Delayed dissociation of in vitro moving actin filaments from heavy meromyosin induced by low concentrations of Triton X-100

1997 ◽  
Vol 67 (1-3) ◽  
pp. 199-210 ◽  
Author(s):  
Miklós S.Z. Kellermayer
Keyword(s):  
Actin Filaments ◽  
Heavy Meromyosin ◽  
Low Concentrations ◽  
Triton X

scholarly journals Overexpression of human fibroblast caldesmon fragment containing actin-, Ca++/calmodulin-, and tropomyosin-binding domains stabilizes endogenous tropomyosin and microfilaments.

1994 ◽  
Vol 125 (2) ◽  
pp. 359-368 ◽  
Author(s):  
K S Warren ◽  
J L Lin ◽  
D D Wamboldt ◽  
J J Lin
Keyword(s):  
Cho Cells ◽  
Actin Filaments ◽  
Actin Binding ◽  
Expression Vectors ◽  
Binding Domains ◽  
Microfilament Bundles ◽  
The Stability ◽  
Triton X

Fibroblast caldesmon is a protein postulated to participate in the modulation of the actin cytoskeleton and the regulation of actin-based motility. The cDNAs encoding the NH2-terminal (aa.1-243, CaD40) and COOH-terminal (aa.244-538, CaD39) fragments of human caldesmon were subcloned into expression vectors and we previously reported that bacterially produced CaD39 protein retains its actin-binding properties as well as its ability to enhance low M(r) tropomyosin (TM) binding to actin and to inhibit TM-actin-activated HMM ATPase activity in vitro (Novy, R. E., J. R. Sellers, L.-F. Liu, and J. J.-C. Lin. 1993. Cell Motil. Cytoskeleton. 26:248-261). Bacterially produced CaD40 does not bind actin. To study the in vivo effects of CaD39 expression on the stability of actin filaments in CHO cells, we isolated and characterized stable CHO transfectants which express varying amounts of CaD39. We found that expression of CaD39 in CHO cells stabilized microfilament bundles as well as endogenous TM. CaD39-expressing clones displayed an increased resistance to cytochalasin B and Triton X-100 treatments and yielded increased amounts of TM-containing actin filaments in microfilament isolation procedures. In addition, analysis of these clones with immunoblotting and indirect immunofluorescence microscopy with anti-TM antibody revealed that stabilized endogenous TM and enhanced TM-containing microfilament bundles parallel increased amounts of CaD39 expression. The increased TM observed corresponded to a decrease in TM turnover rate and did not appear to be due to increased synthesis of endogenous TM. Additionally, the phenomenon of stabilized TM did not occur in stable CHO clones expressing CaD40. Therefore, it is likely that CaD39 can enhance TM's binding to F-actin in vivo, thus reducing TM's rate of turnover and stabilizing actin microfilament bundles.

scholarly journals 110OSMOTIC STRESS ON THE CELLULAR ACTIN FILAMENT ORGANIZATION OF IN VITRO PRODUCED PORCINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 177 ◽  
Author(s):  
H. Men ◽  
Y. Agca ◽  
S.F. Mullen ◽  
E.S. Critser ◽  
J.K. Critser
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Developmental Competence ◽  
Tween 20 ◽  
Embryonic Cells ◽  
Sucrose Solutions ◽  
The Status ◽  
Osmotic Treatment ◽  
Triton X

Disruption of the actin cytoskeleton is one of the leading causes in low survival of pig embryos after cryopreservation (Dobrinsky et al., 2000 Biol. Reprod. 62, 564–570). In this study, the effect of osmotic stress on cellular actin filament organization in porcine embryos produced in vitro was studied. Excellent quality Day 6 (fertilization=Day 0) porcine blastocysts were randomly exposed to 6 different anisosmotic sucrose solutions (75, 150, 210, 600, 1200, 2400mOsm) for 10min. Embryos were then returned to embryo culture medium (NCSU-23) after washing with NCSU-23, and cultured under 38.5°C, 5% CO2 in air with maximal humidity for them to recover. Blastocysts cultured in NCSU-23 medium (280mOsm) served as a control for embryos with intact actin filament organization. Blastocysts treated with 7.5μgmL−1 cytochalasin-b for 60min served as a control for embryos with F-actin depolymerization. Eighteen hours post-anisosmotic treatments, all blastoysts were fixed in 3.7% paraformaldehyde in PBS for 60min and stored in PBS with 0.1% Triton X-100 and 0.2% sodium azide at 4°C. Staining of actin filaments was performed according to procedures described earlier (Wang et al., 1999 Biol. Reprod. 60, 1020–1028). Embryos were blocked in PBS with 20mgmL−1 BSA and 150mM glycine for 30min. After being washed in PBS with 0.1% Tween 20 for 60min, embryos were stained with 10UmL−1 Alexa Fluor 488 phalloidin in PBS with 0.1% Tween 20 at 38.5°C for 60min, and then washed twice in PBS with 0.1% Tween 20 for 60min each. The status of actin filaments in embryonic cells was examined by confocal microscopy. Integrity of cellular actin filaments was classified as either intact or disrupted according to the distribution within embryonic cells. Blastocysts were then classified according to the status of actin filaments in embryonic cells. Data were analyzed using logistic regression. Results from 7 replicates are displayed in Table 1. There was a significant relationship between osmotic treatment levels and the probability of blastocysts with disrupted cellular actin filaments (P<0.0001). These data support the hypothesis that porcine embryos are very sensitive to osmotic changes. Ongoing experiments will assess the extent of actin disruption required to significantly reduce developmental competence of pig blastocysts. This study was supported by Monsanto Company. Table 1 Cellular actin filament integrity of in vitro produced porcine blastocysts after being treated with sucrose solutions with different osmolalities (mOsm)

