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Fig. 3. PtdIns(4,5)P₂ depletion affects peripheral basal body proteins. (A-C) Analysis of the core basal body protein GFP-PACT. (A,B) Fluorescence micrographs of (A) wild-type and (B) SigD-expressing spermatids, showing the morphology of GFP-PACT-containing basal bodies (arrows). Note that some SigD-expressing cells are multinucleate and contain four basal bodies (B, arrow). (C) Analysis of the length of the GFP-PACT signal in wild-type (wt) versus PtdIns(4,5)P₂-depleted (SigD) spermatids indicates no significant difference in the length of GFP-PACT-containing basal bodies (P=0.0957). (D-F) Fluorescence micrographs of spermatids stained for the basal-body-associated protein Centrosomin (Cnn, green, arrows) and dynein heavy chain (DHC, red, arrowheads). (D) In wild-type spermatids, Cnn localizes to puncta that are tightly associated with DHC in the nuclear envelope (arrow and arrowhead). (E) In SigD-expressing spermatids, Cnn localizes to diffuse comet-shaped structures that are not always tightly associated with the nuclear envelope (arrow). DHC localization appears normal (arrowhead). (F) Spermatids co-expressing SigD and Sktl exhibit normal DHC and Cnn localization. (G-I) Spermatids stained for -tubulin (-tub, red) and DNA (blue). (G) Early and late (inset) wild-type spermatids. Note that early spermatids have barely discernible -tub foci (arrow), whereas late spermatids exhibit prominent -tub puncta that closely associate with nuclei (one per nucleus). (H) Early and late (inset) SigD-expressing spermatids show aberrant -tub accumulation (arrow) and loose association of -tub and nuclei (inset). (I) Early and late (inset) spermatids expressing SigD and Sktl show restored -tub localization (arrow). Scale bars, 10 µm (A,B,D-F), 20 µm (G-I).

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