Figure 8 AFF-1::GFP protein dynamics during PVDdendrite regeneration. (A) Superresolution structured illumination microscopy image of alignedAFF-1::GFP (green) vesicles in the seam cells (left,white arrows) and AFF-1::GFP extracellular vesiclesderived from the seam cells (right, white arrowheads) in intact wild-type animals. c, cell body.PVD is labeled in red. (B) Spinning disk confocalimages of AFF-1::GFP expression inside and outsidethe seam cells (Se), and in the vulva (V) of an intactL4 animal (left). AFF-1::GFP (cyan) expression in theseam cells and in vesicles near the seam cells isshown in a two channels merged image (right,PVD in magenta; File S5). Inset shows magnificationof a single AFF-1::GFP vesicle near the seam cells(left, black arrowhead, numbered 1; File S6). (C)AFF-1::GFP expression inside and outside the seamcells (Se), in the anchor cell (AC), and in the vulva (V)of a reconnected PVD L4 animal (left) is shown 2-hrpostdendrotomy in a spinning disk confocal image.AFF-1::GFP is expressed in vesicles inside and outside of the seam cells, in the anchor cell (AC) and inVulA ring (see File S7; right, blue arrowhead, site ofreconnection). Inset shows magnification of areawith multiple vesicles, two are labeled (2 and 3)and shown in (D and E). (D) Two AFF-1::GFP vesicles, numbered 2 and 3, are moving near the seamcells in a PVD dendrotomized animal. Four timepoints are shown (0, 20, 60, and 80 sec; File S8).(E) Vesicle dynamics (1, 2, and 3) is demonstrated ina color-coded graph during 340 sec for vesicles2 and 3 and 620 sec for vesicle number 1. The plotillustrates vast differences between AFF-1::GFP vesicle movements in animals with an intact PVD vs.AFF-1::GFP vesicles in PVD dendrotomized animals(File S6 and File S8). (F) Dot plot of the number ofAFF-1::GFP vesicles outside
aff-1-expressing cells inanimals with intact PVD vs. in animals with dendrotomized PVD. The graph demonstrates statisticalsignificant difference between the mean numberof
aff-1::GFP vesicles outside
aff-1-expressing organs such as seam cells, vulva, anchor cells, andthe uterus. ***P = 0.001, statistics were calculatedusing the nonparametric Mann-Whitney test.