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First published online 12 October 2004
doi: 10.1242/jcs.01416

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Spatial mapping of integrin interactions and dynamics during cell migration by Image Correlation Microscopy

Paul W. Wiseman^1,*, Claire M. Brown^2,*,, Donna J. Webb², Benedict Hebert¹, Natalie L. Johnson², Jeff A. Squier³, Mark H. Ellisman⁴ and A. F. Horwitz²

¹ Departments of Chemistry and Physics, McGill University, 801 Sherbrooke St. W. Montreal, Quebec H3A 2K6, Canada
² Department of Cell Biology, University of Virginia, PO Box 800732, Charlottesville, VA 22908, USA
³ Department of Physics, Colorado School of Mines, 1523 Illinois Street, Golden, CO 80401, USA
⁴ Departments of Neurosciences and Bioengineering, and National Center for Microscopy and Imaging Research, University of California, San Diego, CA 92093, USA

View larger version (87K): [in a new window]   Fig. 1. Cells expressing both paxillin-CFP and 5-YFP show visibly organized paxillin but not 5-integrin. (A-D) Expressed paxillin-CFP in a protrusion at various time points of a confocal image time series of MEF cells during adhesion formation and turnover. The pairwise corresponding images of 5-YFP expression for the same regions of the cell are shown in E-H. Cells were 24 hours post-transfection and were plated on 1 µg ml–1 fibronectin for an hour before imaging at 37°C. Scale bar, 10 µm.

View larger version (69K): [in a new window]   Fig. 2. Average cluster size for 5-integrin in CHO B2 cells. Confocal images of a CHO K1 cell expressing GAP-GFP and plated on coverslips coated with 2 µg ml–1 fibronectin (A), or CHO B2 cells expressing 5-GFP plated on coverslips coated with either 200 µg ml–1 poly-D-lysine (B) or 2 µg ml–1 fibronectin (C). Data were corrected for the fact that the EGFP is 1.4 times brighter than the GAP-GFP (S65A). The plot shown is the average number of integrins per cluster calculated from many regions on many cells (D), 166 areas, 44 cells for GAP-GFP, 102 areas, 18 cells for 5-GFP on poly-D-lysine, and 137 areas, 30 cells for 5-GFP on fibronectin. The DA values were calculated from the spatial correlation function x-axis fit because the GAP-GFP was moving during the time the laser beam moved from one row of the image to the next. For consistency the integrin DA values were also calculated from the x-axis fit. The DA of 6.02±0.07 for GAP-GFP was used to normalize the data and calculate the number of proteins per cluster for 5-integrin. The break for low and high expressers was set at an average intensity of below or above 300 intensity units. Error bars are s.e.m. Scale bars, 5 µm.

View larger version (65K): [in a new window]   Fig. 3. Integrins are concentrated in nascent adhesions demarked by the presence of paxillin-containing adhesions. MEF or CHO cells were transfected with both paxillin-CFP (A) and 5-YFP (B). Confocal image time series were collected 24 hours after transfection and 1 hour after cells were plated on coverslips coated with 1 µg ml–1 fibronectin. The % increase in the intensity of 5-YFP in nascent adhesive areas relative to adjacent areas was calculated and plotted as a histogram (C; 144 adhesions, 17 cells). In 5% of the adhesions measured, 5-integrin showed an equal or slightly lower intensity of fluorescence in areas adjacent to paxillin containing adhesions. (D) The relative concentration of 5-integrin in adhesive areas is higher for lower-expressing cells. For the two image frames shown there is a 27% increase in 5 intensity under paxillin based on measurements taken on 20 adhesions. Scale bar, 5 µm.

View larger version (46K): [in a new window]   Fig. 4. FRAP of 5-integrin shows that it is less mobile in adhesive regions of the cell. Confocal images of 5-GFP expressed in a CHO B2 cell plated on 2 µg ml–1 fibronectin for 3 hours at 37°C. The first image in the time series (A), the first image after bleaching (B) and the last image (C) in the time series are shown. (D) MEF cells transfected with paxillin-CFP and 5-YFP about 1 hour after plating on 1 µg ml–1 fibronectin. The lower panel shows where the 5-YFP was bleached both under paxillin and in a more central area of the cell. The paxillin-CFP was not bleached. Normalized FRAP curves showing the recovery of 5-GFP from the highlighted regions in A-C (E) or of 5-YFP (F) from the highlighted regions in D. The plots show the normalized intensity of fluorescence following bleaching of organized (open triangles) or nonorganized 5-integrin (filled circles) in either case. Scale bar, 5 µm.

