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Dynamics of integrin clustering at focal contacts of endothelial cells studied by multimode imaging microscopy

¹ Department of Physiology, Nagoya University Graduate School of Medicine, 65 Tsurumai Showa-ku, Nagoya Aichi 4668550, Japan
² Department of Physical Therapy, Nagoya University School of Health Sciences, 1-1-20 Daikominami Higashi-ku, Nagoya Aichi 4618673, Japan
³ ICORP, Cell Mechanosensing Project, Japan Science and Technology Corporation, 65 Tsurumai Showa-ku, Nagoya Aichi 4668550, Japan

View larger version (109K): [in a new window]   Fig. 1. Integrin clustering during FC formation. (A) HUVECs were fixed at 30 minutes (a-c), 1 hour (d-f) and 6 hours (g-i) after plating. The left-hand column shows EpiF images, the middle column TIRF images and the right-hand column DIC images of the cells at different time after plating; images in each line are from the same cell. At 30 minutes after plating (a-c), a uniform distribution of integrins is observed in >90% of the bottom area of the cell (b). At 1 hour (d-f), a dappled distribution of integrins (the region surrounded by a broken line in e) and clusters of integrin are seen. Arrows show typical clusters of integrins in the region surrounded by a continuous line in e. At 6 hours after plating (g-i), clusters of integrins cover the entire bottom surface of the cell (four typical examples are indicated by arrows in h); arrows in g and h show the same integrin clusters. Integrins in the bottom surface of the cell were imaged more clearly by TIRF microscopy than by EpiF microscopy. Arrowheads in a, d and g show the punctate spots of integrins near the cell nucleus. (B) Integrin clusters observed by TIRF (arrows in b) correspond to the dark areas in the RIC image (arrows in a) of a HUVEC at 2 hours after plating. (C) Integrin clusters observed by EpiF (a) correspond to the vinculin clusters in the EpiF image (b) of the same HUVECs at 2 hours after plating. Bars, 20 µm.

View larger version (58K): [in a new window]   Fig. 2. Analysis of the elongation of integrin clusters at FCs. (A) A composite TIRF image of 12 cells with superimposed arrows that indicates the direction of elongation of the integrin clusters. Each TIRF image was taken from a region of the cell where active elongation of integrin clusters at FCs was seen. Each arrow shows the gross shape and length of a cluster analyzed and the direction of its elongation. The brightness of the arrow indicates the elongation rate (ER) of the cluster. For example, white arrows show the FCs that elongated with a rate greater than 0.4 µm minute-1 and black arrows a slower elongation rate (<0.2 µm minute-1). (B) Time courses of elongation of integrin clusters at FCs located at the marginal and central regions of the cells. The elongation rate at the marginal region was significantly higher than that in the central region at 12 minutes and 15 minutes. (C) The relative direction () of the elongation was measured with respect to the horizontal axis that approximates a tangent to the cell margin nearest to the cluster. (D) Histogram of the direction distribution of the elongation. Each bar indicates the numbers of elongating clusters with different directions () defined in (C).

View larger version (67K): [in a new window]   Fig. 3. Time-lapse TIRF images of the elongation of individual integrin clusters at FCs. (A) Time-lapse TIRF images of integrin clustering for 15 minutes of recording. The left-hand column shows a series of TIRF images of an elongating integrin cluster in the central region of the cell. The right-hand column shows the corresponding fluorescence intensity profiles (arbitrary unit; a.u.) along the elongation path. Arrows show the tip of the integrin cluster, extending to the right. (B) A series of TIRF images of an elongating integrin cluster at the FC near the marginal region of the cell. Arrows point to the tip of an integrin cluster. The right-hand column indicates the corresponding fluorescence intensity profiles of the integrin cluster, showing that the extending cluster is brighter near the tip (arrowheads) and grows faster than those at the central region of the cell.

