Isolation of focal contact membrane using saponin

Neyfakh, Alexander A.; Svitkina, T.M.

doi:10.1016/0014-4827(83)90369-5

Cited by 31 publications

(22 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Several different procedures have been used to isolate material designated either as cellular contact sites, cell substratum-attached material, foot pads, or focal contact membranes (5,7,9,15,37,40,47). These procedures include use of chelating agents, low concentrations of nonionic or zwitterionic detergents, or mechanical extraction.…”

Section: Discussionmentioning

confidence: 99%

“…Saponin, which we used as a cholesterol-removing detergent, has also been used to isolate acethylcholine receptor clusters from cultured rat myotubes (8). In addition, using saponin and mechanical extraction Neyfakh et al (47) found Mr 51,000 and 47,000 proteins in isolated focal contact membranes of mouse and chick fibroblasts, respectively. Interestingly, Bayley and Rees (7) detected a major new protein component with an Mr 50,000-55,000 in fibroblast focal adhesion preparations that remains attached to a glass substratum after cells are mechanically removed.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells

Pöllänen¹,

Saksela²,

Salonen³

et al. 1987

The Journal of Cell Biology

296

127

View full text Add to dashboard Cite

Abstract. We studied the immunocytochemical localization of urokinase-type plasminogen activator (u-PA) and the type 1 plasminogen activator inhibitor (PAI-1) in human fibroblasts and sarcoma cells, using both polyclonal and monoclonal antibodies. The u-PA was found to be located at discrete cell-substratum contact sites, and also at areas of cell-cell contacts, whereas PAI-1 was distributed as a homogenous carpet excluding strialike areas on the substrate under the cells. To confirm the extracellular localization of u-PA and PAIl, we stained the cells live at 0°C before fixation. A double-labeling experiment showed different distribution of u-PA and PAI-1 under the cells, and especially their peripheral parts. The staining pattern of u-PA and PAI-1 resisted treatment with 0.2% saponin followed by mechanical removal of cells, a method previously reported to isolate focal contact membranes of fibroblasts. We further demonstrated the deposition of u-PA to the contact areas of cells obtained by saponin treatment by zymography, and that of PAI-1 by metabolic labeling, reverse zymography, immunoblotting, and immunoprecipitation. Fibronectin was also present in the preparations. The deposition of both PAI-1 and fibronectin by the sarcoma cells was enhanced, after treating the cells with 10 -6 M dexamethasone. The confinement of u-PA to discrete contact sites and the more uniform distribution of PAI-1 on the cell substratum may explain how cells producing large amounts of enzyme inhibitors can produce PAmediated focal proteolysis.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells

Pöllänen¹,

Saksela²,

Salonen³

et al. 1987

The Journal of Cell Biology

296

127

View full text Add to dashboard Cite

show abstract

“…Other proteins with molecular masses similar to p52 have been localized to the cell-substratum contact region. Neyfakh & Svitkina (1983) identified proteins of 51 and 47 kDa as enriched components of saponin-prepared focal contacts in chick and mouse fibroblasts respectively. Bayley & Rees (1982), by using stream-dislodgement of cultured rat fibroblasts to generate focal adhesions, resolved actin, a-actinin and vimentin, in addition to a protein of 50-55 kDa, in such structures but failed to find vinculin, a major cytoplasmic-face protein of focal contacts (Burridge, 1986).…”

Section: Discussionmentioning

confidence: 99%

“…Cell-substrate contact sites were also prepared in the present study using the saponinextraction procedure. Such extractions generate peptide patterns (Pollanen et al, 1987) and subcellular structures (Neyfakh & Svitkina, 1983) that are simpler than those encountered in SAM preparations and that probably represent predominantly focal-type contact regions (Neyfekh & Svitkina, 1983). Electrophoretic comparison of SAM-and SAP-fraction proteins indicated that SAP preparations of NRK cells contained augmented levels of p52 (relative to SAM-fraction p52 content) and that this enrichment appeared to be selective.…”