scholarly journals Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos.

1983 ◽  
Vol 97 (4) ◽  
pp. 963-973 ◽  
Author(s):  
P C Letourneau
Keyword(s):  
Growth Cone ◽  
Actin Filaments ◽  
Electron Microscopic Observation ◽  
Neurite Growth ◽  
Growth Cones ◽  
Chick Embryos ◽  
Heavy Meromyosin ◽  
Electron Microscopic ◽  
Cytochemical Localization ◽  
Triton X

Sensory neurons from chick embryos were cultured on substrata that support neurite growth, and were fixed and prepared for both cytochemical localization of actin and electron microscopic observation of actin filaments in whole-mounted specimens. Samples of cells were treated with the detergent Triton X-100 before, during, or after fixation with glutaraldehyde to determine the organization of actin in simpler preparations of extracted cytoskeletons. Antibodies to actin and a fluorescent derivative of phallacidin bound strongly to the leading margins of growth cones, but in neurites the binding of these markers for actin was very weak. This was true in all cases of Triton X-100 treatment, even when cells were extracted for 4 min before fixation. In whole-mounted cytoskeletons there were bundles and networks of 6-7-nm filaments in leading edges of growth cones but very few 6-7-n filaments were present among the microtubules and neurofilaments in the cytoskeletons of neurites. These filaments, which are prominent in growth cones, were identified as actin because they were stabilized against detergent extraction by the presence of phallacidin or the heavy meromyosin and S1 fragments of myosin. In addition, heavy meromyosin and S1 decorated these filaments as expected for binding to F-actin. Microtubules extended into growth cone margins and terminated within the network of actin filaments and bundles. Interactions between microtubule ends and these actin filaments may account for the frequently observed alignment of microtubules with filopodia at the growth cone margins.

scholarly journals Acceleration of the sliding movement of actin filaments with the use of a non-motile mutant myosin in in vitro motility assays driven by skeletal muscle heavy meromyosin

PLoS ONE ◽  
2017 ◽  
Vol 12 (7) ◽  
pp. e0181171 ◽  
Author(s):  
Kohei Iwase ◽  
Masateru Tanaka ◽  
Keiko Hirose ◽  
Taro Q. P. Uyeda ◽  
Hajime Honda
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Heavy Meromyosin ◽  
In Vitro Motility

Hydrolysis of membrane-bound liver alkaline phosphatase by GPI-PLD requires bile salts

1996 ◽  
Vol 271 (4) ◽  
pp. G655-G663 ◽  
Author(s):  
J. T. Deng ◽  
M. F. Hoylaerts ◽  
M. E. De Broe ◽  
V. O. van Hoof
Keyword(s):  
Alkaline Phosphatase ◽  
Bile Salt ◽  
Bile Salts ◽  
Small Range ◽  
Liver Sinusoid ◽  
Gpi Anchor ◽  
Low Concentrations ◽  
Membrane Bound ◽  
Triton X

