Visualization of cellular focal contacts using a monoclonal antibody to 80 kD serum protein adsorbed on the substratum

Neyfakh, Alexander A.; Tint, I.S.; Svitkina, T.M.; Bershadsky, Alexander D.; Gelfand, Vladimir I.

doi:10.1016/0014-4827(83)90351-8

Cited by 39 publications

(14 citation statements)

References 19 publications

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“…By this technique the smallest recognizable focal contacts fall in the size range of 0.25 by 1-2 ~tm (Izzard and Lochner, 1980). Vinculin foci in the leading edge can be much smaller than this and from the results of the antibody exclusion method of Neyfakh and coworkers (Neyfakh et al, 1983) these very small sites appear to be close enough to the substrate to be regarded as focal contacts of a primordial type.…”

Section: Discussionmentioning

confidence: 93%

Contact formation during fibroblast locomotion: involvement of membrane ruffles and microtubules.

Rinnerthaler

Geiger

Small

1988

The Journal of Cell Biology

164

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Abstract. We have correlated the motility of the leading edge of fibroblasts, monitored by phase-contrast cinematography, with the relative distributions of several cytoskeletal elements (vinculin, tubulin, and actin) as well as with the contact patterns determined by interference reflection microscopy. This analysis has revealed the involvement of both ruffles and microspikes, as well as microtubules in the initiation of focal contact formation. Nascent vinculin sites within the leading edge or at its base, taken as primordial cell-substrate contacts, were invariably colocalized with sites that showed a history of transient, prolonged, or cyclic ruffling activity. Extended microspike structures, often preceded the formation of ruffles. Immunofluorescent labeling indicated that some of these primordial contacts were in close apposition to the ends of microtubules that penetrated into the leading edge. By fluorescence and electron microscopy short bundles of actin filaments found at the base of the leading edge were identified as presumptive, primordial contacts. It is concluded that ruffles and microspikes, either independently or in combination, initiate and mark the sites for future contact. Plaque proteins then accumulate (within 10-30 s) at the contact site and, beneath ruffles, induce localized bundling of actin filaments. We propose that all primordial contacts support traction for leading edge protrusion but that only some persist long enough to nucleate stress fiber assembly. Microtubules are postulated as the elements that select, stabilize, and potentiate the formation of these latter, long-lived contacts.

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Section: Discussionmentioning

confidence: 93%

Contact formation during fibroblast locomotion: involvement of membrane ruffles and microtubules.

Rinnerthaler

Geiger

Small

1988

The Journal of Cell Biology

164

View full text Add to dashboard Cite

show abstract

“…Interference reflection microscopy (IRM) was performed basically as described (Izzard and Lochner, 1976) and was used to identify focal adhesions (FAs) in the same field as the two color fluorescence. FAs were also localized by the antibody exclusion technique (Neyfakh et al, 1983;Carter et al, 1990a).…”

Section: Immunofluorescence Interference Reflection and Antibody-exmentioning

confidence: 99%

Human keratinocytes express a new CD44 core protein (CD44E) as a heparan-sulfate intrinsic membrane proteoglycan with additional exons.

Brown

Bouchard

John

et al. 1991

The Journal of Cell Biology

352

182

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Abstract. We previously identified a 90-kD (GP90), collagen-binding, membrane glycoprotein, termed extracellular matrix receptor 1II (ECMR HI), that is homologous to the lymphocyte homing receptor and CD44 antigen (Gallatin, W. M., E. A. Wayner, P. A. Hoffman, T. St. John, E. C. Butcher, and W. G. Carter. 1989. Proc. Natl. Acad. Sci. USA. 86:4654-4658). CD44 is abundantly expressed in many epithelial tissues, and is localized predominantly to filopodia in cultured keratinocytes. Here we establish CD44 as a polymorphic family of related membrane proteoglycans and glycoproteins possessing extensive diversity in both glycosylation and core protein sequence. Human neonatal foreskin keratinocytes (HFKs) and QG56 lung squamous carcinoma cells express an alternatively spliced form of the CD44 core protein (termed CD44E) that contains an additional 132 amino acids in the carbohydrate attachment region of the extracellular domain. HFKs, HTI080 fibrosarcoma and QG56 cells, as well as many other human cells, contain varying ratios of GP90 and structurally related, higher molecular mass forms of CD44 that express the following characteristics: (a) each form reacted with anti-CD44 (mAbs) P1G12, P3H9, and P3H5. Each of these mAbs recognized a distinct, nonovedapping epitope present on each CD44 form. (b) Differences in mass were due primarily to variation in carbohydrate moieties, including sulfated aspargine-linked glycopeptides (GP), chondroitin sulfate (CS), and heparan sulfate (HS) glycosaminoglycans, as well as O-linked mucin and polylactosamine structure(s). The major polymorphic forms were designated HT1080 GP90 and CS180, QG56 GP230, and HFK HS/CS250, based on dominant carbohydrate moieties and relative mass. (c) The polymorphic forms use CD44 and CD44E core proteins, each containing a unique set of potential attachment sites for O-and N-glycosides and glycosaminoglycans. (d) Immunofluorescence microscopy, differential extraction with Triton-X-114 detergent, and incorporation into liposomes indicated that all the forms were membrane bound glycoconjugates. These results define CD44 as a structurally diverse, but immunologically related, set of intrinsic membrane macromolecules, and suggests that these structurally varied forms might be expected to manifest multiple functions.

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“…Initial attachment and spreading patterns have been studied, and various techniques have been introduced to quantify the cell adhesion strength [3][4][5][6][7]. Morphology and topographical distribution of focal adhesions have been investigated in real-time manner with confocal microscopy and immuno-staining techniques [8,9].…”

Section: Introductionmentioning

confidence: 99%

Quartz crystal microbalance and electrochemical cytosensing on a chitosan/multiwalled carbon nanotubes/Au electrode

Jia

Tan

Xie

et al. 2008

Sensors and Actuators B: Chemical

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Visualization of cellular focal contacts using a monoclonal antibody to 80 kD serum protein adsorbed on the substratum

Cited by 39 publications

References 19 publications

Contact formation during fibroblast locomotion: involvement of membrane ruffles and microtubules.

Contact formation during fibroblast locomotion: involvement of membrane ruffles and microtubules.

Human keratinocytes express a new CD44 core protein (CD44E) as a heparan-sulfate intrinsic membrane proteoglycan with additional exons.

Quartz crystal microbalance and electrochemical cytosensing on a chitosan/multiwalled carbon nanotubes/Au electrode

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