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Fig. 4. Migration of monolayer cells exposed to wound-healing and EF signals. (A) EF application after wounding hastens initiation and biases motility direction of monolayer cells. Wounded 3T3 cell monolayers were placed in the indicated EFs as rapidly as practical after wounding (
30 minutes) with the length of wounds oriented perpendicular to the EF. Cell movements in a 1.5 hour interval (i.e. 0.5-2.0 hours after wounding) with the EF (towards cathode) and against the EF (towards anode) were plotted as absolute values of directional velocity. Notice that EFs increased initial speed toward the cathode significantly (P<0.05, marked with 
, for comparing 2 V cm-1 and 0 V cm-1). EFs also biased motility towards the cathode (P<0.05, marked with * for comparing cathode- and anode-directed velocities at both EF strengths). EFs >2 V cm-1 were not used; they caused cells to round up and motility was compromised. More than 20 cells were quantified per direction, and three experiments per condition. (B) Cells in monolayers simultaneously wounded and placed in EFs migrate preferentially towards the cathode. 3T3 cell monolayers placed in a 0.6 V cm-1 EF 30 minutes after wounding migrated towards the anode (+) and cathode (-) during a 3-hour recording; black line traces the initial wound edges. Notice that cells migrated farther with than against the EF (A). Scale bar, 40 µm. Also, see supplementary movie of wound-healing migration Wound_2V_3hr (http://jcs.biologists.com/supplemental/). (C) Applying EFs to pre-polarized cells directionally biases wound-healing motility. Confluent 3T3 cell monolayers were wounded and incubated for 2.5 hours to allow reorientation of cytoskeletal components toward the wound edge and were then observed for 1.5 hours (i.e. 3.0-4.5 hours after wounding) with EFs indicated. Directional velocities show that wound-healing speed of cells toward the cathode was unaffected by applied voltage (0.6 V cm-1 and 2.0 V cm-1 were equivalent to 0 V cm-1, P>0.05), but speed toward the anode was decreased by the EF (P<0.05 at 0.6 V cm-1). Statistical significance, and *, shown as in (A). (D) MT cytoskeleton reorients toward wound edge with and against the EF. 3T3 cell monolayers treated as in (C) were placed in an EF (0.6 V cm-1) 0.5-2.5 hours after wounding. (a,b) Glu and (c,d) total tubulin antibody staining of cells migrating against (a,c) or with (b,d) the EF shows that cells oriented MTOCs (white arrows) and stable Glu MTs towards the wound edge, regardless of their directional velocity (C) and of whether they faced the cathode or anode. Short black arrows show the wound edge for each pair of micrographs; long black arrow shows the EF direction in a-d. Scale bar, 10 µm.
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