PI4P and Fwd colocalize on Golgi membranes. (A and B) Phase–contrast (phase) and corresponding fluorescence micrographs of live squashed wild-type or fwd mutant (fwd/Df) spermatocytes expressing RFP-PH-FAPP (PI4P) with Fws-GFP (A) or with GFP-Fwd or GFP-Fwd^KD (B). Colocalization (arrows) of Fws-GFP, GFP-Fwd, or GFP-Fwd^KD (green) and RFP-PH-FAPP (magenta) appears white (overlay). Bar, 20 µm. Note the diffuse RFP-PH-FAPP signal in fwd/Df (A) and GFP-Fwd^KD; fwd/Df (B) spermatocytes (bottom panels). (C) Phase–contrast (phase) and corresponding fluorescence micrographs of a squashed dividing spermatocyte coexpressing GFP-Fwd and RFP-PH-FAPP (PI4P). Note the presence of PI4P and absence of GFP-Fwd at the midzone (arrowheads). Bar, 20 µm. (D) Phase–contrast (phase) and corresponding fluorescence micrographs showing time-lapse images of a dividing spermatocyte expressing GFP-Fwd. Fluorescence micrographs are inverted for clarity. Times are in min:sec. GFP-Fwd is primarily in puncta at the poles (t = 08:00–20:00) and fails to accumulate at the midzone (arrows). Note background mitochondrial autofluorescence (elongated dark structures) due to long exposure times. Bar, 10 µm.