Paxillin Dynamics Measured During Adhesion Assembly and Disassembly by Correlation Spectroscopy

Overview

Journal Biophys J

Publisher Cell Press

Specialty Biophysics

Date 2007 Nov 13

PMID 17993500

Citations 83

Authors

Michelle A Digman

Claire M Brown

Alan R Horwitz

William W Mantulin

Enrico Gratton

Affiliations

Soon will be listed here.

Abstract

Paxillin is an adaptor molecule involved in the assembly of focal adhesions. Using different fluorescence fluctuation approaches, we established that paxillin-EGFP is dynamic on many timescales within the cell, ranging from milliseconds to seconds. In the cytoplasmic regions, far from adhesions, paxillin is uniformly distributed and freely diffusing as a monomer, as determined by single-point fluctuation correlation spectroscopy and photon-counting histogram analysis. Near adhesions, paxillin dynamics are reduced drastically, presumably due to binding to protein partners within the adhesions. The photon-counting histogram analysis of the fluctuation amplitudes reveals that this binding equilibrium in new or assembling adhesions is due to paxillin monomers binding to quasi-immobile structures, whereas in disassembling adhesions or regions of adhesions, the equilibrium is due to exchange of large aggregates. Scanning fluctuation correlation spectroscopy and raster-scan image correlation spectroscopy analysis of laser confocal images show that the environments within adhesions are heterogeneous. Relatively large adhesions appear to slide transversally due to a treadmilling mechanism through the addition of monomeric paxillin at one side and removal of relatively large aggregates of proteins from the retracting edge. Total internal reflection microscopy performed with a fast acquisition EM-CCD camera completes the overall dynamic picture and adds details of the heterogeneous dynamics across single adhesions and simultaneous bursts of activity at many adhesions across the cell.

Citing Articles

YAP phosphorylation within integrin adhesions: Insights from a computational model.

Jafarinia H, Shi L, Wolfenson H, Carlier A Biophys J. 2024; 123(21):3658-3668.

PMID: 39233443 PMC: 11560305. DOI: 10.1016/j.bpj.2024.09.002.

A flexible loop in the paxillin LIM3 domain mediates its direct binding to integrin β subunits.

Baade T, Michaelis M, Prestel A, Paone C, Klishin N, Herbinger M PLoS Biol. 2024; 22(9):e3002757.

PMID: 39231388 PMC: 11374337. DOI: 10.1371/journal.pbio.3002757.

Delayed cortical development in mice with a neural specific deletion of β1 integrin.

Rashid M, Olson E Front Neurosci. 2023; 17:1158419.

PMID: 37250402 PMC: 10213249. DOI: 10.3389/fnins.2023.1158419.

Correlative light and electron microscopy reveals fork-shaped structures at actin entry sites of focal adhesions.

Legerstee K, Sueters J, Abraham T, Slotman J, Kremers G, Hoogenboom J Biol Open. 2022; 11(11).

PMID: 36409550 PMC: 9836080. DOI: 10.1242/bio.059417.

Kinetic Changes of Ptdins (3,4,5) P3 within Fast and Slow Turnover Rates of Focal Adhesion.

Al-Fahad D, Alyaseen F, Al-Amery A, Singh G, Srinath M, Rehman H Rep Biochem Mol Biol. 2022; 11(2):262-269.

PMID: 36164635 PMC: 9455192. DOI: 10.52547/rbmb.11.2.262.

References

Webb D, Parsons J, Horwitz A . Adhesion assembly, disassembly and turnover in migrating cells -- over and over and over again. Nat Cell Biol. 2002; 4(4):E97-100. DOI: 10.1038/ncb0402-e97. View

Ballestrem C, Hinz B, Imhof B, Wehrle-Haller B . Marching at the front and dragging behind: differential alphaVbeta3-integrin turnover regulates focal adhesion behavior. J Cell Biol. 2002; 155(7):1319-32. PMC: 2199321. DOI: 10.1083/jcb.200107107. View

Schroeder M, Webb D, Shabanowitz J, Horwitz A, Hunt D . Methods for the detection of paxillin post-translational modifications and interacting proteins by mass spectrometry. J Proteome Res. 2005; 4(5):1832-41. DOI: 10.1021/pr0502020. View

