Paxillin dynamics measured during adhesion assembly and disassembly by correlation spectroscopy

被引:99
作者
Digman, Michelle A. [1 ]
Brown, Claire M. [2 ]
Horwitz, Alan R. [2 ]
Mantulin, William W. [1 ]
Gratton, Enrico [1 ]
机构
[1] Univ Calif Irvine, Dept Biomed Engn, Fluorescence Dynam Lab, Irvine, CA 92717 USA
[2] Univ Virginia, Sch Med, Dept Cell Biol, Charlottesville, VA 22908 USA
关键词
D O I
10.1529/biophysj.107.104984
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
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.
引用
收藏
页码:2819 / 2831
页数:13
相关论文
共 31 条
  • [1] Marching at the front and dragging behind:: differential α-Vβ3-integrin turnover regulates focal adhesion behavior
    Ballestrem, C
    Hinz, B
    Imhof, BA
    Wehrle-Haller, B
    [J]. JOURNAL OF CELL BIOLOGY, 2001, 155 (07) : 1319 - 1332
  • [2] 2-PHOTON FLUORESCENCE CORRELATION SPECTROSCOPY - METHOD AND APPLICATION TO THE INTRACELLULAR ENVIRONMENT
    BERLAND, KM
    SO, PTC
    GRATTON, E
    [J]. BIOPHYSICAL JOURNAL, 1995, 68 (02) : 694 - 701
  • [3] Probing the integrin-actin linkage using high-resolution protein velocity mapping
    Brown, Claire M.
    Hebert, Benedict
    Kolin, David L.
    Zareno, Jessica
    Whitmore, Leanna
    Horwitz, Alan Rick
    Wiseman, Paul W.
    [J]. JOURNAL OF CELL SCIENCE, 2006, 119 (24) : 5204 - 5214
  • [4] Brown CM, 1998, J CELL SCI, V111, P271
  • [5] Paxillin: Adapting to change
    Brown, MC
    Turner, CE
    [J]. PHYSIOLOGICAL REVIEWS, 2004, 84 (04) : 1315 - 1339
  • [6] The photon counting histogram in fluorescence fluctuation spectroscopy
    Chen, Y
    Müller, JD
    So, PTC
    Gratton, E
    [J]. BIOPHYSICAL JOURNAL, 1999, 77 (01) : 553 - 567
  • [7] Fluctuation correlation spectroscopy with a laser-scanning microscope: Exploiting the hidden time structure
    Digman, MA
    Sengupta, P
    Wiseman, PW
    Brown, CM
    Horwitz, AR
    Gratton, E
    [J]. BIOPHYSICAL JOURNAL, 2005, 88 (05) : L33 - L36
  • [8] Measuring fast dynamics in solutions and cells with a laser scanning microscope
    Digman, MA
    Brown, CM
    Sengupta, P
    Wiseman, PW
    Horwitz, AR
    Gratton, E
    [J]. BIOPHYSICAL JOURNAL, 2005, 89 (02) : 1317 - 1327
  • [9] Cell adhesion strengthening:: Contributions of adhesive area, integrin binding, and focal adhesion assembly
    Gallant, ND
    Michael, KE
    García, AJ
    [J]. MOLECULAR BIOLOGY OF THE CELL, 2005, 16 (09) : 4329 - 4340
  • [10] Stick and grip - Measurement systems and quantitative analyses of integrin-mediated cell adhesion strength
    Garcia, AJ
    Gallant, ND
    [J]. CELL BIOCHEMISTRY AND BIOPHYSICS, 2003, 39 (01) : 61 - 73