View larger version (117K): [in a new window]   Fig. 5. A spatial map of the dynamics of 5-integrin or -actinin across the cell. ICM results for a confocal image time series of 5-GFP expressed in a CHO-B2 cell plated for 1 hour on 2 µg ml–1 fibronectin (A), or for a two-photon microscopy image time series of -actinin-GFP on a CHO-B2 cell plated for 24 hours on 5 µg ml–1 fibronectin (B), (C) and (D). Both image series were collected at 37°C. A temporal ICM analysis was performed on each of the highlighted regions of size 1282, 642 or 322 pixels. Image stacks were with 100 frames at 0.111 µm (A) or 0.118 µm (C,D) pixel size and a frame interval of 5 seconds. The temporal correlation functions were fit to Equations 11, 12 and 13 and the fit with the best R2 value was used. The circles represent the root mean square average diffusion distance from the center of the circle for a 10 minute period based on the measured average diffusion coefficient for each region. The vectors represent the mean translation distance and direction over a 10 minute period based on the measured velocities for regions exhibiting flowing integrin populations. The colored bars depict the proportion of immobile (green), flowing (yellow) and diffusing (cyan) integrin or -actinin within each region. For the 5-GFP image series some areas appear to be off of the cell (e.g. area 1). If this was the case the analysis was limited to the image frames where the region of interest was completely on the cell. Region 6 was too small for the directional correlation analysis. Correlation velocity mapping was done for small areas around a retracting microspike just below region 3 in Fig. 5B. The arrows show the direction of the flow component of the correlation function and the size of the arrow is proportional to the magnitude of the velocity in that area of the cell. (D) Correlation analysis of images 70-100 of the image time series showing diffusion of -actinin after adhesion disassembly. (E) Spatial-temporal correlation functions for -actinin in area 1 shown in Fig. 5C. Notice the center of the correlation function peak moves from the center of the axis in the direction shown by the arrow as things flow towards the upper left quadrant. Scale bars, 5 µm (A,C,D) or 10 µm (B).

View larger version (98K): [in a new window]   Fig. 6. Disassembly of 5-integrin in a retracting region of the cell. Confocal images of CHO B2 cells stably expressing 5-GFP 3 hours after plating on 2 µg ml–1 fibronectin. The first (A) and the last image in the 17 minute time series (B) are shown. Plots of the average intensity of the four bright adhesions (filled circles) and of a nonadhesive area (open triangles, region 8) within the boxed region are shown. A single exponential fit was made to the data to determine the rate of adhesion disassembly. (D,E) Outlines of individual adhesions from the boxed region that were analyzed by intensity to provide a measure of integrin density within adhesions (Table 2). Scale bars, 10 µm.

View larger version (15K): [in a new window]   Fig. 7. Temporal ICCM Correlation Functions for 5-integrin-YFP correlated with -actinin-CFP or paxillin-CFP. Temporal ICCM correlation functions were calculated from two-photon microscope image time series of CHO B2 cells expressing 5-integrin-YFP and -actinin-CFP (A) or 5-integrin-YFP and paxillin-CFP (B). The integrin-paxillin data were calculated from images in a central region of the cell where there was no organized paxillin. The temporal integrin autocorrelation function (closed circles), -actinin or paxillin autocorrelation function (open triangles) and the cross-correlation function (closed squares) are shown. Cross-correlation functions and the YFP autocorrelation function were corrected for bleed through of the CFP signal into the YFP channel (see Materials and Methods).

View larger version (77K): [in a new window]   Fig. 8. ICCM analysis of 5-YFP and -actinin-CFP. Two-photon fluorescence microscopy images collected at 37°C of -actinin-CFP (A-C) and 5-YFP (D-F) 2.5 hours after plating on 10 µg ml–1 fibronectin. ICM and ICCM were conducted on each highlighted region of the cell. The distribution of -actinin, 5-integrin or colocalized species was determined via spatial autocorrelation or cross-correlation analysis. The dynamics of -actinin, 5-integrin or complexes moving together was determined via a temporal autocorrelation or cross-correlation analysis. Spatial functions were fit to Equation 4, and temporal functions were fit to one of Equations 11, 12 or 13. Equation 17 was used to estimate the fraction of each population that was immobile. The green bars show the fraction of a given species (diffusing, flowing or immobile) that is interacting with the second fluorescently tagged protein within a complex. The white part of the bar is the fraction noninteracting – i.e. the green bar in a region of the cell in Fig. 8A would represent the fraction of -actinin that is diffusing together with 5-integrin, whereas the green bar in Fig. 8D would represent the fraction of 5-integrin that is diffusing together with -actinin. Similarly, the green bar in Fig. 8B and 8E show the fractions of the respective proteins that are flowing together, and Fig. 8C and 8F represent the fraction of each protein population that is immobile and colocalized. Scale bars, 10 µm.

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