Circulating liver plasma membrane fragments (LPMF) were purified from human serum by means of a monoclonal antileucine aminopeptidase antibody, AD-1. This was done by immunoaffinity chromatography or by incubating the sera with AD-1-coated nitrocellulose disks. Alkaline phosphatase (ALP, EC 3.1.3.1) is bound to these LPMF through a glycosylphosphatidylinositol (GPI) anchor and is referred to as membrane-bound liver ALP (Mem-LiALP). Low concentrations of Triton X-100 or high bile salt concentrations released GPI anchor-bearing LiALP (Anch-LiALP) from purified LPMF; once released, Anch-LiALP was slowly and progressively converted to hydrophilic dimeric LiALP [soluble LiALP (Sol-LiALP)], free from its GPI anchor. Low levels of GPI-specific phospholipase D (GPI-PLD) activity were measured in the pure LPMF. Apparently, this membrane-associated GPI-PLD was released by the action of detergents and contributed to the spontaneous conversion of Anch-LiALP to Sol-LiALP. In the absence of detergents, GPI-PLD had little effect on Mem-LiALP, both in purified form as well as in serum. In vitro, isolated Anch-LiALP was converted to Sol-LiALP by both GPI-specific phospholipase C and GPI-PLD. Sol-LiALP in serum, however, appeared to be the product of GPI-PLD activity only. Five- to tenfold higher concentrations of Triton X-100 were needed to release Anch-LiALP from LPMF in serum, compared with those required in a solution of purified LPMF. In serum, as well as in purified conditions, only a small range of detergent of bile salt concentrations permitted the conversion of Mem-LiALP to Sol-LiALP. A model is proposed for the release in the circulation of Mem-LiALP, Anch-LiALP, and Sol-LiALP, involving both LPMF-associated GPI-PLD and liver sinusoid bile salts.

Association of the gamma12 subunit of G proteins with actin filaments

1997 ◽  
Vol 110 (13) ◽  
pp. 1503-1511
Author(s):  
H. Ueda ◽  
S. Saga ◽  
H. Shinohara ◽  
R. Morishita ◽  
K. Kato ◽  
...  
Keyword(s):  
G Proteins ◽  
Actin Filaments ◽  
Calf Serum ◽  
C6 Glioma Cells ◽  
Heterotrimeric G Proteins ◽  
Differential Distribution ◽  
Functional Differences ◽  
Gamma Subunits ◽  
Triton X

Recent studies have suggested an association between heterotrimeric G proteins, which play a major role in transmembrane signal transduction, and intracellular components. We therefore examined the subcellular localization of isoforms of G protein gamma subunits in Swiss 3T3 and C6 glioma cells, mainly containing the gamma5 and gamma12 subunits. Immunocytochemical double staining with phalloidin showed co-localization of the gamma12 subunit with actin filaments (F-actin), while the gamma5 co-localized with vinculin, suggesting an association with focal adhesion. Pretreatment of cells with Triton X-100 eliminated the gamma5 but not the gamma12 staining. Co-localization of gamma12 and F-actin was preserved when F-actin was disorganized with cytochalasin D or reorganized using fetal calf serum. Large amounts of gamma12 were recovered in the vimentin- and tubulin-free F-actin-rich fraction prepared from crude cytoskeleton preparations by double depolymerization-repolymerization. Co-localization of Gi2alpha, beta and gamma12 in the F-actin-rich fraction suggested the existence of gamma12 as a betagamma or heterotrimeric complex. Furthermore, purified betagamma12 was found to associate with F-actin in vitro more tightly than betagamma5. These results strongly suggest that the gamma12 subunit associates with F-actin in cells. The observed differential distribution of gamma12 and gamma5 implies functional differences for the two gamma subunits.

scholarly journals Subtilisin cleavage of actin inhibits in vitro sliding movement of actin filaments over myosin.

1990 ◽  
Vol 111 (2) ◽  
pp. 465-470 ◽  
Author(s):  
D H Schwyter ◽  
S J Kron ◽  
Y Y Toyoshima ◽  
J A Spudich ◽  
E Reisler
Keyword(s):  
Atpase Activity ◽  
Linear Dependence ◽  
Actin Filaments ◽  
Structural Integrity ◽  
Heavy Meromyosin ◽  
Dynamic Changes ◽  
In Vitro Motility Assay ◽  
In Vitro Motility ◽  
Coated Surface

Subtilisin cleaved actin was shown to retain several properties of intact actin including the binding of heavy meromyosin (HMM), the dissociation from HMM by ATP, and the activation of HMM ATPase activity. Similar Vmax but different Km values were obtained for acto-HMM ATPase with the cleaved and intact actins. The ATPase activity of HMM stimulated by copolymers of intact and cleaved actin showed a linear dependence on the fraction of intact actin in the copolymer. The most important difference between the intact and cleaved actin was observed in an in vitro motility assay for actin sliding movement over an HMM coated surface. Only 30% of the cleaved actin filaments appeared mobile in this assay and moreover, the velocity of the mobile filaments was approximately 30% that of intact actin filaments. These results suggest that the motility of actin filaments can be uncoupled from the activation of myosin ATPase activity and is dependent on the structural integrity of actin and perhaps, dynamic changes in the actin molecule.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

1989 ◽  
Vol 61 (02) ◽  
pp. 254-258 ◽  
Author(s):  
Margaret L Rand ◽  
Peter L Gross ◽  
Donna M Jakowec ◽  
Marian A Packham ◽  
J Fraser Mustard
Keyword(s):  
Cyclic Amp ◽  
Inhibitory Effect ◽  
Rabbit Platelet ◽  
Inhibitory Effects ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
In Vitro Effects ◽  
Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

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