Section: Discussionmentioning

confidence: 99%

“…p52 could not be resolved in the corresponding fraction of KNRK cells, but was inducible in transformed fibroblasts by exposure to NaB, an agent which initiates cell spreading (Ryan & Higgins,1 988a) and substrate-contact site formation (Altenburg et al, 1976 Total cell extracts were prepared by scraping cells into lysis buffer 20 (v/v) Ampholytes, pH 7-9, 100 mM-dithiothreitol] (Bravo, 1984), followed by vortex agitation and clarification at 13 000 g. For preparation of the detergent-resistant cytoskeletal fraction, EDTA-released cells (0.2 g of EDTA/ 1 Mg2+ and Ca2+-free HBSS) (Rosen & Culp, 1977;Rheinwald et al:, 1987) were collected by centrifugation and extracted in TN/Triton buffer (140 mM-NaCl, 10 mMTris/HCl, pH 7.6, 1 % Triton X-100) (Franke et al, 198ia,b;Higgins, 1986) (Rosen & Culp, 1977;Lark et al, 1985) remaining after EDTA-release of adherent cells, was solubilized directly in either lysis buffer (for two-dimensional gel electrophoresis) or sample buffer (for separation on onedimensional gels). Cell-substratum contact regions were prepared with saponin [0.20 in Ca2"/Mg2+-free phosphate-buffered saline (PBS)] (Neyfakh & Svitkina, 1983) and the saponin residual material (SAP) solubilized in sample buffer. For electrophoresis on twodimensional gels (Bravo, 1984), 5 x 104-5 x I05 c.p.m.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Biochemical localization of the transformation-sensitive 52 kDa (p52) protein to the substratum contact regions of cultured rat fibroblasts. Butyrate induction, characterization, and quantification of p52 in v-ras transformed cells

Higgins

Ryan

1989

Biochemical Journal

View full text Add to dashboard Cite

A 52 kDa protein (p52) was identified, using differential extraction and electrophoretic criteria, as a major extracellular and substrate-associated component of normal rat kidney (NRK) fibroblasts. Cells transformed with Kirsten murine sarcoma virus (KNRK cells) did not express p52 constitutively, but were inducible for both p52 production -and its substrate association during culture in sodium butyrate (NaB)-supplemented growth medium. Comparative analysis of the relative molecular mass, subcellular distribution, and isoelectric complexity (five variants ranging in pl from 5.4 to 6.2) of the 52 kDa species constitutively and inducibly expressed by NRK and KNRK/NaB cells respectively, indicated that they were, indeed, the same protein. p52 selectively localized to cellular fractions enriched in substrate focal contact sites and associated ventral undersurface components. NaB induction of p52 in KNRK cells occurred before cell spreading; other polar compounds, such as dimethyl sulphoxide, which did not induce KNRK cell spreading, similarly failed to elicit p52 production. p52 accumulated more rapidly in (and was quickly released from) the focalcontact-enriched protein fraction of NRK cells compared with its time course of appearance in the medium. These data collectively suggest that p52 is one of a relatively small number of proteins the synthesis of which is either involved in determination of cell shape or regulated as a consequence of cell-shape changes.

show abstract

Complex protein composition of isolated focal adhesions: A two‐dimensional gel and database analysis

et al. 1994

View full text Add to dashboard Cite

Current ideas about the composition of the focal adhesion complexes in cultured cells are based mainly on indirect immunocytochemical data. We here report a two-dimensional (2-D) gel electrophoresis analysis of the focal adhesion associated-structures that remain in the growth substrate after removal of cells by mechanical shearing. Many proteins additional to the known adhesion proteins, and in higher abundance, could be identified. Using selective extraction procedures, employing detergent or gelsolin, these could be classified as either membrane-associated, actin-associated or both. Cross correlation of these polypeptode patterns with a 2-D gel database allowed identification of some proteins, not previously considered as resident of focal adhesions. The data point to a more complex make up of focal adhesions than formerly supposed.

show abstract

Isolation of focal contact membrane using saponin

Cited by 31 publications

References 16 publications

Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells

Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells

Biochemical localization of the transformation-sensitive 52 kDa (p52) protein to the substratum contact regions of cultured rat fibroblasts. Butyrate induction, characterization, and quantification of p52 in v-ras transformed cells

Complex protein composition of isolated focal adhesions: A two‐dimensional gel and database analysis

Contact Info

Product

Resources

About