JCS02830 Supplementary Material

Files in this Data Supplement:

• Movie 1 - Dynamic imaging of fibronectin in a day 2 FRC culture. Composite frame, showing the DIC movie of cells (top left), fluorescence movie of fibronectin fibrils (bottom left) and the merged movie (right) in 2 day FRC cultures. In the merged movie, the fibronectin fibrils are pseudocolored red. Alexa Fluor 488-labeled plasma fibronectin was used as the probe. Images were collected every 15 minutes for 18 hours and assembled into QuickTime movies, at 30 frames per second (fps).  Note the large amount of cell movement that causes continual stretching and contracting of the fibronectin fibrillar network. Refer to figure 1 for locations of specific fibrils that show dramatic dynamic motions  (frame rate = 30 fps, corresponding figure = Fig. 1).

• Movie 2 - Example of fibronectin fibril dynamics in a day 2 FRC culture. This movie shows a cropped area from Movie 1. The DIC movie of the cells (left), fluorescence movie of the fibronectin fibrils (middle) and the merged movie (right) are shown simultaneously. In the merged movie, the fibronectin fibrils are pseudocolored red. Note here two fibronectin fibrils (A and B from figure 2) that are joined initially, but gradually pull apart until they are completely separated. Visualization of the same fibrils in the merged image (right) shows that these fibrils are lying on cellular processes, and become separated in response to retraction of these processes. This movie also shows the superimposed trajectories of three individual cells (blue, green and red) that are outlined in Figure 2. Trajectories were obtained by tracking the nucleus of each cell through the timelapse image series using the ‘Manual Tracking’ plug-in in the Image-J software.  Note also that the entire fibrillar network shows distortions and dynamic motion during the imaging period (frame rate = 15fps, corresponding figure = Fig. 2).

• Movie 3 - Example of ECM fibril shunting and fibril exchange mediated by motile cells. This movie shows another cropped area from Movie 1. The DIC movie of the cells (left), fluorescence movie of the fibronectin fibrils (middle) and the merged image (right) are shown simultaneously. In the merged movie, the fibronectin fibrils are pseudocolored red.  Note here a piece of fibrillar material (fibril C from figure 3) that  moves upwards and to the left to join up with another fibril. The joined unit then behaves as a single fibril. Also note another fibril (D from figure 3) that breaks off from one fibril, contracts, then moves upwards and to the left to join with a different fibril. Again, the entire fibrillar network shows continual distortions and dynamic motion during the imaging period (frame rate = 15 fps, corresponding figure = Fig. 3)

• Movie 4 - Example of addition of ‘globules’ of fibrillar material.  DIC movie of the cells (left), fluorescence movie of the fibronectin fibrils (middle) and merged movie (right) in a day 2 FRC culture. In the merged movie, the fibronectin fibrils are pseudocolored red. Alexa Fluor 488-labeled plasma fibronectin was used as the probe. Note here a globule of fibrillar material (indicated by an arrowhead in fig 4) that moves across and gets added on to the end of a Y-shaped fibril (indicated by an arrow in fig 4). Also note that this ‘globule’ appears to be carried along by a cell moving in the same direction (frame rate = 15 fps, corresponding figure = Fig. 4). 

• Movie 5 - Dynamic dual imaging of fibronectin and LTBP1 in day 2 FRC culture. Composite frame, showing a fluorescence movie of LTBP1 (top left), fluorescence movie of fibronectin fibrils (bottom left) and the merged movie (right) in 2 day FRC cultures. In the merged movie, the fibronectin fibrils are pseudocolored green and LTBP1 fibrils are pseudocolored red. Alexa Fluor 488-labeled plasma fibronectin was used as the probe for fibronectin and a Cy3-labeled anti-LTBP1 antibody was used as the probe for LTBP1. Note that in these 2 day cultures, the fibronectin and LTBP1 are predominantly colocalized in similar fibrillar networks (yellow fluorescence in merged picture). The LTBP1 probe also gives some cytoplasmic staining, that enables the cells to be visualized. Note the large amount of cell movement that causes continual stretching and contracting of the fibronectin and LTBP1 fibrillar networks. Due to their colocalization, the LTBP1 and fibronectin fibrils appear to show identical dynamic movements.  (frame rate = 30 fps, corresponding figure = Fig. 5)

• Movie 6 - Dual imaging of LTBP1 and fibronectin fibrils in a day 12 FRC culture. Composite frame, showing a fluorescence movie of LTBP1 (top left), fluorescence movie of fibronectin fibrils (bottom left) and the merged movie (right) in 12 day FRC cultures. In the merged movie, the fibronectin fibrils are pseudocolored green and LTBP1 fibrils are pseudocolored red. Alexa Fluor 488-labeled plasma fibronectin was used as the probe for fibronectin and a Cy3-labeled anti-LTBP1 antibody was used as the probe for LTBP1. Note that even in these 12 day postconfluent cultures, there is still a large amount of cell movement that causes continual stretching and contracting of the ECM fibrillar networks.   In contrast to day 2 cultures, LTBP1 and fibronectin are localized in separate fibrillar networks in these day 12 FRC cultures. The LTBP1 is localized in long parallel fibrils in a layer above the fibronectin fibrils and shows less dynamic movement as compared to the underlying fibronectin fibrils (frame rate = 30 fps, corresponding figure = Fig. 6A).

• Movie 7 - . Fibronectin and LTBP1 show different dynamic movements in mature FRC cultures. This movie shows a cropped area from Movie 6 (day 12 culture). The composite frame includes; a fluorescence movie of the LTBP1 fibrils (left), a fluorescence movie of the fibronectin fibrils (middle) and the merged movie (right) in a day 12 FRC culture. In the merged image, LTBP1 is pseudocolored red and fibronectin is pseudocolored green. Alexa Fluor 488-labeled plasma fibronectin was used as the fibronectin probe and Cy3-labeled anti-LTBP1 antibody was used for LTBP1. Note here a green fibronectin fibril (indicated by the arrow in figure 6B) that stretches and contracts to a large extent, whereas the overlying red LTBP1 fibrils in the same region show less dynamic movement (frame rate = 15fps, corresponding figure = Fig. 6B).