Phosphorylated paxillin and FAK constitute subregions within focal adhesions

Integrin-mediated adhesions are convergence points of multiple signaling pathways. Their inner structure and their diverse functions can be studied with super-resolution microscopy. We used structured illumination microscopy (SIM) to analyze spatial organization of paxillin phosphorylation (pPax) within adhesions. We found that pPax and focal adhesion kinase (FAK) form spot-like, spatially defined clusters within adhesions in several cell lines. In contrast, other adhesion proteins showed no consistent organization in such clusters. Live-cell super-resolution imaging revealed that pPax-FAK clusters persist over time but modify distance to each other dynamically. Moreover, we show that the distance between separate clusters of pPax is mechanosensitive. Thus, in this work we introduce a new structural organization within focal adhesions and demonstrate its regulation and dynamics.

contrast, other adhesion proteins showed no consistent organization in such clusters. Live-23 cell super-resolution imaging revealed that pPax-FAK clusters persist over time but modify 24 distance to each other dynamically. Moreover, we show that the distance between separate 25 clusters of pPax is mechanosensitive. Thus, in this work we introduce a new structural 26 organization within focal adhesions and demonstrate its regulation and dynamics. 136 integrin, talin-1, and kindlin-2 -revealed a behavior somewhat between the two cases 137 mentioned above. This is highlighted by the zoom-in for talin-1 in Figure 1 which shows focal 138 adhesions with continuous and spot-like organization of talin-1 next to each other.

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Thus, SIM revealed a spatial organization within focal adhesions that differed not only 140 between different adhesome proteins but even relied on phosphorylation status, as shown for 141 pPax-Y118 compared to paxillin.

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Phosphorylated paxillin is spatially restricted to separated clusters 144 Surprised by the difference in paxillin vs. pPax-Y118 organization we decided to 145 analyze their spatial organization in detail and in a quantitative manner. We cultured REF cells

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on cover slips and stained endogenous paxillin as well as pPax-Y118 ( Fig. 2A). Magnifications 147 of single focal adhesions confirmed our earlier observation that paxillin is spread rather 148 homogenously throughout adhesions, while pPax-Y118 shows a spatial organization in 149 discrete clusters (Fig. 2B-D). To analyze the pPax-Y118 spacing in more detail, we developed 150 a custom-written Matlab code that measures the center-to-center distance of protein clusters 151 based on the position of their intensity maxima (Fig. S1). The analysis revealed a distribution 152 of pPax-Y118 cluster distances with an average separation around 508 nm (Fig. 2E, G). The 153 algorithm also allowed to extract an average cluster diameter of 210 +/-70 nm (Fig. 2F). 154 While the size of these clusters is close to the resolution limit of most microscopes, 155 spacing between spots should be well resolved by diffraction-limited microscopes. Therefore,

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we imaged paxillin and pPax-Y118 in the same focal adhesions with confocal laser scanning Leterrier, 2020)). AiryScan, as SIM, was able to resolve an organization of pPax-Y118 in spots 165 (Fig. S2C). Thus, resolution improvement of at least 1.7x consistently indicates clusters of 166 pPax-Y118 within single adhesions independent of the specific microscopic technique used.

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Furthermore, to confirm that differences in spatial organization are not induced by 168 staining pitfalls, like unspecific antibody binding, we performed titration experiments of primary 169 antibodies for paxillin (Fig. S4) and pPax-Y118 (Fig. S3). We could not detect a concentration 170 dependent effect of antibodies on the appearance of paxillin compared to pPax-Y118.

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Phosphorylated tyrosines in focal adhesions organize in clusters 177 Besides pPax (Y31 and Y118), we observed that FAK also organizes in discrete 178 clusters with a near-regular spacing (Fig. 1). Interestingly, FAK was reported to complex

Spatial organization of pPax and FAK is dynamic while remaining in clusters 209
To understand the spatial organization of pPax and FAK in more detail we set out to 210 analyze the temporal evolution of this pattern in focal adhesions. First, we cultured REF cells

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on cover slips and fixed them at different time points after cell seeding, followed by 212 immunostainings for paxillin and pPax-Y118 (Fig. 4 A,B). Starting with culturing cells for 2 hrs, 213 we observed focal adhesions with the same spatial organization as described above: paxillin 214 is spread throughout adhesions while pPax-Y118 is organized in distinct spatial clusters.

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Quantitative analysis revealed that the spacing between neighbored pPax-Y118 clusters is 216 preserved over time and always remains around 501 nm (Fig. 4C). This experiment, however, 217 measured pPax-Y118 spacing as an average for many focal adhesions from different cells.

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Thus, the possibility remained that single clusters of pPax are dynamic and change their 219 spacing over time. Live-cell SIM would be ideal to analyze the origin and development of pPax 220 clusters in more detail. However, live-cell imaging of protein phosphorylation is challenging.

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Instead, we made advantage of the fact that FAK also showed an organization in discrete

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Paxillin phosphorylation has also been linked to vinculin recruitment (Pasapera et al., 250 2010). Our initial analysis indicated that vinculin is not organized in discrete clusters within 251 focal adhesions (Fig. 1). However, this does not preclude that vinculin is relevant for the spatial 252 organization of pPax. To test this, we cultured mouse embryonic fibroblasts from vinculin 253 knockout mice (MEF vin -/-) and analyzed pPax-Y118 organization in these cells compared to 254 MEF wt cells (Fig. 7). As shown before, paxillin appeared spread throughout focal adhesions 255 while pPax-Y118 remained in separated clusters. Quantitative analysis of pPax-Y118 spot 256 distance revealed an increase of cluster distance in absence of vinculin (Fig. 7E). Additionally,

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we tested the impact of cellular contractility in MEF cells (Fig. 7C, D) as we did before for REF 258 cells (Fig. 6). These experiments again confirmed that reduced cellular contractility decreases 259 pPax-Y118 spacing (Fig. 7E).

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To conclude, acto-myosin contractility affects the spacing of pPax-Y118 clusters within

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GTPase signaling contributes to more exploratory lamellipodia in the cell periphery. Paxillin 298 phosphorylated at these sites was also discussed to recruit vinculin in a contractility-dependent 299 manner . Indeed, we observed that chemically reducing cell contractility 300 as well as a knockout of vinculin both affect pPax-Y118 spacing (Fig. 6, 7). Concerning the 301 organization of pPax/FAK in separated clusters, it is interesting to consider alternative spatial

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Another interesting question is the molecular mechanism that organizes and keeps

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In summary, we described here new features of lateral adhesion organization based 332 on super-resolution microscopy techniques. It was surprising to us how consistently 333 pPax/(p)FAK/pTyr are organized in spatially separated clusters while some kind of "spotty 334 appearance" of these stainings is surely well known to everyone working with these stainings.

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Importantly, however, we found this organization in clusters in different cell lines and overall to 336 be conserved under a range of conditions. Moreover, experiments with reduced cellular 337 contractility and modified vinculin expression indicate an active regulation of spot distance.

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Thus, we expect that this spatial organization will emerge as an important aspect of paxillin