Geiger B, Bershadsky A, Pankov R, Yamada K . Transmembrane crosstalk between the extracellular matrix--cytoskeleton crosstalk. Nat Rev Mol Cell Biol. 2001; 2(11):793-805. DOI: 10.1038/35099066. View

Chen Y, Muller J, So P, Gratton E . The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J. 1999; 77(1):553-67. PMC: 1300352. DOI: 10.1016/S0006-3495(99)76912-2. View

Laukaitis C, Webb D, Donais K, Horwitz A . Differential dynamics of alpha 5 integrin, paxillin, and alpha-actinin during formation and disassembly of adhesions in migrating cells. J Cell Biol. 2001; 153(7):1427-40. PMC: 2150721. DOI: 10.1083/jcb.153.7.1427. View

Palecek S, Schmidt C, Lauffenburger D, Horwitz A . Integrin dynamics on the tail region of migrating fibroblasts. J Cell Sci. 1996; 109 ( Pt 5):941-52. DOI: 10.1242/jcs.109.5.941. View

Rocheleau J, Wiseman P, Petersen N . Isolation of bright aggregate fluctuations in a multipopulation image correlation spectroscopy system using intensity subtraction. Biophys J. 2003; 84(6):4011-22. PMC: 1302981. DOI: 10.1016/S0006-3495(03)75127-3. View

Webb D, Brown C, Horwitz A . Illuminating adhesion complexes in migrating cells: moving toward a bright future. Curr Opin Cell Biol. 2003; 15(5):614-20. DOI: 10.1016/s0955-0674(03)00105-4. View

10.

Garcia A, Gallant N . Stick and grip: measurement systems and quantitative analyses of integrin-mediated cell adhesion strength. Cell Biochem Biophys. 2003; 39(1):61-73. DOI: 10.1385/CBB:39:1:61. View

11.

Springer T, Wang J . The three-dimensional structure of integrins and their ligands, and conformational regulation of cell adhesion. Adv Protein Chem. 2004; 68:29-63. DOI: 10.1016/S0065-3233(04)68002-8. View

12.

Brown M, Turner C . Paxillin: adapting to change. Physiol Rev. 2004; 84(4):1315-39. DOI: 10.1152/physrev.00002.2004. View

13.

Webb D, Donais K, Whitmore L, Thomas S, Turner C, Parsons J . FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat Cell Biol. 2004; 6(2):154-61. DOI: 10.1038/ncb1094. View

14.

Ridley A, Schwartz M, Burridge K, Firtel R, Ginsberg M, Borisy G . Cell migration: integrating signals from front to back. Science. 2003; 302(5651):1704-9. DOI: 10.1126/science.1092053. View

15.

Lauffenburger D, Horwitz A . Cell migration: a physically integrated molecular process. Cell. 1996; 84(3):359-69. DOI: 10.1016/s0092-8674(00)81280-5. View

16.

Wiseman P, Brown C, Webb D, Hebert B, Johnson N, Squier J . Spatial mapping of integrin interactions and dynamics during cell migration by image correlation microscopy. J Cell Sci. 2004; 117(Pt 23):5521-34. DOI: 10.1242/jcs.01416. View

17.

Brown C, Hebert B, Kolin D, Zareno J, Whitmore L, Horwitz A . Probing the integrin-actin linkage using high-resolution protein velocity mapping. J Cell Sci. 2006; 119(Pt 24):5204-14. DOI: 10.1242/jcs.03321. View

18.

Yamada K, Pankov R, Cukierman E . Dimensions and dynamics in integrin function. Braz J Med Biol Res. 2003; 36(8):959-66. DOI: 10.1590/s0100-879x2003000800001. View

19.

Brown C, Petersen N . An image correlation analysis of the distribution of clathrin associated adaptor protein (AP-2) at the plasma membrane. J Cell Sci. 1998; 111 ( Pt 2):271-81. DOI: 10.1242/jcs.111.2.271. View

20.

Hebert B, Costantino S, Wiseman P . Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells. Biophys J. 2005; 88(5):3601-14. PMC: 1305507. DOI: 10.1529/biophysj.104.054874. View