scholarly journals Role of gelsolin in actin depolymerization of adherent human neutrophils.

1997 ◽  
Vol 8 (1) ◽  
pp. 121-128 ◽  
Author(s):  
J S Wang ◽  
J P Coburn ◽  
A I Tauber ◽  
K S Zaner
Keyword(s):  
Soluble Fraction ◽  
Signal Transduction Pathway ◽  
Insoluble Fraction ◽  
Actin Binding ◽  
Human Neutrophils ◽  
Nonreceptor Tyrosine Kinase ◽  
Actin Depolymerization ◽  
Herbimycin A ◽  
Triton X

Human neutrophils generally function adherent to an extracellular matrix. We have previously reported that upon adhesion to laminin- or fibronectin-coated, but not uncoated, plastic there is a depolymerization of actin in neutrophils. This phenomenon was not affected by inhibitors of the more well-studied components of the signal transduction pathway, specifically, pertussis toxin, an inhibitor of G-proteins, H-7 or staurosporine, inhibitors of protein kinase C, or herbimycin A, an inhibitor of nonreceptor tyrosine kinase. We therefore focused our attention on actin-binding proteins and measured the changes in the partitioning of gelsolin between the Triton X-100-soluble and -insoluble cellular fractions which occur upon neutrophil adhesion by means of quantitating anti-gelsolin antibody binding to aliquots of these fractions. It was found that approximately 90% of the total cellular gelsolin was found in the Triton X-100-soluble fraction in suspended cells, but that upon adherence to either fibronectin- or laminin-coated plastic about 40% of the soluble gelsolin could be detected in the insoluble fraction. This effect was not observed in cells adherent to uncoated plastic, wherein more than 90% of the gelsolin was found in the soluble fraction. Results of immunofluorescence microscopy of these cell preparations was consistent with this data. A gelsolin translocation to the insoluble cellular actin network may account for a part of the observed actin depolymerization.

scholarly journals In vitro fibrillogenesis of collagen II from pig vitreous humour

1995 ◽  
Vol 306 (3) ◽  
pp. 871-875 ◽  
Author(s):  
C Yang ◽  
H Notbohm ◽  
Y Açil ◽  
R Heifeng ◽  
S Bierbaum ◽  
...  
Keyword(s):  
Cross Sections ◽  
Soluble Fraction ◽  
Unit Length ◽  
Insoluble Fraction ◽  
Vitreous Humour ◽  
Collagen Types ◽  
Collagen Molecules ◽  
Collagen Ii

Collagen from pig vitreous humour was fractionated into a soluble and an insoluble fraction by centrifugation. Most of the collagen II in the soluble fraction was present as pN-collagen II (procollagen II without the C-terminal propeptide), besides smaller quantities of procollagen II, collagen II and two as yet unidentified alpha-chains of collagen II. Other collagen types may be present only in trace amounts. Collagen II of the insoluble fraction, which is mostly deposited in fibrillar aggregates, consists of both pN-collagen II and collagen II. To determine the possible role of collagen II precursors in the formation of the extracellular matrix of the vitreous humour these collagen molecules were purified and in vitro fibrillogenesis was used to demonstrate that pN-collagen II could form fibrils in mixtures with collagen II. These fibrils have a reduced mass per unit length depending on the content of pN-collagen in the mixture. Cross-sections of the newly formed fibrillar aggregates revealed a flattened shape. The incomplete processing of the precursors of collagen II may be part of regulatory mechanisms possibly controlling the formation of a translucent scaffold as is required in the vitreous humour.

