DOI: 10.1126/science.1064829. Taking Cell-Matrix Adhesions to the Third Dimension

Taking Cell-Matrix Adhesions to the Third Dimension

10.1126/science.1064829

Crossref journal-article

American Association for the Advancement of Science (AAAS)

Science (221)

Abstract

Adhesions between fibroblastic cells and extracellular matrix have been studied extensively in vitro, but little is known about their in vivo counterparts. Here, we characterized the composition and function of adhesions in three-dimensional (3D) matrices derived from tissues or cell culture. “3D-matrix adhesions” differ from focal and fibrillar adhesions characterized on 2D substrates in their content of α 5 β 1 and α v β 3 integrins, paxillin, other cytoskeletal components, and tyrosine phosphorylation of focal adhesion kinase (FAK). Relative to 2D substrates, 3D-matrix interactions also display enhanced cell biological activities and narrowed integrin usage. These distinctive in vivo 3D-matrix adhesions differ in structure, localization, and function from classically described in vitro adhesions, and as such they may be more biologically relevant to living organisms.

Bibliography

Cukierman, E., Pankov, R., Stevens, D. R., & Yamada, K. M. (2001). Taking Cell-Matrix Adhesions to the Third Dimension. Science, 294(5547), 1708â1712.

Authors 4

Edna Cukierman (first)
Roumen Pankov (additional)
Daron R. Stevens (additional)
Kenneth M. Yamada (additional)

References 29 Referenced 2,498

B. M. Jockusch et al. Annu. Rev. Cell Dev. Biol. 11 379 (1995). (10.1146/annurev.cb.11.110195.002115)
10.1016/S0955-0674(97)80155-X
10.1006/excr.2000.5043
10.1007/PL00000864
10.1016/0092-8674(92)90115-S
10.1146/annurev.cb.11.110195.003001
10.1038/13043
E. Zamir et al. J. Cell Sci. 112 1655 (1999). (10.1242/jcs.112.11.1655)
R. Pankov et al. J. Cell Biol. 148 1075 (2000). (10.1083/jcb.148.5.1075)
10.1139/o95-046
10.1083/jcb.54.3.626
10.1007/s000180050498
10.1038/sj.onc.1203877
10.1016/S1357-2725(98)00062-4
Cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% fetal bovine serum (Hyclone) penicillin (100 U/ml) and streptomycin (100 μg/ml). Immunofluorescence was performed as described (24) with minor modifications: For permeabilization 0.5% Triton X-100 was added to the fixing solution for 5 min and the phosphate-buffered saline washes contained 0.05% Tween 20. Cryostat sections of heads of mouse embryos (embryonic day E8.5 to E13.5) 10 μm thick were stained after use of the M.O.M. blocking reagent (Vector Laboratories). Routine controls for staining and fluorescence channel specificity were performed. Laser scanning images (maximum projection views) were obtained with Leica and Zeiss confocal microscopes. Antibodies were obtained as described (25) and from Pharmingen (β 1 integrin mAb 9EG7 β 3 integrin mAb VI-PL2 and mouse α 5 integrin mAb CD49e) Transduction Laboratories (paxillin and tensin) Upstate Biotechnology (FAK) Biosource International (phospho-FAK 397 and phospho-paxillin 31 ) Chemicon International (α v β 3 integrin LM609) and Jackson Immunoresearch (species-specific secondary antibodies).
Cell attachment after 10 min was determined after nuclear staining with bisenzimide H3342 fluorochrome by determining numbers of nuclei with the use of MetaMorph 4.5.3 software (Universal Imaging); the average value obtained on fibronectin was normalized to 1.0 arbitrary unit. For measurement of cell morphology human fibroblast plasma membranes were stained in suspension with 1 1′-dioctadecyl-3 3 3′ 3′-tetramethylindocarbocyanine perchlorate (DiI; 4 μg/ml) and cultured and the elliptical form factor (EFF = length/breadth) was digitally determined using MetaMorph 4.5.3. Cell migration was measured as described (26). For measurement of proliferation fibroblasts (6 × 10 4 cells/ml) were cultured overnight with bromodeoxyuridine (BrdU; 27 μg/ml) fixed and double-stained with antibodies to lamin B (to determine total cell number) and to BrdU (to identify replicating cells). For morphometric comparisons of 3D and 2D adhesions see (18).
Cover slips were coated with fibronectin laminin-1 vitronectin or collagen I at 5 μg/ml. Collagen gels (Collagen Biomaterials) were 1 mg/ml. Cell-free 3D matrices were prepared as described (27) except that 10- to 15-μm sections of mouse craniofacial tissue or heavily confluent 5-day cultures of NIH-3T3 cells were used; dextran and precoating of surfaces with gelatin were omitted from the protocol. Local regions of 3D matrices were mechanically compressed by applying a weight of 158 g to an area of 113 mm 2 for 2 min to generate a flattened “2D matrix.” Alternatively cell-derived 3D matrices were solubilized in 5 M guanidine containing 10 mM dithiothreitol and 5 mM phenylmethylsulfonyl fluoride. The dissolved matrix components (“2D mix”) were coated on cover slips at concentrations of 5 to 50 μg/ml. Fibrillar fibronectin deposits were prepared as described (28) without the use of urea. All cover slips were blocked with 2% heat-denatured bovine serum albumin before use. Rigidified cell-derived matrices were produced by covalent fixation with 1% glutaraldehyde and blocking with 1 M ethanolamine.
For supplementary material see Science Online (www.sciencemag.org/cgi/content/full/294/5547/ 1708/DC1).
E. Cukierman et al. data not shown.
B. Z. Katz et al. Mol. Biol. Cell 11 1047 (2000).
10.1242/jcs.112.8.1149
D. Ilic et al. Am. J. Pathol. 159 93 (2001). (10.3917/comm.093.0159)
10.1146/annurev.cellbio.12.1.463
10.1083/jcb.126.5.1287
10.1126/science.7846531
J. Gu et al. J. Cell Biol. 146 389 (1999). (10.1083/jcb.146.2.389)
I. Vlodavsky Curr. Protocols Cell Biol. 1 10.4.1 (1999). (10.1002/0471143030.cb1004s01)
10.1016/0012-1606(83)90052-0
We thank H. Imai for help with in vivo studies C. Galbraith for suggestions K. Clark and M. Hoffman for comments K. Matsumoto and S. Hahn for technical assistance and H. Grant for proofreading.

Dates

Type	When
Created	23 years, 1 month ago (July 27, 2002, 5:47 a.m.)
Deposited	1 year, 7 months ago (Jan. 9, 2024, 4:46 p.m.)
Indexed	1 day, 18 hours ago (Aug. 26, 2025, 3:09 a.m.)
Issued	23 years, 9 months ago (Nov. 23, 2001)
Published	23 years, 9 months ago (Nov. 23, 2001)
Published Print	23 years, 9 months ago (Nov. 23, 2001)

Funders 0

None

BibTeX

@article{Cukierman_2001, title={Taking Cell-Matrix Adhesions to the Third Dimension}, volume={294}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.1064829}, DOI={10.1126/science.1064829}, number={5547}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Cukierman, Edna and Pankov, Roumen and Stevens, Daron R. and Yamada, Kenneth M.}, year={2001}, month=nov, pages={1708–1712} }

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