(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. Note that Figure 2C and 4B are from the same experiment and to the same scale, normalized to the initial S1 average intensity value. (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). CID localization in primary spermatocytes at stages S1, S4, S5, S6, and M1a of meiosis I and stage M4 of interphase II are shown. Larval testes were fixed and stained with anti-CID antibody (green), and DNA is stained with DAPI (red). (B) Changes in the amount of CID at centromeres during meiosis I. Standard nomenclature used is described in Cenci et al. Further maturation and differentiation of spermatids (T1–T5+) over a period of days give rise to mature spermatozoa. In the second meiotic division, secondary spermatocytes divide again to form a cyst of 64 spermatids (M10–M11). In the first meiotic division, each cell in a 16 cell cyst divides synchronously to form a cyst of 32 secondary spermatocytes (M4–M9). At late prophase I of meiosis (M1), DNA condenses into three distinct domains, each corresponding to an autosomal pair. 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 the tip of the testes at the germinal proliferation center, a single germline stem cell divides mitotically producing a primary spermatogonial cell. Time elapsed in minutes after anaphase onset is shown on the x-axis, and fold increase in total centromeric GFP-CID intensity per nucleus is shown on the y-axis. (D) Quantification of total centromeric GFP-CID fluorescence intensity per nucleus from live imaging of dividing nonstem brain cells in larvae ( n = 9 movies). Circle indicates the initiation of CID assembly between 6 and 12 min after anaphase onset.
(C) Live imaging of a nonstem brain cell from anaphase into early G1 phase expressing GFP-CID (green) and the chromatin marker H2Av-RFP (red). N = 171, interphase 35, prophase 52, metaphase 21, anaphase 39, telophase/early G1. Values are normalized to the interphase average. Condensed chromatin at metaphase and anaphase results in a reduction in antibody penetration.
(B) Quantification of total centromeric CID fluorescence intensity per nucleus in stages of mitosis in dividing nonstem brain cells. Larval brains were fixed and stained with anti-CID antibody (green) and DNA is stained with DAPI (red). (A) Changes in the amount of CID at centromeres during mitosis in nonstem brain cells. Cell cycle timing of CID assembly in mitotic tissues.
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