Fig. 3: Pfs230, Pfs48/45, and Pfs230-Pfs48/45 subcellular localization in P. falciparum macrogametes and microgametes by confocal microscopy under live and/or fixed conditions.

a Live emerged macrogametes were labeled with Pfs230D1-specific mAbs (mAb) as listed at the left. To demonstrate the presence of Pfs230, macrogametes were fixed and labeled with Pfs230D1-specific rabbit polyclonal (p) Abs (Rbα230). The third column shows the same cells by differential interference contrast (DIC) microscopy. 4F12 reacted with the surfaces of live macrogametes, but that 5G3, 5H1, and 7G4 did not. b Live emerged macrogametes were labeled with pAbs (Rbα230) then fixed and stained with the same Pfs230D1-specific mAbs as panel A. After fixation all mAbs reacted with the surface of the macrogametes. c Live microgametes were labeled with Pfs230D1-specific mAbs as listed at left. To demonstrate the presence of Pfs230, they were fixed and labeled with Pfs230D1-specific pAbs (Rbα230). 4F12 and 5H1 recognized live microgametes, unlike 3G2. d Fixed microgametes were labeled with pAbs (Rbα230) and with Pfs230D1-specific mAbs. After fixation, all mAbs recognize Pfs230. Only 4F12 recognized live macrogametes in A while both 4F12 and 5H1 recognized live microgametes in c. e Pfs230 and Pfs48/45 colocalize on the macrogamete surface membrane with live staining using the Pfs230-specific pAbs (RbαPfs230, green) and mAb 3E12 (MsαPfs48/45_3E12, red). f inset showing DIC of e. g Three-dimensional surface distribution of the Pfs230 and Pfs48/45 proteins. h Staining of wild-type (WT) NF54 emerged macrogametes labelled live with rabbit Pfs230D1-specific pAbs (green, RbαPfs230) and mouse Pfs48/45 domain 3 pAbs (red, MsαPfs48/45) show distinct staining patterns. At left, two macrogametes stain with Pfs230-specific mAb 4F12 (arrows). In the middle, only the lower of the two macrogametes is recognized by Pfs48/45-specific pAbs (arrows). At right, DIC shows the overlay of both stainings. i Pfs230 appears to be membrane associated without Pfs48/45. Live NF54 Pfs48/45 knockout (KO) macrogamete labels with rabbit Pfs230D1-specific pAbs (green, RbαPfs230), but is not labelled with mouse Pfs48/45 domain 3 pAbs (red, MsαPfs48/45). DIC shows both Abs overlaid. Scale bars in a–d and g represent 2 µm, h–i represent 3 µm, while in e and f represent 20 µm.