Multiparametric Analysis of Focal Adhesions in Bidimensional Substrates
Abstract
Focal adhesions in planar substrates constitute an excellent cellular resource to evaluate different parameters related to cell morphology, cytoskeletal organization, and adhesive strength. However, their intrinsic heterogeneity in terms of size, molecular composition, orientation, and so on complicates their analysis. Here, we describe a simple and straightforward ImageJ/Fiji-based method to quantify several parameters that describe the morphology and relative composition of focal adhesions. This type of analysis can be implemented in various ways and become useful for drug and shRNA screenings.
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Vinculin or paxillin immunostaining were used interchangeably as valid markers for focal adhesion measurements.
“using vinculin or paxillin immunostaining interchangeably, as the validity of both markers for these measurements was reported in the same study.”
From Paclitaxel induces <scp>NM2</scp> ‐dependent cellular contraction through <scp>GEF</scp> ‐ <scp>H1</scp> dissociation from microtubules and <scp>RhoA</scp> / <scp>ROCK</scp> activation in cancer cells (2026) · cites this paper as [31]
Section 2.2 (Fixation and staining; page not provided) describes vinculin immunostaining for focal-adhesion measurements and says that several antibodies have been validated. Note 4 recommends a Paxillin-pEGFP plasmid for visualizing focal adhesions, but the copy does not establish that paxillin immunostaining was validated or used interchangeably with vinculin immunostaining. (LLM, conf=0.88)
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Research works printed but not linked: 12
- [1]1. Horwitz AR (2012) The origins of the molecular era of adhesion research. Nat Rev Mol Cell Biol 13 (12):805811. doi:10.1038/nrm3473 · DOI
- [2]2. Lazarides E, Burridge K (1975) Alpha-actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells. Cell 6 (3):289-298
- [3]3. Zaidel-Bar R, Itzkovitz S, Ma'ayan A, Iyengar R, Geiger B (2007) Functional atlas of the integrin adhesome. Nat Cell Biol 9 (8):858-867. doi:10.1038/ncb0807-858 · DOI
- [4]4. Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G, Parsons JT, Horwitz AR (2003) Cell migration: integrating signals from front to back. Science 302 (5651):1704-1709
- [5]5. Humphries JD, Paul NR, Humphries MJ, Morgan MR (2015) Emerging properties of adhesion complexes: what are they and what do they do? Trends Cell Biol 25 (7):388-397. doi:10.1016/j.tcb.2015.02.008 · DOI
- [6]6. Kubow KE, Conrad SK, Horwitz AR (2013) Matrix microarchitecture and myosin II determine adhesion in 3D matrices. Curr Biol 23 (17):1607-1619. doi:10.1016/j.cub.2013.06.053 · DOI
- [7]7. Kubow KE, Horwitz AR (2011) Reducing background fluorescence reveals adhesions in 3D matrices. Nat Cell Biol 13 (1):3-5; author reply 5-7. doi:10.1038/ncb0111-3 · DOI
- [8]8. Fraley SI, Feng Y, Krishnamurthy R, Kim DH, Celedon A, Longmore GD, Wirtz D (2010) A distinctive role for focal adhesion proteins in three-dimensional cell motility. Nat Cell Biol 12 (6):598-604. doi:10.1038/ncb2062 · DOI
- [9]9. Kim DH, Wirtz D (2013) Focal adhesion size uniquely predicts cell migration. FASEB J 27 (4):1351-1361. doi:10.1096/fj.12-220160 · DOI
- [11]11. Berginski ME, Vitriol EA, Hahn KM, Gomez SM (2011) High-resolution quantification of focal adhesion spatiotemporal dynamics in living cells. PLoS One 6 (7):e22025. doi:10.1371/journal.pone.0022025 · DOI
- [3]3. doi:10.1002/0471143030.cb1005s60 · DOI
- [13]13. Wurm CA, Neumann D, Schmidt R, Egner A, Jakobs S (2010) Sample preparation for STED microscopy. Methods Mol Biol 591:185-199. doi:10.1007/978-1-60761-404-3\_11 · DOI
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Asensio‐Juárez, G., Pérez‐Díaz, R., Ramos‐Solano, H., Garrido‐Casado, M., Talayero, V. C., & Vicente‐Manzanares, M. (2026) · Molecular Oncology · doi:10.1002/1878-0261.70333
Self-citation
- Authors
- Talayero, V. C., & Vicente-Manzanares, M.
- Affiliations
- Consejo Superior de Investigaciones Científicas (ES)
- ORCID
- Talayero, Vanessa C.
- Venue
- Methods in Molecular Biology
- Publisher
- Springer US
- Published
- 2020-11-20
- Volume
- 2217
- Pages
- 27-37
- ISSN
- 1064-3745 (ISSN-L)
- 1940-6029
- OpenAlex
- W3105481742
- Cited by
- 7
- References
- 0
- Citations by year
- 2026: 3 · 2025: 3 · 2021: 1
- FWCI
- 0.42
- Type
- book chapter
- Language
- en
- Open access
- closed
- Locations
- Methods in molecular biology (publishedVersion)
- PubMed (publishedVersion)
- DIGITAL.CSIC (Spanish National Research Council (CSIC)) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular Mechanics and Interactions
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
- Health Sciences › Medicine › Immunology and Allergy › Cell Adhesion Molecules Research
- Keywords
- focal adhesions, planar substrates, cell morphology, cytoskeletal organization, adhesion strength, drug screening, shRNA screening
- MeSH terms
- Actins, Animals, Cell Adhesion, Cricetulus, Extracellular Matrix, Fibronectins, Humans, Image Processing, Computer-Assisted, Actin Cytoskeleton, Osteoblasts, Phalloidine, CHO Cells, Focal Adhesions, NIH 3T3 Cells, Cell Line, Tumor, Mice, Molecular Imaging
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