INTERACTION OF MEMBRANE GLYCOPROTEIN GPIIb AND Ilia WITH CYTOSKELETAL PROTEINS DURING PLATELET ACTIVATION

1987 ◽  
Author(s):  
K Fujimura ◽  
T Fujimoto ◽  
M Takemoto ◽  
K Oda ◽  
S Maehama ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Soluble Fraction ◽  
Insoluble Fraction ◽  
Cytoskeletal Proteins ◽  
Molecular Weight Protein ◽  
High Molecular Weight Protein ◽  
Weight Protein ◽  
Triton X ◽  
Molecular Weight Protein Fraction

Experiments were designed and performed to analyse the cytoskeleton assembly and the interaction of glycoprotein (GP)IIb, IIIa and cytoskeletal proteins during platelet activation. A23187 stimulated 125I labeled platelets were solubilised with Triton X-100 solution and centrifuged. The insoluble fraction were analysed by two dimensional electrophoresis and the soluble fraction were fractionated with 5-25% sucrose gradient centrifugation and analysed by SDS PAGE. In Triton X-100 insoluble fraction, high molecular weight protein fraction(MW > 106) was present after stimulation which were consisted of actin binding protein(ABP), myosin heavy chain(MHC), actin and GPIIb and IIIa. And some of the ABP and MHC formed dimer. ABP and actin in this fraction were decreased with 1 mM CaCl2 treatment but the reduction of ABP was inhibited by leupeptm. In Triton X-100 soluble fraction after stimulation, some of the ABP, MHC, P235 protein, actin and small amount of GPIIb, IIIa were sedimented in the same high density fraction but most proteins were sedimented as a monomer form or GPIIb-IIIa complex form. The GPIIb, IIIa incorporation in high molecular weight protein fraction or high density fraction was absent in Ca++ chelating condition or the presence of competitive fibrinogen binding inhibitor which blocked the platelet aggregation. It is concluded that cytoskeletal proteins and GPIIb, IIIa are assembled each other and formed high molecular weight protein fraction or dimer formation during activation. In stimulated platelets these assembled cytoskeletal proteins containing GPIIb, IIIa were also found in Triton X-100 soluble fraction as a precursor of high molecular weight fraction in Triton X-100 insoluble fraction. The binding of fibrinogen to GPIIb-IIIa complex induce the linkage of GPIIb, IIIa to assembled cytoskeletal proteins.

Thrombopoietin stimulates cortactin translocation to the cytoskeleton independently of tyrosine phosphorylation

2001 ◽  
Vol 356 (3) ◽  
pp. 875-881 ◽  
Author(s):  
Isabelle LOPEZ ◽  
Véronique DUPREZ ◽  
Josiane MELLE ◽  
François DREYFUS ◽  
Sylviane LÉVY-TOLÉDANO ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Shape Change ◽  
Insoluble Fraction ◽  
Actin Binding ◽  
Syk Kinase ◽  
Tyrosine Phosphorylated Protein ◽  
Triton X ◽  
Cortactin Tyrosine Phosphorylation ◽  
Platelet Shape

Cortactin is an F-actin-binding protein expressed in platelets. During aggregation by thrombin, cortactin associates with Src, is tyrosine phosphorylated, and then translocates to the cytoskeleton. It is also found to associate with Syk during platelet shape change. Since cortactin undergoes tyrosine phosphorylation in platelets activated by thrombopoietin (TPO) that exhibit neither shape change nor aggregation, we investigated whether it could also relocalize to the detergent-insoluble fraction. We demonstrate that cortactin was present as a tyrosine-phosphorylated protein and co-localized with Syk in the Triton X-100-insoluble fraction of TPO-activated platelets. TPO stimulated Syk activation and association with cortactin. Conversely, cortactin associated with the kinases, Syk and Src. Cortactin tyrosine phosphorylation was blocked by Syk kinase inhibitor, piceatannol or Src family kinase inhibitor, PP2, suggesting that it depends on these two kinases. However, piceatannol or PP2 did not prevent cortactin translocation to the detergent-insoluble fraction. These data suggest that tyrosine phosphorylation is not required for cortactin translocation to the detergent-insoluble compartment. Furthermore, TPO activates, through its receptor c-Mpl, a signalling pathway to the cytoskeleton.

