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The Cell Cycle Timing of Centromeric Chromatin Assembly in Drosophila Meiosis Is Distinct from Mitosis Yet Requires CAL1 and CENP-C

Figure 2

CID assembly in male meiosis I.

(A) Spermatogenesis in Drosophila males. At the tip of the testes at the germinal proliferation center, a single germline stem cell divides mitotically producing a primary spermatogonial cell [62]. Primary spermatogonia complete four mitotic divisions and generate a cyst of 16 primary spermatocytes, which then replicate their DNA and undergo a 25-fold increase in volume in prophase I of meiosis, which is a developmentally specialized extended G2 phase (stages S1 to S6). At late prophase I of meiosis (M1), DNA condenses into three distinct domains, each corresponding to an autosomal pair. In the first meiotic division, each cell in a 16 cell cyst divides synchronously to form a cyst of 32 secondary spermatocytes (M4–M9). In the second meiotic division, secondary spermatocytes divide again to form a cyst of 64 spermatids (M10–M11). Further maturation and differentiation of spermatids (T1–T5+) over a period of days give rise to mature spermatozoa [38]. Standard nomenclature used is described in Cenci et al. [38]. (B) Changes in the amount of CID at centromeres during meiosis I. Larval testes were fixed and stained with anti-CID antibody (green), and DNA is stained with DAPI (red). CID localization in primary spermatocytes at stages S1, S4, S5, S6, and M1a of meiosis I and stage M4 of interphase II are shown. Outlines of nuclei are circled in white. Scale bar: 5 µM. (C) Quantification of total centromeric CID fluorescence intensity per nucleus in primary spermatocytes during stages S1 to S6 (prophase I), M1a–M1b (late prophase/early prometaphase I), and stage M4 (interphase II). Bars, standard errors. N = 157 total cells. N = 48, S1; 42, S4; 18, S5; 17, S6; 17, M1a–b; 32, M4a–b. Note that Figure 2C and 4B are from the same experiment and to the same scale, normalized to the initial S1 average intensity value. (D) Live imaging of primary spermatocytes expressing GFP-CID (green) and H2Av-RFP (red) at stages S1, S4, S6, and M1b (late prophase/early prometaphase) and M4 (interphase II) of meiosis I. Outlines of nuclei are circled in white. Scale bar: 15 µM. (E) Quantification of total centromeric GFP-CID fluorescence intensity per nucleus during S1 (n = 20), S4 (n = 18), and S6 (n = 26) of prophase I, M1a–M1b (late prophase/early prometaphase, n = 18), and M4a–M4b (interphase II, n = 23). Bars, standard errors. (F) Live imaging of a primary spermatocyte expressing GFP-CID (green) and H2Av-RFP (red) at early prometaphase of meiosis I (M1b). Time elapsed is in minutes. White arrows indicate centromeres shown in enlarged windows. Scale bar: 15 µM.

Figure 2