The fibronexus: a transmembrane association of fibronectin-containing fibers and bundles of 5 nm microfilaments in hamster and human fibroblasts

Singer, Irwin

doi:10.1016/0092-8674(79)90040-0

Cited by 510 publications

(227 citation statements)

References 33 publications

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“…1B,C), in agreement with previously published images of such artificial Fn fibers (Ejim et al, 1993;Wojciak-Stothard et al, 1997). Indeed, cryo-scanning electron microscopic images suggest that Fn fibers exist as 'cables' comprised of individual fibrous strands of ~5-15 nm in diameter and larger (Chen et al, 1978;Dzamba and Peters, 1991;Peters et al, 1998;Singer, 1979) which are proposed to be held together by hydrogen bonds, intermolecular beta-strand swapping (Briknarova et al, 2003;Litvinovich et al, 1998), disulfide bonds which are potentially formed by cryptic disulfide isomerase activity (Langenbach and Sottile, 1999), and other weak electrostatic interactions (Chen and Mosher, 1996;Morla et al, 1994). Although the exact location and properties of these bonds are unknown, it has been observed that they are strong enough to render cell-derived Fn fibers irreversibly insoluble in 1% de-oxycholate (McKeown-Longo and Mosher, 1983), which is a phenomenon we observed with manually deposited Fn fibers as well (data not shown).…”

Section: Manually Deposited Fn Fibers Bundle Into Fiber Cables Similasupporting

confidence: 89%

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

et al. 2008

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In response to growing needs for quantitative biochemical and cellular assays that address whether the extracellular matrix (ECM) acts as a mechanochemical signal converter to co-regulate cellular mechanotransduction processes, a new assay is presented where plasma fibronectin fibers are manually deposited onto elastic sheets, while force-induced changes in protein conformation are monitored by fluorescence resonance energy transfer (FRET). Fully relaxed assay fibers can be stretched at least 5-6 fold, which involves Fn domain unfolding, before the fibers break. In native fibroblast ECM, this full range of stretch-regulated conformations coexists in every field of view confirming that the assay fibers are physiologically relevant model systems. Since alterations of protein function will directly correlate with their extension in response to force, the FRET vs. strain curves presented herein enable the mapping of fibronectin strain distributions in 2D and 3D cell cultures with high spatial resolution. Finally, cryptic sites for fibronectin's N-terminal 70-kD fragment were found to be exposed at relatively low strain, demonstrating the assay's potential to analyze stretch-regulated protein-rotein interactions.

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Section: Manually Deposited Fn Fibers Bundle Into Fiber Cables Similasupporting

confidence: 89%

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

et al. 2008

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“…It features a codistribution of fibronectin, its plasma membrane receptor, actin, vinculin, and alpha-actinin throughout the entire contact site and is located at central regions of the cell rather than at its margins (11,15). Fibronexuses show very close transmembrane associations of actin microfilaments and fibronectin fibrils (42,43). …”

mentioning

confidence: 99%

“…The so-called close contacts exhibit a greater substrate separation distance, do not contain vinculin, and probably represent a more labile type of contact (23). A third kind of substrate adhesion site termed the fibronexus (42) or extracellular matrix contact site (9) is characteristic of well-spread and stationary fibroblasts. It features a codistribution of fibronectin, its plasma membrane receptor, actin, vinculin, and alpha-actinin throughout the entire contact site and is located at central regions of the cell rather than at its margins (11,15).…”

mentioning

confidence: 99%

Ultrastructural localization of plasma membrane-associated urokinase-type plasminogen activator at focal contacts.

Pöllänen

Hedman

Nielsen

et al. 1988

The Journal of Cell Biology

297

117

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Abstract.We have recently shown that urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 are both found extracellularly beneath cultured human skin fibroblasts and HT-1080 sarcoma cells, but in distinct localizations. Here, the ultrastructural distribution of u-PA was studied using immunoferritin electron microscopy. In HT-1080 cells, u-PA on the extracellular aspect of the plasma membrane was detected at sites of direct contact of the cell with the growth substratum beneath all parts of the ventral cell surface. The ferritin-labeled adhesion plaques, which were enriched in submembraneous microfilaments, were frequently seen at the leading lamellae of the cells as well as in lamellipodia and microspikes. Besides the cell-substratum adhesion plaques, ferritin label was detected at cell-cell contact sites. Double-label immunofluorescence showed a striking colocalization of u-PA and vinculin in both HT-1080 cells and WI-38 lung fibroblasts, which is consistent with u-PA being a focal contact component. The u-PAcontaining focal contacts of WI-38 cells had no direct codistribution with fibronectin fibrils. In WI-38 cells made stationary by cultivation in a medium containing 0.5 % FCS, vinculin plaques became highly elongated and more centrally located, whereas u-PA immunolabel disappeared from such focal adhesions. These findings show that plasma membrane-associated u-PA is an intrinsic component of focal contacts, where, we propose, it enables directional proteolysis for cell migration and invasion. rIESIVE interactions of cells with the extracellular matrix and their reversal are critical events for morphogenetic movements during embryonic development and for cancer cell invasion during metastasis (30, 49). The molecular structures at the sites where cultured cells contact each other and the growth substratum are thus of great interest. Focal contacts, the sites of closest cell-substratum apposition, can be visualized either by electron microscopy (1) or by interference reflection microscopy (23). Focal contacts are localized at the termini of actin-containing microfilament bundles, and contain vinculin (7, 19) and talin (6) on the cytoplasmic side, but apparently do not have fibronectin on the extracellular side (9). The so-called close contacts exhibit a greater substrate separation distance, do not contain vinculin, and probably represent a more labile type of contact (23). A third kind of substrate adhesion site termed the fibronexus (42) or extracellular matrix contact site (9) is characteristic of well-spread and stationary fibroblasts. It features a codistribution of fibronectin, its plasma membrane receptor, actin, vinculin, and alpha-actinin throughout the entire contact site and is located at central regions of the cell rather than at its margins (11,15). Fibronexuses show very close transmembrane associations of actin microfilaments and fibronectin fibrils (42,43).