Increased transglutaminase in the aortas of cholesterol-fed rabbits: occurrence of buffer soluble and insoluble forms and an inhibitor

1991 ◽  
Vol 69 (12) ◽  
pp. 821-827 ◽  
Author(s):  
Rick I. Wiebe ◽  
Alan H. Tarr ◽  
J. Michael Bowness
Keyword(s):  
Soluble Fraction ◽  
Tissue Transglutaminase ◽  
Insoluble Fraction ◽  
Peanut Oil ◽  
Insoluble Residue ◽  
Transglutaminase Activity ◽  
Atherosclerotic Lesions ◽  
Triton X ◽  
Two Peaks

Rabbits were fed for 10–12 weeks on a normal pellet diet or on the same diet containing 1% cholesterol and 6% peanut oil. The animals were killed and the aortas divided into three layers which were homogenized and extracted. The extracts and the insoluble residues were assayed for transglutaminase activity and tissue transglutaminase antigen. When compared with normal aortas, the inner and middle layers of aortas with atherosclerotic lesions from cholesterol-fed rabbits showed higher transglutaminase activities in the buffer-soluble fraction without a corresponding increase in antigen. The buffer extracts showed two peaks (I and II) of activity and antigen on DE 52 chromatography; peak I was also found, together with lipid, in Triton X-100 extracts of the buffer-insoluble residue. The Triton X-100 insoluble fraction showed higher concentrations of both activity and antigen in the inner and middle layers of atherosclerotic aortas than in normal aortas, but the activity per nanogram of antigen was lower than in the buffer-soluble fraction. The activity in this insoluble residue was largely extracted, together with an inhibitor, by an NaCl – sucrose – dithiothreitol – Triton X-100 solution. DE 52 chromatography of this extract showed a third peak of activity and antigen (peak III) and an inhibitor peak that was distinct from the activity peaks.Key words: aorta, transglutaminase, inhibitor, cholesterol, atherosclerosis.

scholarly journals EFA6, Exchange Factor for ARF6, Regulates the Actin Cytoskeleton and Associated Tight Junction in Response to E-Cadherin Engagement

2004 ◽  
Vol 15 (3) ◽  
pp. 1134-1145 ◽  
Author(s):  
Frédéric Luton ◽  
Stéphanie Klein ◽  
Jean-Paul Chauvin ◽  
André Le Bivic ◽  
Sylvain Bourgoin ◽  
...  
Keyword(s):  
Tight Junction ◽  
Insoluble Fraction ◽  
Actin Remodeling ◽  
Exchange Factor ◽  
Selective Retention ◽  
The Stability ◽  
Triton X ◽  
Darby Canine Kidney ◽  
E Cadherin

We addressed the role of EFA6, exchange factor for ARF6, during the development of epithelial cell polarity in Madin-Darby canine kidney cells. EFA6 is located primarily at the apical pole of polarized cells, including the plasma membrane. After calcium-triggered E-cadherin–mediated cell adhesion, EFA6 is recruited to a Triton X-100–insoluble fraction and its protein level is increased concomitantly to the accelerated formation of a functional tight junction (TJ). The expression of EFA6 results in the selective retention at the cell surface of the TJ protein occludin. This effect is due to EFA6 capacities to promote selectively the stability of the apical actin ring onto which the TJ is anchored, resulting in the exclusion of TJ proteins from endocytosis. Finally, our data suggest that EFA6 effects are achieved by the coordinate action of both its exchange activity and its actin remodeling C-terminal domain. We conclude that EFA6 is a signaling molecule that responds to E-cadherin engagement and is involved in TJ formation and stability.

scholarly journals Phototactic Migration ofDictyosteliumCells Is Linked to a New Type of Gelsolin-related Protein

1999 ◽  
Vol 10 (1) ◽  
pp. 161-178 ◽  
Author(s):  
Susanne Stocker ◽  
Mary Hiery ◽  
Gerard Marriott
Keyword(s):  
Functional Characterization ◽  
Coiled Coil ◽  
Insoluble Fraction ◽  
Actin Binding ◽  
Related Protein ◽  
Sequence Elements ◽  
Extractable Protein ◽  
New Type ◽  
Triton X ◽  
Related Proteins

