JCS00684 Movies

Files in this Data Supplement:

• Movie 1 - (see Fig. 4) Cells lacking myosin II heavy chain were imaged under 0.5% agarose while moving in a folate gradient. The cells were allowed to move up the folate gradient for approximately 4 hours before imaging was started. Images were acquired every 5 seconds and then processed to create a Quicktime™ movie (Fig. 4A). The cells can be seen to stretch and fragment as they attempt to move up the gradient. A magnified view of a single cells fragmenting is shown in the second movie (4B).

• Movie 2 - (see Fig. 5) The localization of F-actin in myosin II mutant cells moving under agarose. MhcA^– cells expressing GFP-ABD120 were used to visualize F-actin filaments while cells were moving under 0.5% agarose. The cell shown is in the process of fragmenting. The GFP probe can be seen to accumulate in the uropod that will eventually be lost when the cell fragments. This loss of actin from the cell may explain why they eventually cease moving up the gradient.

• Movie 3 - (see Fig. 6) The movement of myosin II mutant cells at the edge of a 2% agarose trough. MhcA^– cells do not move out of troughs when the concentration of agarose is 1% or above. Cells were visualized by time lapse imaging at 5 second intervals near the trough edge. The movie shows the cells moving back and forth along the trough edge, periodically protruding a pseudopod underneath the agarose. However, unlike wild-type cells, the cell body of the myosin mutants is never able to flatten and continue to move up the gradient under the agarose.

• Movie 4 - (see Fig. 7) The localization of myosin and F-actin in wild-type and mlcE^– mutant cells moving under agarose. Both wild-type cells and mutants lacking the essential light chain (mELC) are able to move under agarose. In order to examine the cytoskeletal structure, cells expressing either GFP-ABD120 or GFP-myosin II were imaged while moving under agarose. The folate gradient was established using a glass bottom Petri dish (Willco Wells) and the cells were imaged at 5 second intervals by confocal microscopy using a 100´ oil immersion lens. Wild type and mlcE^– cells show a similar dynamic distribution of myosin and F-actin while moving under agarose.