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Fig. 6. Increased spreading time alters morphology and speed of cells migrating in response to an EF. 3T3 cells plated on acid-washed glass coverslips at T=0 were allowed to attach and spread for 1-24 hours. (A) Morphology of cells undergoing EF-mediated motility is altered by spreading time. Micrographs excerpted from time-lapse images of cells migrating after spreading for 1 hour (a) show that cells are less flat than after 3 hours (b) and 24 hours (c). The cathode (-) and 40 µm bar (a) are indicated. (B) Increased spreading time alters cell footprint and substratum contact area. TIRF (a-c) and corresponding immunofluorescence (a'-c') micrographs of cells spread for 1 hour (a,a'), 3 hours (b,b') and 24 hours (c,c'). TIRF micrographs (a-c) were used to quantify the cell surface area directly contacting substratum. Immunofluorescence micrographs (a'-c') were used to show that cell surface area (`footprint') and proportion of cells with asymmetric lamellipodia both increased with spreading time (more than 12 cells in each of two experiments). Scale bar, 10 µm. (C) Increased spreading time alters adhesion strength. The number of cells per field remaining after centrifugation (1500 g or 6400 g) was compared with the control (not centrifuged) for cells plated 1 hour, 3 hours and 24 hours before fixation. Centrifugation detached >80% of 1-hour-plated cells but did not significantly detach 3-hour- or 24-hour-plated cells. (D) EF-motility speed is altered by spreading time. Average speeds (µm hour-1) of cells plated for specified times were quantified from time-lapse recordings of EF motility (6 V cm-1; 1.5 hours); speeds were normalized to the 24-hour-plated cells (100%) (at least 18 cells in each of three experiments). Migration of 1-hour-plated cells (*) was significantly faster than 3-hour- and 24-hour-plated cells (P<0.05); the latter two were indistinguishable.
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