View larger version (97K): [in a new window]   Fig. 4. Photobleaching and recovery of the fluorescence. The two columns show a photobleaching and recovery process in two cells (A-D) and that of two integrin clusters at FCs (E-H), respectively. A and E show control TIRF images; B and F are those just after photobleaching; C and G are those after 15 minutes of recovery. D and H show the result of subtracting B from C and F from G, respectively. Two arrows in A point out the two cells, separated by a bright border. The arrow in G shows the intensive recovery of integrin fluorescence at the extending tip of the integrin cluster, and the arrowhead in G demonstrates the relatively weak recovery of fluorescence in the preformed cluster. I and J show the fluorescence intensity profile along the broken lines (a-c) shown in F and G. The three profiles in I were measured immediately after photobleaching and those in J after 15 minutes of recovery. Arrows in I and J show the intensity at the extending tip of the integrin cluster recovered in G, arrowheads in I and J show a preformed part of the integrin cluster, and double arrowheads show the background level of fluorescence near the tip. Upon photobleaching, the fluorescence decreased to 60 counts from the original 150 counts. A strong recovery was then seen at the tip of the integrin cluster (Jc, arrow), whereas the recovery was relatively small at the middle of the integrin cluster (Jb, arrowhead) and in the background (Jc, double arrowhead). (K) Control images (a,c) and images immediately after photobleaching (b,d); all images are from the same cell. The TIRF image (b) shows that the FITC anti-ß1-integrin antibody fluorescence at the ventral surface of the cell was faded after photobleaching. The EpiF image (d) shows that the intracellular FITC fluorescence remains intact after photobleaching.

View larger version (92K): [in a new window]   Fig. 5. Simultaneous time-lapse imaging of integrin clusters at FCs by TIRF and CLSF microscopy. (A) A TIRF image of integrin clusters at 90 minutes after plating. The area surrounded by a rectangle was magnified and their dynamics were imaged by time-lapse TIRF and CLSF microscopy for 15 minutes as shown in B. Elongation of an integrin cluster at an FC was observed in the upper series of TIRF images (an arrow in each frame shows the tip of an extending integrin cluster), and a punctate spot of integrins in the intracellular space (an arrow in the lower series) was located slightly (1 µm) above the tip of the integrin cluster at the FC. Bars, 10 µm (A) and 5 µm (B).

View larger version (70K): [in a new window]   Fig. 6. Time-lapse imaging of exocytosis and elongation of FCs. HUVECs were double stained with FM4-64, a marker for transport vesicles, and fluorescent anti-ß1-integrin antibody for 30 minutes before plating. (A) EpiF images observed at 60 minutes (a,b) and 90 minutes (c,d) after plating; (a,c) fluorescence spots from vesicles; (b,d) fluorescence spots from integrins. Arrowheads in a and b indicate the spots positive for both vesicles and integrins. The number of fluorescence spots decreased gradually, suggesting the progress of exocytosis (c). (B) TIRF images were observed 60 minutes (a,b) and 63 minutes (c,d) after plating. (a,c) The disappearance of fluorescence spots from vesicles (arrows). (b,d) The fluorescent spot from integrins and the spread of the bright integrin cluster. (C) TIRF images of the elongation of FCs. Images were taken at 60 minutes (a) and 75 minutes after plating (b). Red dots in b show the location of vesicle disappearance (putative exocytosis) during 15 minutes of observation.

View larger version (127K): [in a new window]   Fig. 7. Colocalization of integrins and stress fibers imaged by CLSF and TIRF microscopy. (A) HUVECs were fixed 30 minutes (a-c), 1 hour (d-f) and 6 hours (g-i) after plating. The left-hand column shows the images of integrin distribution (a,d,g); the centre column shows stress fiber distribution (b,e,h); the right-hand column shows superimposed images of the corresponding images in the left and centre columns (c,f,i). These images were taken 1 µm above the basal plane of the cell with a CLSF microscope. At 30 minutes after plating, integrins were colocalized with F-actin at the marginal region of the cell (arrows in c). Arrowheads in e and f indicate a typical stress fiber. At 1 hour after plating, the integrin clusters were located along the stress fibers (two arrows in d and f show typical cases). The same trend was observed in integrin cultures at 6 hours after plating (g-i). (B) HUVECs were fixed at 1 hour after plating and observed by TIRF microscopy. (a) A TIRF image of integrin clusters at FCs; (b) stress fibers; (c) superimposed images of a and b. Bright images of integrin clusters can be seen at the ends of stress fibers (arrows in a-c). Bar, 20 µm.

View larger version (23K): [in a new window]   Fig. 8. Hypothetical model of integrin transport and the clustering at FCs. Surface integrins are first internalized, probably by endocytosis (1), and accumulated around the nucleus; this occurs 30 minutes to 1 hour after plating the cell. Integrins are transported to the ventral side of the cell by an actomyosin-dependent vesicle transport system (2) and are recruited to the tip of extending FC, probably via exocytosis (3) (red arrows show the sequence of these events). The direction of FC elongation in this study was towards the cell centre in most observations (the direction is shown by blue arrow). Elongation ceased when FCs grew to 7 µm.