Wounds induce epithelial syncytia via cell fusion.

A) Syncytia form within 2 h post wounding, evident by the clustering of multiple nuclei within cell borders. B) The number of nuclei per syncytia increases over time after wounding. Number of nuclei was estimated based on area and nuclear density (see text) for the 3 largest syncytia of 3 different wounds, mean and SD. C-D) Larger wounds generate larger syncytia. Images are at 3 h post wounding, single syncytium outlined. E) Apical area of three largest syncytia is proportional to initial wound size, mean and SD. F) A time course of six cells fusing within 30 min after wounding. Apical borders are lost (white arrowhead) as syncytia form. Original cells are numbered. G) All borders lost to cell fusion (white) mapped to cells in the first frame after wounding. The leading edge of wound closure will form at dashed line; cells within the shaded area were damaged by the wound and will be dismantled. H) Distance from the wound center vs time for all border breakdowns in 3 wounds. Each symbol represents a cell border. Leading-edge locations indicated by solid lines. I) Cytoplasmic GFP is expressed in cell 1 before wounding and mixes with neighboring cells 2-4 by 2 min after wounding. Cytoplasmic sharing is followed by the lagging fusion indicator of visible border breakdown (white arrow). The fates of cells 3 and 4 are shown at later times in Fig. 2D. Maximum intensity projections in A, C-D, G; single Z slices in F, I. Scales: A-D,G = 20 µm, F,I = 10 µm. W and red star indicate wound.

Cell fusion often appears as cell shrinking.

A) An epithelial cell (yellow arrow) shrinks in the epithelial plane after wounding. (B) Same sample as A, showing the shrinking cell’s nucleus (asterisk) entering a neighboring syncytium, outlined in Biv. C) Cell 1 expresses actin-GFP before wounding. After wounding, cell 1 fuses with cells 2-6 evidenced by GFP sharing, then cell 1 shrinks. D) Two cells shrink in the X-Y plane (white and yellow arrows in Di-Diii). X-Z projections are shown for each (Div-Dvi for the top yellow line; Dvii-Dix for the lower white line). Rather than extrude, the shrinking cells’ cytoplasm moves to the right, to join with the neighboring wound-proximal syncytium, via basal connections. These frames are a continuation of the sample shown in Fig. 1I. E) All shrinking cells mapped to first frame after wounding. F) All shrinking cells and border breakdowns were tracked in 3 wounds. Border breakdown fusions happen sooner after wounding than shrinking fusions. A, B, C, Di - iii, E show maximum intensity projections. Div − ix show X-Z projections. Scales: A, B, C, Di-iii, E = 10 µm; Div - ix = 5 µm.

Half the cells near the wound fuse to form syncytia, demonstrated by tracking individual cell fates.

A) All GFP labeled cells in the region of fusion (80 µm) were tracked in 5 wounds over 6.5 h after wounding to determine frequency of fusion (GFP mixing) or persistence (no GFP mixing). Untrackable cells lost GFP, see Fig. S2A. B) Fusion is common within 70 µm. C) Shrinking-fusion and border-breakdown fusion occur at similar distances from the wound.

Syncytia outpace smaller cells.

A) Before wounding, two clusters of cells are labeled with GFP, 1-2 and 3-4 (white numbers). After wounding, cells 2 and 3 fuse with neighbors 5-9 to form a syncytium (dashed leading edge in Aiii), which advances toward the wound, passing unfused cell 4 (arrow). B-C) Unfused cell (outlined in orange and white) with corresponding nucleus (asterisk in C) is replaced at leading edge by neighboring syncytia (S and yellow outline, yellow arrows). D) All small cells are excluded from leading edge by syncytia well before the wound closes 20-160 min later. E) Images of sample 1 from the graph D. Unfused cells (arrows) were tracked over the course of wound closure and mapped back to the 30 min timepoint. F) At 120 min, the last unfused cell is ejected from leading edge (arrow). The wound closed at 140 min. G) Percent of leading-edge perimeter occupied by syncytia increases to 100%. Images are single Z slices in Ai-Aii and maximum intensity projections in Aiii-F. Scale: A,E-F = 20 µm, B-C = 10 µm.

Radial border fusions reduce the number of wound proximal intercalations.

A) Illustration of tangential vs radial border breakdown. B) More tangential than radial borders break down after wounding. C) Radial border fusions reduce intercalation at a wound. D) Quantification of intercalations and fusions around the wounds of Fig. 4D,G.

Syncytia concentrate pooled resources at the leading edge.

Random scattered cells expressing Actin-GFP were generated by heat-shock mediated flip-out expression of Gal4. A-B) Labeled actin is expressed in cell 1 before wounding (Ai). Actin-GFP rapidly equilibrates between cells 1 and 2 by 28 minutes after wounding, demonstrating cytoplasmic fusion. At 180 min, cell 2 shrunk into cell 1. The resulting syncytiuym has no access to the leading edge, and actin remains equilibrated, as shown in the kymograph (Biii) generated from actin-GFP intensity over time at the yellow line in Bii. C-D) Labeled actin is expressed in cell 1 before wounding (Ci) and equilibrates between cells 1 and 2 by 6 minutes after wounding, demonstrating cytoplasmic fusion (Cii). The resulting syncytium contacts the leading edge, and by 28 min after wounding actin from cell 1 is redistributed to the wound margin by 28 min after wounding, as shown in the kymograph (Div) of actin intensity over time at the yellow line in Diii. At 55 min after wounding, cell 1 shrunk into cell 2. E-F) Before wounding, actin-GFP in cell 1 is three cells away from the future leading edge. After wounding, fusion of cells 1-4 allows actin-GFP to move to and accumulate at the leading-edge actin cable. Border breakdown occurs between cells 1-2 (Ev) which later shrink into cells 3,4 (see Movie S4). G) Mean profile plot of actin-GFP comparing the syncytia in Fv at 30 min after wounding (dark line) with the cells in Fi before wounding (dotted line) demonstrating that nearly all actin-GFP has been redistributed to the leading edge from its starting position 20-30 µm away. Single Z slices for Ei - ii, Fi - ii; maximum intensity projections for A-D, Eiii - v, Fiii - v. Scale A-Bii, Ci-Diii = 5 µm, E-F = 10 µm.