The molecular and functional characterization of a 125-kDa Ca2+-extractable protein of the Triton X-100–insoluble fraction of Dictyostelium cells identified a new type of a gelsolin-related molecule. In addition to its five gelsolin segments, this gelsolin-related protein of 125 kDa (GRP125) reveals a number of unique domains, two of which are predicted to form coiled-coil regions. Another distinct attribute of GRP125 concerns the lack of sequence elements known to be essential for characteristic activities of gelsolin-like proteins, i.e. the severing, capping, or nucleation of actin filaments. The subcellular distribution of GRP125 to vesicular compartments suggests an activity of GRP125 different from actin-binding, gelsolin-related proteins. GRP125 expression is tightly regulated and peaks at the transition to the multicellular pseudoplasmodial stage of Dictyostelium development. GRP125 was found indispensable for slug phototaxis, because slugs fail to correctly readjust their orientation in the absence of GRP125. Analysis of the GRP125-deficient mutant showed that GRP125 is required for coupling photodetection to the locomotory machinery of slugs. We propose that GRP125 is essential in the natural environment for the propagation of Dictyostelium spores. We also present evidence for further representatives of the GRP125 type inDictyostelium, as well as in heterologous cells from lower to higher eukaryotes.

scholarly journals Vimentin Filaments in Fibroblasts Are a Reservoir for SNAP23, a Component of the Membrane Fusion Machinery

2000 ◽  
Vol 11 (10) ◽  
pp. 3485-3494 ◽  
Author(s):  
Wolfgang Faigle ◽  
Emma Colucci-Guyon ◽  
Daniel Louvard ◽  
Sebastian Amigorena ◽  
Thierry Galli
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Human Fibroblasts ◽  
Insoluble Fraction ◽  
Protein Receptors ◽  
Syntaxin 4 ◽  
Triton X ◽  
Protein Attachment ◽  
Vimentin Filaments

Soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors (SNAREs) are core machinery for membrane fusion during intracellular vesicular transport. Synaptosome-associated protein of 23 kDa (SNAP23) is a target SNARE previously identified at the plasma membrane, where it is involved in exocytotic membrane fusion. Here we show that SNAP23 associates with vimentin filaments in a Triton X-100 insoluble fraction in fibroblasts in primary culture and HeLa cells. Upon treatment of human fibroblasts withN-ethyl-maleimide, SNAP23 dissociates from vimentin filaments and forms a protein complex with syntaxin 4, a plasma membrane SNARE. The vimentin-associated pool of SNAP23 can therefore be a reservoir, which would supply the plasma membrane fusion machinery, in fibroblasts. Our observation points to a yet unexplored role of intermediate filaments.

Binding of Human Platelet Glycoprotein lb and Actin to Fragments of Actin-Binding Protein

1992 ◽  
Vol 67 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Anne M Aakhus ◽  
J Michael Wilkinson ◽  
Nils Olav Solum
Keyword(s):  
Actin Filaments ◽  
High Speed ◽  
Binding Protein ◽  
Protein A ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Actin Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Calpain Inhibitors

SummaryActin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein lb (GP lb) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP lb peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP lb. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP lb was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 × g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

The Signaling Pathways in Nitric Oxide Production by Neutrophils Exposed to N-nitrosodimethylamine

2018 ◽  
Vol 16 (2) ◽  
pp. 194-199
Author(s):  
Wioletta Ratajczak-Wrona ◽  
Ewa Jablonska
Keyword(s):  
Nitric Oxide ◽  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
Polymorphonuclear Neutrophils ◽  
Microbial Pathogens ◽  
Human Neutrophils ◽  
No Production ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Background: Polymorphonuclear neutrophils (PMNs) play a crucial role in the innate immune system’s response to microbial pathogens through the release of reactive nitrogen species, including Nitric Oxide (NO). </P><P> Methods: In neutrophils, NO is produced by the inducible Nitric Oxide Synthase (iNOS), which is regulated by various signaling pathways and transcription factors. N-nitrosodimethylamine (NDMA), a potential human carcinogen, affects immune cells. NDMA plays a major part in the growing incidence of cancers. Thanks to the increasing knowledge on the toxicological role of NDMA, the environmental factors that condition the exposure to this compound, especially its precursors- nitrates arouse wide concern. Results: In this article, we present a detailed summary of the molecular mechanisms of NDMA’s effect on the iNOS-dependent NO production in human neutrophils. Conclusion: This research contributes to a more complete understanding of the mechanisms that explain the changes that occur during nonspecific cellular responses to NDMA toxicity.

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