Analyzing focal adhesion structure by atomic force microscopy
- PMID: 16263758
- DOI: 10.1242/jcs.02653
Analyzing focal adhesion structure by atomic force microscopy
Abstract
Atomic force microscopy (AFM) can produce high-resolution topographic images of biological samples in physiologically relevant environments and is therefore well suited for the imaging of cellular surfaces. In this work we have investigated focal adhesion complexes by combined fluorescence microscopy and AFM. To generate high-resolution AFM topographs of focal adhesions, REF52 (rat embryo fibroblast) cells expressing YFP-paxillin as a marker for focal adhesions were de-roofed and paxillin-positive focal adhesions subsequently imaged by AFM. The improved resolution of the AFM topographs complemented the optical images and offered ultrastructural insight into the architecture of focal adhesions. Focal adhesions had a corrugated dorsal surface formed by microfilament bundles spaced 127+/-50 nm (mean+/-s.d.) apart and protruding 118+/-26 nm over the substratum. Within focal adhesions microfilaments were sometimes branched and arranged in horizontal layers separated by 10 to 20 nm. From the AFM topographs focal adhesion volumes could be estimated and were found to range from 0.05 to 0.50 microm(3). Furthermore, the AFM topographs show that focal adhesion height increases towards the stress-fiber-associated end at an angle of about 3 degrees . Finally, by correlating AFM height information with fluorescence intensities of YFP-paxillin and F-actin staining, we show that the localization of paxillin is restricted to the ventral half of focal adhesions, whereas F-actin-containing microfilaments reside predominantly in the membrane-distal half.
Similar articles
-
Rho-kinase dependent organization of stress fibers and focal adhesions in cultured fibroblasts.Genes Cells. 2007 May;12(5):623-38. doi: 10.1111/j.1365-2443.2007.01073.x. Genes Cells. 2007. PMID: 17535253
-
Differential transmission of actin motion within focal adhesions.Science. 2007 Jan 5;315(5808):111-5. doi: 10.1126/science.1135085. Science. 2007. PMID: 17204653
-
Light-microscopical analysis of focal adhesions of retinal pigmented epithelial cells.Invest Ophthalmol Vis Sci. 1986 Nov;27(11):1622-33. Invest Ophthalmol Vis Sci. 1986. PMID: 3771144
-
AFM: a nanotool in membrane biology.Biochemistry. 2008 Aug 5;47(31):7986-98. doi: 10.1021/bi800753x. Epub 2008 Jul 11. Biochemistry. 2008. PMID: 18616288 Review.
-
Atomic force microscopy of actin.Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2003 Jun;35(6):489-94. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2003. PMID: 12796807 Review.
Cited by
-
Force-dependent focal adhesion assembly and disassembly: A computational study.PLoS Comput Biol. 2023 Oct 6;19(10):e1011500. doi: 10.1371/journal.pcbi.1011500. eCollection 2023 Oct. PLoS Comput Biol. 2023. PMID: 37801464 Free PMC article.
-
Mechanics of tensegrity cell units incorporating asymmetry and insights into mollitaxis.J R Soc Interface. 2023 May;20(202):20230082. doi: 10.1098/rsif.2023.0082. Epub 2023 May 17. J R Soc Interface. 2023. PMID: 37194274 Free PMC article.
-
Organization, dynamics and mechanoregulation of integrin-mediated cell-ECM adhesions.Nat Rev Mol Cell Biol. 2023 Feb;24(2):142-161. doi: 10.1038/s41580-022-00531-5. Epub 2022 Sep 27. Nat Rev Mol Cell Biol. 2023. PMID: 36168065 Free PMC article. Review.
-
Adhesion of Triple-Negative Breast Cancer Cells under Fluorescent and Soft X-ray Contact Microscopy.Int J Mol Sci. 2021 Jul 6;22(14):7279. doi: 10.3390/ijms22147279. Int J Mol Sci. 2021. PMID: 34298899 Free PMC article.
-
LIM domain proteins in cell mechanobiology.Cytoskeleton (Hoboken). 2021 Jun;78(6):303-311. doi: 10.1002/cm.21677. Epub 2021 Jun 10. Cytoskeleton (Hoboken). 2021. PMID: 34028199 Free PMC article. Review.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Miscellaneous