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“…We also identified a second, smaller class of aggregates of membrane particles that contained/~-integrin but not vinculin or talin and that were not associated with actin microfilaments. Our results indicate that vinculin, talin, and ~-integrin are assembled into distinctive structures that mediate multiple lateral interactions between microfilaments and the membrane at focal contacts.F OCAL contacts are specialized structures where actin filaments converge and terminate at the plasma membrane (16,32,44,63,65). Interactions between the membrane and microfilaments involve several proteins that are concentrated at focal contacts, including integrin (e.g., references 17, 21, 22; reviewed in 1, 61) and cytoskeletal proteins such as vinculin (26), talin (13), fimbrin (9), tensin (71), paxillin (68), zyxin (5), and ot-actinin (40).…”

mentioning

confidence: 99%

“…F OCAL contacts are specialized structures where actin filaments converge and terminate at the plasma membrane (16,32,44,63,65). Interactions between the membrane and microfilaments involve several proteins that are concentrated at focal contacts, including integrin (e.g., references 17, 21, 22; reviewed in 1, 61) and cytoskeletal proteins such as vinculin (26), talin (13), fimbrin (9), tensin (71), paxillin (68), zyxin (5), and ot-actinin (40).…”

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confidence: 99%

Structures linking microfilament bundles to the membrane at focal contacts

Samuelsson

Luther

Pumplin

et al. 1993

The Journal of Cell Biology

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Abstract. We used quick-freeze, deep-etch, rotary replication and immunogold cytochemistry to identify a new structure at focal contacts. In Xenopus fibroblasts, elongated aggregates of particles project from the membrane to contact bundles of actin microfilaments. Before terminating, a single bundle of microfilaments interacts with several aggregates that appear intermittently over a distance of several microns. Aggregates are enriched in proteins believed to mediate actin-membrane interactions at focal contacts, including B~-integrin, vinculin, and talin, but they appear to contain less t~-actinin and filamin. We also identified a second, smaller class of aggregates of membrane particles that contained/~-integrin but not vinculin or talin and that were not associated with actin microfilaments. Our results indicate that vinculin, talin, and ~-integrin are assembled into distinctive structures that mediate multiple lateral interactions between microfilaments and the membrane at focal contacts.F OCAL contacts are specialized structures where actin filaments converge and terminate at the plasma membrane (16,32,44,63,65). Interactions between the membrane and microfilaments involve several proteins that are concentrated at focal contacts, including integrin (e.g., references 17, 21, 22; reviewed in 1, 61) and cytoskeletal proteins such as vinculin (26), talin (13), fimbrin (9), tensin (71), paxillin (68), zyxin (5), and ot-actinin (40). Biochemical studies have suggested several ways in which these proteins could anchor microfilaments to the cytoplasmic surface of the plasma membrane at focal contacts. Early studies suggested that particular isoforms of integrin ("CSAT'; 47) bound talin, and that this complex then bound vinculin (35; reviewed in 15). Later studies showed that integrins could also bind tx-actinin (53). Thus, integrin could anchor actin filaments indirectly through the actin-binding protein, tx-actinin, as well as through talin and vinculin. If both of these mechanisms are important for actin-membrane interactions, one would expect all these proteins to be concentrated at focal contacts.Structural studies also suggest two models for microfilament-membrane interactions at focal contacts. Focal contacts are several microns long, and individual actin filaments course over this entire distance before terminating (8). These filaments could bind to the membrane laterally as they approach their termini. In this case, vinculin, talin, integrin, and other proteins of focal contacts would be expected to concentrate at many points along the filament length. Alter- natively, filaments could attach only at their termini. In this case, the proteins of focal contacts would be expected to occur only at the ends of microfilaments.Predictions of the relationship between actin microfilaments and the membrane, and of the distribution of proteins at focal contacts, can be tested by ultrastructural methods coupled with immunocytochemistry. We exposed the cytoplasmic surfaces of ventral membrane by mechanical shearing, the...

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The fibronexus: a transmembrane association of fibronectin-containing fibers and bundles of 5 nm microfilaments in hamster and human fibroblasts

Cited by 510 publications

References 33 publications

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

Ultrastructural localization of plasma membrane-associated urokinase-type plasminogen activator at focal contacts.

Structures linking microfilament bundles to the membrane at focal contacts

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