications

(1 citation statement)

References 16 publications

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“…EGTA and ethylenediamine-tetraacetic acid (EDTA) are added in buffers to inhibit the activation of gelsolin by Ca+ + (30)(31)(32)(33). Protease inhibitors such as trypsin inhibitor and leupeptin are also added in buffers to inhibit the degradation of gelsolin (2,34,35). Of the several inhibitors studied to determine whether they could inhibit the degradation of gelsolin, only leupeptin has been found effective (36).…”

Section: Discussionmentioning

confidence: 99%

Monoclonal Antibody to Human Plasma Gelsolin

Hiyoshi

Terano

Tatsumi

et al. 1992

JJMSB

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SUMMARY: Human plasma gelsolin was purified by column chromatography. The method yielded a protein of high purity and activity. Using this protein, we produced monoclonal antibody (Mab H6B11) against human plasma gelsolin by somatic cell fusion. This monoclonal antibody reacted in a dose-dependent manner with gelsolin derived from human plasma and platelets and neutralized depolymerizing activity to F-actin. It differed from the commercially available substance (Mab G4896; Sigma) in that the time required for the reaction between the antigen and antibody in the enzyme-linked immunosorbent assay could be shortened by one-third.The antibody was judged to be useful in assays for elucidating the physiological role of plasma gelsolin.

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Section: Discussionmentioning

confidence: 99%

Monoclonal Antibody to Human Plasma Gelsolin

Hiyoshi

Terano

Tatsumi

et al. 1992

JJMSB

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Direct observation of actin filament severing by gelsolin and binding by gCap39 and CapZ.

Bearer

1991

The Journal of Cell Biology

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Abstract. Dynamic behavior of actin filaments in cells is the basis of many different cellular activities . Remodeling of the actin filament network involves polymerization and depolymerization of the filaments . Proteins that regulate these behaviors include proteins that sever and/or cap actin filaments . This report presents direct observation of severing of fluorescency-labeled actin filaments . Coverslips coated with gelsolin, a multi-domain, calcium-dependent capping and severing protein, bound rhodamine-phalloidin-saturated filaments along their length in the presence of EGTA. Upon addition of calcium, attached filaments bent as they broke. Actophorin, a low molecular weight, monomer sequestering, calcium-independent severing protein did not sever phalloidin-saturated filaments .N intact and dynamic actin-based cytoskeleton is necessary for a wide range ofcellular functions. However, I A. although a number of proteins that sever actin filaments have been isolated and their in vitro activity described, the precise molecular mechanisms ofhow these proteins depolymerize actin filaments is as yet unknown . The traditional methods by which the severing of actin filaments have been studied rely upon indirect means, such as measuring the viscosity ofa solution containing actin filaments (Yin and Stossel, 1979), or measuring the rate of decrease in fluorescence of pyrene-actin diluted below the critical concentration (Walsh et al., 1984). These methods do not permit direct observation of the effect of any protein upon an individual actin filament . In addition, in these assays the activity of any one protein could be obscured by the activity of some other protein with an opposite effect on the filaments when a mixture of proteins is used. To overcome this obstacle, I decided to develop an in vitro optical assay in which I could directly observe the effects of purified proteins and mixtures of proteins upon individual actin filaments. Actin filaments are too small to be visualized directly by light microscopy. However, direct observations of their dynamic behavior in the presence of various binding proteins have recently become possible through the use of fluorescent labeling (Yanagida, et al., 1984 ;Kron and Spudich, 1986) . Actin filaments saturated with rhodamine-labeled phalloidin have been used to observe the interactions between myosin

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HIV Type 1 Gag and Nucleocapsid Proteins: Cytoskeletal Localization and Effects on Cell Motility

Ibarrondo

Choi²,

Geng³

et al. 2001

AIDS Research and Human Retroviruses

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Cell motility is likely to play a pivotal role in HIV infection by promoting the dissemination of infected cells. On the basis of observations indicating an interaction between HIV-1 Gag and target cell filamentous actin, we hypothesized that these interactions would promote cell motility of HIV-infected cells. Indeed, we have found that HIV-1 infection enhances the chemotactic response of macrophages. To specifically investigate the significance of the interactions between Gag and cellular actin, we transfected NIH 3T3 fibroblasts and HeLa cells with a construct that permits the expression of HIV-1 Gag in the absence of any other viral protein. Fractionation experiments showed that Gag was present in cytoskeletal fraction containing long actin filaments and in a high-speed postcytoskeletal fraction with short actin filaments. We have also localized HIV-1 Gag to the lamellipodia of chemoattractant-stimulated cells. Significantly, the motility of Gag-expressing cells was enhanced in chemotaxis assays. In vitro mutagenesis experiments showed that HIV-1 Gag binds filamentous actin through the nucleocapsid domain (NC). An NC-green fluorescent protein fusion had the same cellular distribution as the complete protein, and its expression increased cell motility. These data suggest that interactions between HIV-1 Gag and actin in infected cells enhance cell motility. Ultimately this enhanced motility of infected cells could promote the dissemination of virus into the brain and other tissues.

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[1] Preparation and assay of cytoplasmic and secreted gelsolin

Cited by 3 publications

References 16 publications

Monoclonal Antibody to Human Plasma Gelsolin

Monoclonal Antibody to Human Plasma Gelsolin

Direct observation of actin filament severing by gelsolin and binding by gCap39 and CapZ.

HIV Type 1 Gag and Nucleocapsid Proteins: Cytoskeletal Localization and Effects on Cell Motility

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