Centriole Remodeling during Spermiogenesis in Drosophila Atul Khire, Kyoung H. Jo, Dong Kong, Tara Akhshi, Stephanie Blachon, Anthony R. Cekic, Sarah Hynek, Andrew Ha, Jadranka Loncarek, Vito Mennella, Tomer Avidor-Reiss  Current Biology  Volume 26, Issue 23, Pages 3183-3189 (December 2016) DOI: 10.1016/j.cub.2016.07.006 Copyright © 2016 Elsevier Ltd Terms and Conditions

Current Biology 2016 26, 3183-3189DOI: (10.1016/j.cub.2016.07.006) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Poc1 Is Enriched in the Modified Sperm Centrioles (A) Poc1BGFP and Ana1tdTomato label early spermatids (EE), but only Poc1BGFP labels spermatozoa (S) centrioles. (B) Poc1BGFP is enriched during spermatogenesis whereas AslGFP and Ana1tdTomato are reduced. A, almost needle spermatid; C, spermatocyte; G, spermatogonia; R, round spermatid; S, spermatozoa. (C) In early elongated spermatid with round nucleus (N), the PCL with a central tubule (white arrow) is found near the GC inside the CA. (D) Correlative light (left) and electron microscopy (right) find that Ana1GFP labels the microtubule base GC and a novel PCL structure without microtubules in an intermediate elongated spermatid. (E and F) A model (E) and TEM (F) of PCL in intermediate-elongated spermatid. (G) TEM and a model of PCL in late spermatid. (H) Correlative light (last panel) and electron microscopy find that Poc1BGFP (gPoc1ABGFP) labels a modified PCL structure in the spermatozoon. (I) TEM and a model of PCL in spermatozoa. (J) Summary of GC and PCL proteins during spermiogenesis. -, undetectable; EE, elongated spermatid; IE, intermediate elongated spermatid; IN, initial spermatid; P, presence; R, round spermatid. See also Figure S1 and Movies S1 and S2. Current Biology 2016 26, 3183-3189DOI: (10.1016/j.cub.2016.07.006) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Poc1A and Poc1B Have Differential Roles in Centriole Formation and Sperm Motility (A) Poc1 codes for two isoforms, Poc1A and B, that share seven N-terminal-WD domains coded by exon 1 and a middle segment coded by exon 2 but differ in their C termini; coded by the end of exon 2 in Poc1A or exon 3 in Poc1B. Poc1 gene structure (black), the two Poc1 protein isoforms (green), the location of poc1 mutations (blue), and two anti-Poc1 antibodies (red) are shown. (B) poc1W87X and poc1c06059 have the shortest GC labeling, poc1k245 has mild shortening, and poc1W45R has no effect (n = 20). In these mutants, AslGFP and Ana2GFP labeled both the GC (square brackets) and PCL (line), but Ana1GFP/tdTomato selectively labeled the GC. (C) The GC (square brackets) and PCL (line) of poc1W45R sperm expressing gPoc1ABGFP have Ana1tdTomato-labeled PCL. ∗∗∗p < 0.001; ∗∗p < 0.01. (D) Maximum intensity projections and quantification of 3DSIM micrographs of sperm centrioles containing GFPAna1, Ana1GFP, and Poc1BGFP. p = 0.0001. Poc1BGFP and GFPAna1 (N-terminal Ana1) are found in the PCL center, whereas Ana1GFP (C-terminal Ana1) is found in the PCL periphery. (E) A TEM and model of PCL in intermediate-elongated spermatid of poc1W87X that has disorganized electron-dense wall and is missing the central tubule. See also Figure S2. Current Biology 2016 26, 3183-3189DOI: (10.1016/j.cub.2016.07.006) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Atypical Centrioles Are Essential for Normal Fertility and Embryogenesis (A) 0- to 3-min-old poc1W45Rpat embryos have abnormal PCL function at the decondensed male pronuclear (PN) stage. Whereas Cnn and Asl label both the GC and PCL of poc1W45Rpat embryos, D-PLP labels only the GC. (B) 0- to 3-min-old poc1pat embryos have abnormal centrosome function. Microtubules are not formed around the GC and PCL. (C) 0- to 3-min-old poc1W45Rpat embryos have monopolar spindles, whereas control and poc1W45Rpat zygotes expressing gPoc1ABGFP have bipolar spindles. (D) The pole of 0- to 3-min-old poc1W45Rpat zygotes has an Asl-labeled GC and maternally expressed Sas-6 tdTomato-labeled daughter centriole (DC). (E) Unlike 1-hr-old control embryos (cont) or poc1W45Rpat embryos expressing gPoc1ABGFP, poc1pat embryos have delayed embryo development. (F) 72-hr-old poc1W45Rpat embryos are arrested at cleavage stage with two to four nuclei and post-blastoderm stages (see representative figure). (G) Poc1pat embryos have reduced larval hatching. Mean ± SD; ∗∗∗p < 0.001. In all of these experiments, the female had WT Poc1. See also Figure S3. Current Biology 2016 26, 3183-3189DOI: (10.1016/j.cub.2016.07.006) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Paternal Poc1 Enrichment Is Essential for Normal Fertility and Embryogenesis (A) gPoc1ABGFP, cPoc1AGFP, or the combination of cPoc1AGFP and cPoc1BGFP (cPoc1ABGFP), but not cPoc1BGFP, expressed in poc1W87X restored sperm motility, indicating that Poc1A is essential for sperm axoneme architecture. (B) In poc1W87X mutants background, Poc1A and B label specifically the base of the GC and PCL, respectively, in the round spermatids, and Poc1B also labels the base of the GC in almost needle spermatids. (C) Both Poc1A and B are essential for normal GC length. (D) poc1W87Xpat embryos expressing various Poc1 constructs have differential effects on embryo development and spindle formation. Paternal Poc1B is essential for embryo development. (E) BamGAL4 expression of UASPoc1 RNAi affects the PCL, but not GC length. (F) Paternal BamGAL4 and UASPoc1 RNAi reduce early embryo development. (G) Interpretive model: during spermatogenesis, the centrosome composition changes, with the levels of some proteins decreasing (centrosome reduction) and others increasing (Poc1 enrichment). This remodeling eliminates the pericentriolar material (PCM) and produces centrioles with atypical structure, which after fertilization form atypical centrosomes by recruiting PCM. The atypical centrosomes are essential for the formation of typical daughter centrioles, pronuclear migration, zygotic cell division, and embryo development. The typical daughter centrioles form normal centrosomes at a later stage of embryogenesis. See also Figure S4. Current Biology 2016 26, 3183-3189DOI: (10.1016/j.cub.2016.07.006) Copyright © 2016 Elsevier Ltd Terms and Conditions