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Fig. 1. Localization of condensin I and II during the cell cycle. (A-E) Analysis of NRK cells stably expressing EGFP–Flag–Kleisin-
. (A) Incorporation of ectopically expressed EGFP–Flag–Kleisin- into condensin I complexes. Extracts of NRK cells expressing EGFP–Flag–Kleisin- (lane 1) or parental NRK cells (lane 2) were analyzed by SDS-PAGE and immunoblotting using antibodies to Kleisin- (upper panel), to GFP (middle panel) and to Smc4 (lower panel). The same extracts were also subjected to immunoprecipitations with antibodies to Flag-epitope (lane 3), Smc4 (lane 4), Kleisin- (lanes 5 and 7), or control IgG (lane 6) and analyzed by immunoblotting, as indicated. (B) Extracts of EGFP–Flag–Kleisin--expressing NRK cells were fractionated by 5-20% sucrose-density-gradient centrifugation. Fractions were analyzed by immunoblotting using antibodies against Kleisin-, Smc4 or a subunit of the proteasome. (C) Extracts of exponentially proliferating (log), hydroxyurea treated (HU) or nocodazole treated (Noc) NRK cells expressing EGFP–Flag–Kleisin- were separated by centrifugation into pellet (P) and supernatant (S). Endogenous and exogenous Kleisin- proteins were detected by immunoblotting using Kleisin- antibodies. EGFP–Flag–Kleisin- was expressed at approximately 10% of endogenous protein levels, estimated by quantifying the intensity of the bands. (D, E) Localization of EGFP-Kleisin- in live interphase and metaphase NRK cells. DNA was stained with Hoechst 33342 (red in overlay). (D) Mitotic time-lapse imaging of EGFP–Kleisin--expressing cells stained with Hoechst 33342 (red). Time from start of filming is indicated in minutes. Bar, 10 µm. (F) Localization of CAP-D2 and CAP-D3 by IF microscopy. Logarithmically proliferating HeLa cells were fixed with acetone-methanol solution, incubated with CAP-D2 and CAP-D3 antibodies and stained with Alexa 488 (upper panels). Merged signals of Alexa 488 (green) and DAPI (blue) are shown in the lower panels. (Left to right) Representative cells in interphase, prophase, prometaphase, metaphase, anaphase and telophase. (G) Smc2 is located on chromosomes in prophase. HeLa cells were fixed with 4% paraformaldehyde either after pre-extraction (c,d) or without preextraction (a, b), and stained with Smc2 antibodies (green). DNA was stained with DAPI (blue). Representative cells in interphase (a, c) and in prophase (b, d). Notice, staining of Smc2 on axial chromosome structures can be seen in prophase after pre-extraction, although condensed chromosomes are not seen as clearly after pre-extraction treatment.
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