Movies

W. Matthew Petroll, Mridula Vishwanath and Lisha Ma

Files in this Data Supplement:

• Movie 1 - The effects of needle micromanipulation on the ECM. DIC image of a collagen matrix without cells being compressed by pushing with a glass microneedle from right to left.
• Movie 2 - Time-lapse DIC images of a rabbit corneal fibroblast following axial insertion of needle. Pseudopodial extension occurred at the front of the cell (right), whereas the rear was much less active and underwent intermittent retractions.
• Movie 3 - Rabbit corneal fibroblast 1 day after plating inside collagen matrix. Cellular contraction was observed following a 25 mm needle push. This initial contraction was followed by cell spreading (yellow arrows at end of sequence) and traction (green circled region). Red tracks show measured ECM displacements (crosses mark position 1 minutes after needle push).
• Movie 4 - Rabbit corneal fibroblast 2 days after plating inside collagen matrix. Pushing the ECM 45 mm towards the cell induced rapid cellular contraction (36% shortening) and ECM compression along the cell body. This initial contraction was followed by the extension of pseudopodia (yellow arrows at end of sequence) and rapid pulling in of the ECM (traction) at both ends of the cell (green circled regions). Red tracks show measured ECM displacements (crosses mark position 1 minutes after needle push).
• Movie 5 - Addition of Y-27632 60 minutes after needle push induced cell elongation and dramatic relaxation of cell induced matrix tension (red tracks, cross marks start position after adding Y-27632).
• Movie 6 - Overlays of GFP-zyxin (green) and DIC images (red) demonstrating the response to ECM compression. During cellular contraction following a 35 mm needle push, ECM deformation correlated with the inward movement of existing focal adhesions toward the cell body. The secondary spreading response was associated with the formation of new focal adhesions at pseudopodial tips, and rearward movement of existing adhesions, which resulted in additional pulling in of the ECM (circled regions, blue tracks).
• Movie 7 - Y-27632 blocks the cellular response to ECM micromanipulation. Human corneal fibroblast 1 day after plating inside collagen matrix. Cell elongation and ECM relaxation was observed after adding Y-27632. Subsequent pushing with a needle (note shadow from needle on right) induced little cell contraction. There was also no secondary spreading or traction following needle push as indicated by minimal ECM displacements (red tracks).
• Movie 8 - Fibroblast response to ECM stretch produced by pulling the ECM away from cells with a glass microneedle. Rabbit corneal fibroblast, 2 days after plating inside collagen matrix. Upon pulling on the ECM in front of the cell with a microneedle, the cell and ECM were stretched. Later, pseudopodia disengaged and retracted, then extended and retracted processes without inducing significant ECM deformation.
• Movie 9 - GFP-zyxin (green) and DIC (red) overlay of rabbit corneal fibroblast, 1 day after plating inside collagen matrix. Upon pulling on the ECM in front of the cell with a microneedle, the cells and ECM were stretched. Focal adhesions initially remained attached to the ECM, since ECM displacement and focal adhesion displacements were highly correlated. Following the initial stretch, pseudopodia disengaged and retracted. Following retraction, repeated extension and retraction of pseudopodia were observed as cells partially respread. New focal adhesions were formed as the cells respread, but no significant ECM displacement was observed (blue tracks).