Paxillin dynamics measured during adhesion assembly and disassembly by correlation spectroscopy

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.

FIGURE 1

Single-point FCS data from CHO k1 cells expressing paxillin-EGFP in the cytosolic regions (A) and on the adhesions (B) were analyzed globally using a two-component fit (Table 1). Blue circles are the autocorrelation function at points selected on adhesions; red squares are the function at points selected in the cytosolic regions.

FIGURE 2

Statistical representation of single-point FCS data. The minimum-maximum data (×); the mean, represented by a circled “×”; and the 5–95% data range (boxes) are shown. (Upper) Fractional contribution from the fast-diffusing component (D_app = 19.6 μm²/s) relative to the slower component from single-point FCS data. (Lower left) Fractional contribution of the aggregated protein (n₂) relative to the total protein (monomeric (n₁) plus aggregated (n₂)) determined from the G(0) correlation function amplitude. (Lower right) Ratio of the brightness of the aggregate protein (ɛ₂) to that of the monomer (ɛ₁).

FIGURE 3

sFCS of paxillin-EGFP in and around an adhesion. Data were sampled at 64 kHz (1 ms/orbit, 64 points/orbit). The circle on the images shows the center position of the laser scanning orbit. The white dot on the orbit path shows the beginning of the orbit. The orbit is scanned clockwise. Intensity carpets are an image generated using the intensity data along each orbit, the x axis, with each successive orbit displayed along the y axis. Plots are also shown for the ACF-extrapolated amplitude (G(0,0), solid squares), the apparent diffusion coefficient in μm²/s (D_app, red circles), PCH analysis of the molecular brightness in kHz/molecule, (ɛ, blue dots), and the number density (N, solid squares) along the scan orbit. The G(0) and diffusion coefficients were calculated with a one-species fit after detrending. Adhesion 1 (upper) is a relatively stationary adhesion in paxillin null MEFs (pax(−/−)). The region at position 35 corresponds to the bright intensity column in the carpet representation. The total time of this measurement was 240 s. Adhesion 2 (middle) is a growing adhesion in CHO-K1 cells stably expressing paxillin-EGFP. The orbit diameter was 0.85 μm, corresponding to a 0.04-μm pixel size, along the orbit. The total acquisition time for this experiment was 540 s. Adhesion 3 (lower) is a growing adhesion in CHO K1 cells stably expressing paxillin-EGFP. The orbit diameter was 0.85 μm, and the total measurement time was 540 s.

FIGURE 4

Line scanning of paxillin-EGFP in CHO-K1 cells captured on an Olympus FV300 laser scanning confocal microscope. Two hundred fifty-six pixels were captured along the x axis with a pixel dwell time of 8 μs/pixel; there are 10,000 lines in the y axis. (A) The cell and scan line. (B) The intensity carpet of the line scan after detrending for photobleaching. The temporal autocorrelation function at each column was calculated from the detrended data, and the extrapolated amplitude (G(0), solid squares) and apparent diffusion coefficient (D_app, red circles) for each column were determined.

FIGURE 5

Temporal pixel correlation analysis of TIRF images. (A) Average intensity for the image time series. (B) Fluctuation amplitude of the autocorrelation function, G(0), at each pixel. The color scale for the G(0) map is from 0 to 0.00065. (C) Spatial plot of the rates obtained by fitting each temporal ACF to an exponential decay; the rates are color-coded, with those >1 s⁻¹ in red and those <1 s⁻¹ in green. (D) Histogram of rates determined from temporal correlation analysis. Scale bar, 5 μm.

FIGURE 6

RICS analysis of CHO K1 cells expressing EGFP-paxillin. (A) The image was analyzed using the RICS method. A small region of 32 × 32 points was sequentially analyzed and moved in the x and y direction by 16 pixels/step. For each step, the spatial ACF was fit to the RICS equations for one diffusion component. (B and C) Maps of the G(0) and apparent diffusion coefficient, respectively. Scale bar, 4 μm.

FIGURE 7

(A–E) Negative (blue) and positive (red) pixel intensity derivatives at selected times; 128 frames were averaged to calculate the derivative at each time point from 41 to 75 s. (F) Intensity versus time traces at different pixel locations within the image time series. The red, blue, and green arrows identify the points in the image (G) corresponding to the intensity traces.