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Figure 4


Fig. 4. PtdIns(4,5)P2 depletion affects axoneme outgrowth. (A) Diagrams showing Unc-GFP (green) localization in developing spermatids. (i) Unc-GFP is found at the basal body in early round spermatids (arrow). Nucleus (white circle) and mitochondrial derivatives (black circle), which are wrapped together at this stage, are shown. (ii-iv) Elongating spermatids. (ii) Protein body stage. (iii) Later stage of elongation. (iv) Late stage of elongation showing nuclear shaping. During elongation, Unc-GFP is found at the basal body (iv, arrow) and at the elongating end (iv, arrowhead). Not to scale. (B-H') Phase-contrast (B-H) and corresponding fluorescence (B'-H') micrographs of spermatids expressing Unc-GFP (green) and stained for DNA (blue). (B,D,F) Wild-type spermatids. (B') Unc-GFP (arrow) is present at the basal body of early round spermatids. (D') As spermatids elongate, Unc-GFP signals split into two parts, a cylindrical structure at the basal body (arrows) and a prominent dot associated with the growing end (arrowheads). In addition, a weak Unc-GFP signal can be seen along the entire length of the elongating sperm tail (inset, between arrow and arrowhead). (F') In later stages, Unc-GFP signals are clearly observed at opposite ends of the elongating cyst [arrow, nuclei (mostly on a different focal plane); arrowhead, growing end]. Note that the contrast of this image was increased by 30% to make the localization of Unc-GFP more obvious. (C,E,G,H) Spermatids expressing SigD. (C') Unc-GFP localization appears comet-shaped (arrow) in SigD-expressing spermatids. Association of Unc-GFP with nuclear DNA is also disrupted. (E') In a rare elongated SigD-expressing cyst, Unc-GFP splits into two parts (arrow, arrowhead), and a weak signal along the sperm tail can be seen. (G') SigD cyst at the protein body stage. Note the failure of Unc-GFP to split into two signals. (H') Later-stage cyst with Unc-GFP signals scattered in the cytoplasm. Scale bars: 10 µm (B,C,G), 20 µm (D,E), 50 µm (F,H). Inset in D is approximately the same scale as G.