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First published online October 22, 2003
doi: 10.1242/10.1242/jcs.00792

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Early molecular events in the assembly of matrix adhesions at the leading edge of migrating cells

Ronen Zaidel-Bar, Christoph Ballestrem, Zvi Kam and Benjamin Geiger*

Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, 76100, Israel



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  		Fig. 1. Formation of focal complexes (FX) and their development into focal adhesions (FA). Porcine aortic endothelial cells were transfected with GFP-ß3-integrin and cultured to confluence. The monolayer was scratched, creating a `wound' and time lapse movies of transfected cells, migrating into the `wound', were made at 1-minute intervals between exposures (see also Movie 1: http://jcs.biologists.org/supplemental/). For easier visualization of the dynamic process captured in this movie we superimposed sequential `current' and `current + 1 minute' images and colored them red and green, respectively. Structures appearing yellow remained unchanged between the two time points; structures in green are new, and structures in red disappeared before the second exposure. Notice that until 7 minutes there is active protrusion, followed by retraction (until 13 minutes), and the sequence ends with the beginning of a new protrusion. During the protrusive phase every minute new FX are formed, while 1- to 2-minute-old complexes tend to fade and disappear. Upon retraction, most of the FX disappear (in solid line oval) while a few FX grow into FA (dashed line oval). The growing FA expands towards the cell center. Scale bar: 5 µm.

 


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  		Fig. 2. Spatial relationships between paxillin, actin, phosphotyrosine (PY), zyxin and ß3-integrin in FA and FX. Endothelial cells migrating into an in-vitro `wound' were fixed and stained. (A) In a cell labeled for paxillin and actin we can distinguish a region that was protruding at the time of fixation (marked by a solid line oval) next to a region that was retracting (marked by a broken line circle). At the base of the protruding lamellipodium a rim of small FX can be seen surrounded by actin meshwork. At the edge of the retracting region large elongated FA extend towards the cell center, connected to stress fibers. (B) Leading edge movements were monitored by time-lapse microscopy prior to fixation and correlated with the pattern of protein distribution. For example here, a cell stained for PY and zyxin is shown along with frames from its corresponding phase-contrast movie. Retracting regions (marked by arrows) contain zyxin-positive FA, while protruding regions (marked by arrowheads) are lined by zyxin-negative FX. (C) A cell expressing GFP-ß3-integrin was labeled for PY. Co-localization of the two labels is nearly complete both in FX (marked by the solid line oval) and in FA (marked by broken line circle) near the leading edge. In more central regions of the cell (not relevant for this work) {alpha}5ß1-integrins replace {alpha}vß3-integrins. Scale bar: phase-contrast images, 10 µm; others, 5 µm.

 


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  		Fig. 3. Differential distribution of FA proteins in FX and FA. Endothelial cells migrating into an in-vitro `wound' were fixed and immunolabeled for PY (left column) and different FA proteins (middle column) as indicated. Analysis of the differential distribution of the different proteins relative to PY was assisted by calculation of pixel-to-pixel ratio images (right column). VASP forms a chain of 1-2 µm dots, located in front of the PY-positive FX while it co-localizes with PY in FA. Paxillin localizes extensively in both FA and FX. Zyxin appears both in a wavy pattern along actin and in FA; however, it is completely absent from FX at the leading edge. Scale bar: 5 µm.

 


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  		Fig. 4. Quantitative analysis of the association of FA molecules with FX. The quantitative analysis was performed on images of immunolabeled cells similar to those shown in Fig. 3. Motile cells were selected for this analysis, based on the time-lapse movies. Manually drawn polygons surrounding either FX at the leading edge or FA in more central positions were used to sample regions along the lamellipodium with different dynamic behavior. To evaluate the degree of localization of the various proteins in FX and in bona fide FA we used PY as an `adhesion site marker' and calculated the degree of overlap between PY and each of the FA proteins. The results of this analysis, done on 12-19 cells, is shown for FA (A) and for FX (B). Horizontal lines indicate means. To compare the density of a particular protein (based on fluorescent labeling intensity) between FX and FA we calculated the ratio between the average intensity in FA and the average intensity in FX in the same cells (C). Bars indicate standard deviations. The intensity of PY, ß3-integrin and talin was found to be 50% higher in FA than in FX, while paxillin, vinculin, FAK, VASP, and {alpha}-actinin are, on average, three times more intense in FA than in FX. To test the effect of local protrusion rate on the degree of co-localization of paxillin with PY in FX we measured the protrusion rate, prior to fixation, around each polygon in all the cells tested. (D) Paxillin labeling in FA (marked by x) is not affected by the protrusion rate, whereas the labeling intensity in FX (marked by triangles) is inversely related to the protrusion rate (linear fit, solid line, P<0.04).

 


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  		Fig. 5. Recruitment of zyxin into FX occurs following local retraction of the lamellipodium. Endothelial cells were cotransfected with YFP-paxillin and CFP-zyxin. Time-lapse two-color movies captured the differential dynamic behavior of these proteins in migrating cells. A series of merged images from such a movie is presented here, where paxillin is shown in red and zyxin in green (see also Movie 2: http://jcs.biologists.org/supplemental/). Arrows mark the direction of leading edge movement and empty circles mark a static state. Notice that while FA contain both paxillin and zyxin (yellow) the array of FX in the advancing lamella contain paxillin alone (red). One minute after the onset of retraction (time point 5') zyxin is seen entering the FX, which grow centripetally to become FA. Scale bar: 5 µm.

 


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  		Fig. 6. FX induced by active Rac do not contain zyxin. PAE cells were transfected with dominant active Rac (L61) and double-labeled for PY and zyxin. Transfected cells have a round morphology, and while maintaining some FA they show a substantial accumulation of FX around the cell periphery, as seen by the PY staining. Zyxin is associated with FA and with actin filaments, but is absent from FX. Scale bar: 5 µm; insets are enlargements (3x) of the boxed areas.

 


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  		Fig. 7. Inhibition of contractility using the Rho-kinase inhibitor Y27632, leads to the disappearance of FA and an enrichment of zyxin-free FX. Migrating endothelial cells were treated for 30 minutes with Y27632 or with the serine/threonine kinase inhibitor H-7 (not shown), which block acto-myosin contractility. FA and SF disappear following this treatment, and at the cell periphery, behind an exceptionally broad {alpha}-actinin-rich band, a conspicuous array of FX is apparent. These FX contain paxillin, but not zyxin. Scale bar: 5 µm.

 

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© The Company of Biologists Ltd 2003