First published online 14 March 2006
doi: 10.1242/jcs.02830
Journal of Cell Science 119, 1350-1360 (2006)
Published by The Company of Biologists 2006
New insights into extracellular matrix assembly and reorganization from dynamic imaging of extracellular matrix proteins in living osteoblasts
Pitchumani Sivakumar1,
Andras Czirok2,
Brenda J. Rongish2,
Vivek P. Divakara1,3,
Yu-Ping Wang3 and
Sarah L. Dallas1,*
1 Department of Oral Biology, UMKC School of Dentistry, 650 E 25th Street, Kansas City, MO 64108, USA
2 Department of Anatomy and Cell Biology, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA
3 School of Computing and Engineering, UMKC, 5100 Rockhill Road, Kansas City, MO 64 USA
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Fig. 1. Image field from a day 2 FRC culture at the start of dynamic imaging. This image corresponds to the bottom left panel of Movie 1 in supplementary material and represents Alexa Fluor 488-labeled fibronectin fibrils. Arrows labeled A-D indicate individual fibrils that show dramatic motions (see Figs 2 and 3 for description of individual fibril dynamics) Bar, 50 µm.
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Fig. 2. Cell and fibronectin fibril dynamics in a day 2 FRC culture. Time-lapse image series showing the dynamic motion of fibrils A and B from Fig. 1. Corresponding images are shown for the cells (viewed by DIC) and fibronectin (FN) fibrils. Merged images in which the fibronectin fibrils are pseudocolored red are shown on the right. The blue, green and red circles are the outlines of nuclei on individual cells that show significant movement during the 19 hour period. The motion paths (trajectories) of these cells are indicated below the image series. Arrows in the fibronectin images indicate two fibrils (A and B) that appear joined at 0 hours, but gradually pull apart until they are separated by 16 hours. Corresponding arrows on the merged pictures indicate that these fibrils lie on cellular processes and become separated upon retraction of these processes. A movie corresponding to these still images is available as Movie 2 in supplementary material. Bar, 50 µm.
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Fig. 3. Cell-mediated shunting and exchange of fibrillar material. Time-lapse image series showing examples of cell-mediated `shunting' of fibrillar ECM material and `exchange' of fibrillar material from one fibril to another. The image series shows an enlargement of fibrils C and D from Fig. 1. Fibril C moves upwards and to the left and by 4 hours, becomes joined with another fibril indicated by the white arrowhead. Fibril D appears joined at zero hours with a fibril indicated by the open arrowhead. By 1 hour 30 minutes, fibril D has detached from this fibril. It then retracts and moves upwards and to the left to become joined to a different fibril, indicated by the open arrow. See Movie 3 in supplementary material. Bar, 50 µm.
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Fig. 4. Addition of a piece of ECM material to the end of a fibril. Time-lapse image series showing the addition of a small piece of ECM material (arrowhead) onto a Y-shaped fibril (arrow). Note that this piece of fibrillar material is gradually moved upward and added on to the end of the Y-shaped fibril (Bar=25 µm). A movie showing this addition event and the associated cell movement is available as Movie 4 in supplementary material.
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Fig. 5. Dual imaging of fibronectin and LTBP1 in a day-2 FRC culture. Still image frames taken at the start of time-lapse imaging in a day 2 FRC culture using fluorescent probes for fibronectin (FN, green) and LTBP1 (red). Note that the fibrillar staining for LTBP1 is predominantly colocalized with fibronectin, as indicated by the yellow areas in the merged image. However, the LTBP1 probe also gives some staining of the cells. A movie corresponding to these images is available as Movie 5 in supplementary material and shows that both fibrillar networks undergo continual stretching and contraction in response to cell movement. Bar, 100 µm.
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Fig. 6. Dual imaging of fibronectin and LTBP1 in a day-12 FRC culture. (A) Still image frames taken at the start of time-lapse imaging in a day 12 FRC culture using fluorescent probes for fibronectin (green) and LTBP1 (red). Note that the LTBP1 and fibronectin fibrils are localized in separate fibrillar networks. Movie 6 in supplementary material shows that both fibrillar networks undergo continual stretching and contraction in response to cell movement. (B) Time-lapse image series from the boxed area in A. Note a fibronectin fibril (large arrow) that stretches between 15 minutes and 4 hours, then contracts between 4 and 8 hours. The fibril then stretches again to reach its maximal length at 12 hours, then contracts again between 12 and 18 hours. Also note an LTBP1 fibril (small arrow) in the same region that appears to show much less motion compared with the fibronectin fibril. The bottom panel shows the trajectories of these fibrils obtained by tracking the co-ordinates of the end of each fibril in the image series and confirms that the LTBP1 fibril traverses a smaller trajectory. See also Movie 7 in supplementary material. Bar, 100 µm (A); 50 µm (B).
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Fig. 7. Displacement mapping analysis of cell and fibril movement (see Materials and Methods for description of technique). Frequency distribution plots showing displacements of point markers positioned on cell, LTBP1 or fibronectin fibril images during a time interval of 1 hour in day 2 (A) and day 12 (B) FRC cultures. Note that the movement of cells, LTBP1 and fibronectin (FN) fibrils in day 2 cultures are very similar. However, in day 12 cultures, the number of markers that moved distances greater than 6 µm was higher for fibronectin compared with LTBP1. Mean displacements for fibronectin and LTBP1 markers were significantly different at day 12 but not day 2 (see text).
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Fig. 8. Correlation of cell and fibril motion. (A) Vector maps depicting the direction of movement of point markers on cells and fibrils in day 2 FRC cell cultures. These vector maps were generated from still images from Movie 1 in supplementary material. 100 point markers were positioned on features in a still image of cells or fibrils. The same features were identified on an image obtained 1 hour later and the markers repositioned if they had moved. Vectors depicting the displacement of the point markers were generated in which the dots represent the initial position of each point marker, and the end of the line represents the position to which the point marker has moved. Note that the direction of movement of point markers on cell and fibril images appears generally correlated. (B) Graphs showing correlation of the direction of movement of vectors on cell and fibril images from day 2 cultures compared using the `local average' analysis (see Materials and Methods section). This software calculates the average angle of vector movement within paired local neighborhoods within a 30 µm radius from comparison images of fibronectin/LTBP1 or cells/fibronectin. The pairs of angles from equivalent neighborhoods in each image were plotted and the correlation coefficient calculated. Note that there was a strong correlation between the movement of fibronectin and LTBP1 vectors, as expected because these two ECM molecules were colocalized. The movement of vectors on cell images was also significantly correlated with the movement of fibronectin vectors. Similar results were obtained comparing cell and LTBP1 vectors (data not shown).
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© The Company of Biologists Ltd 2006
