Integrin-associated complexes form hierarchically with variable stoichiometry in nascent adhesions

Abstract

Background: A complex network of putative molecular interactions underlies the architecture and function of cell-matrix adhesions. Most of these interactions are implicated from coimmunoprecipitation studies using expressed components, but few have been demonstrated or characterized functionally in living cells.

Results: We introduce fluorescence fluctuation methods to determine, at high spatial and temporal resolution, "when" and "where" molecular complexes form and their stoichiometry in nascent adhesions (NAs). We focus on integrin-associated molecules implicated in integrin activation and in the integrin-actin linkage in NAs and show that these molecules form integrin-containing complexes hierarchically within the adhesion itself. Integrin and kindlin reside in a molecular complex as soon as adhesions are visible; talin, although also present early, associates with the integrin-kindlin complex only after NAs have formed and in response to myosin II activity. Furthermore, talin and vinculin association precedes the formation of the integrin-talin complex. Finally, α-actinin enters NAs periodically and in clusters that transiently associate with integrins. The absolute number and stoichiometry of these molecules varies among the molecules studied and changes as adhesions mature.

Conclusions: These observations suggest a working model for NA assembly whereby transient α-actinin-integrin complexes help nucleate NAs within the lamellipodium. Subsequently, integrin complexes containing kindlin, but not talin, emerge. Once NAs have formed, myosin II activity promotes talin association with the integrin-kindlin complex in a stoichiometry consistent with each talin molecule linking two integrin-kindlin complexes.

Figure 1. Cross-variance of α₅β₁ integrin and kindlin2 in nascent adhesions

(A) α₅ integrin co-localizes with talin1, and kindlin2 in nascent adhesions (arrows) with (B) variable assembly rates in CHOK1 cell co-transfected with mCherry-paxillin and the designated GFP tagged plasmid. (C) TIRF images (average over 50 seconds) of α₅ integrin-mGFP and mCherry-kindlin2 used for cross-variance analysis. (D) Fluorescence intensity time trace of α₅ integrin-mGFP and mCherry-kindlin2 in a selected nascent adhesion and the corresponding cross-variance (B_cc) values calculated from phases of adhesion formation and disassembly (labeled A-E). (E) Box plot of the (B_cc) values from pixel regions corresponding to both adhesions and regions without adhesions. (B_cc) values calculated from areas away (N=17) and between (N=19) adhesions were averaged over 5x5 and 3x3 pixel regions respectively. For areas between adhesions, (B_cc) values corresponding to time segments when no adhesions were visible yet were discarded. (B_cc) values for regions around adhesions were calculated from single square pixel regions surrounding the adhesion. GAP (GAP-mGFP and GAP-mCherry) constitute a negative control. Images were acquired every 500 ms.

Figure 2. Cross-variance of talin-α₅β₁ integrin, and talin-kindlin2 in nascent adhesions

Box plots of (B_cc) values calculated for phases of nascent adhesion formation in CHOK1 cells expressing (A) α₅β₁ integrin-mGFP and mCherry-talin1 and (B) mGFP-talin1 and mCherry-kindlin2. (C) Intensity images (averaged over 3 min) of U2OS cells expressing α₅β₁ integrin-GFP and mCherry-talin1 before and 8 min after treatment with Y27632 drug; (D) note the α₅β₁ integrin-mGFP and mCherry-talin1 containing nascent adhesions do not mature into larger adhesions similar to those highlighted by arrows in (C). (E) Box plot of (B_cc) values measured from nascent adhesions in cells treated with Y27632 drug show abolished associations between α₅β₁ integrin-mGFP and mCherry-talin1, but not between (F) α₅β₁ integrin-mGFP and mCherry-kindlin2. Images were acquired every 500 ms.

Figure 3. Cross-variance of talin1 and vinculin in adhesions

(A) Vinculin-mGFP and mCherry-talin1 localize to nascent (NA) and focal adhesions (FA) in CHOK1 cells. (B) Representative fluorescence time traces and (B_cc) values for vinculin-mGFP and mCherry-talin1 in a nascent adhesion that disassembles. (B) Box plot of the (B_cc) values calculated at different times during nascent adhesion formation and from adhesions that stabilize into larger focal adhesions (FA), inferring that they reside in a common complex. The (B_cc) values for the pixels corresponding to individual FA’s (N=5) were averaged over the time segments. (C) Images from selected time segments in the vinculin time series depict the formation of a nascent adhesion that matures into a focal adhesion by elongating perpendicular to the direction of the protrusion. Different (B_cc) values at the distal versus proximal regions relative to the protrusion suggest a differential exchange of the molecular complex in different regions of the elongating adhesion.

Figure 4. α₅β₁ integrin and α-actinin assembly and association in nascent adhesions

(A) Intensity time trace of α₅ integrin-mGFP and α-actinin-mCherry in a nascent adhesion in CHOK1 cells. Arrows show α-actinin intensity spikes during assembly and the average duration between the intensity spikes. (B) The difference in the intensity derivative during assembly for α₅ integrin-mGFP and α-actinin-mCherry, in comparison to mCherry-paxillin-GFP, indicates that α-actinin assembles at a faster rate relative to α₅β₁ integrin. (C) High positive (B_cc) values correspond to the α-actinin intensity peaks during nascent adhesion assembly, whereas no cross-variance is detected in pixels corresponding to regions between adhesions (Off-adhesions). 25 adhesions were used for quantifications in (A-C). (D) α-actinin knockdown in CHOK1 cells expressing talin1-mGFP and mGFP-paxillin. In contrast to paxillin, cells expressing talin1 show persistent protrusive activity. (E) Effect of α-actinin knockdown on the association of α₅β₁ integrin and talin1 during nascent adhesion formation. For cross-variance analysis, images were acquired every 100 ms for mCherry-paxillin-mGFP, 150 ms for integrin-α-actinin, and 500 ms for integrin-talin1.

Figure 5. The number of α₅β₁ integrins and its partners in adhesions

(A) Number of molecules of α₅β₁ integrin as a function of expression level in nascent adhesions that stabilize. Expression level is classified based on the average intensity of α₅β₁ integrin in protruding regions of CHOB2 cells, which lack endogenous α₅β₁, stably expressing ectopic α₅ integrin-mGFP. (B) Degree of aggregation of α₅β₁ integrin in nascent adhesions. (C) Number of molecules of α₅β₁ integrin as a function of variable fibronectin coating concentrations. Number of molecules of α₅β₁ integrin, kindlin2, talin1, and vinculin in (D) nascent and (E) focal adhesions (as a function of size). (F) α-actinin numbers in nascent adhesions and in the intensity peaks that form during assembly selected from 25 adhesions (see Figure 4). The step size of the α-actinin spikes was measured as the difference between the peak intensity of the n^th spike and the trough intensity of the (n-1)^th peak. For the first intensity peak, we used the pre-assembly intensity levels to determine the step size.