Shugoshin Prevents Dissociation of Cohesin from Centromeres During Mitosis in Vertebrate Cells (original) (raw)
Figure 8
Expression of Nonphosphorylatable Scc3-SA2 Suppresses Sgo1-Depletion Phenotype
(A) Total cell extracts prepared from HeLa cells that, upon doxycycline treatment, inducibly express either a wild-type (SA2-wt) or nonphosphorylatable (SA2–12xA) myc-tagged version of Scc3-SA2 were resolved by SDS-PAGE and probed with anti-SA2 antibody. The lower band represents endogenous Scc3-SA2, the upper band, myc-tagged Scc3-SA2. The first lane contains total cell extract prepared from untagged HeLa cells.
(B) Expression of nonphosphorylatable Scc3-SA2 suppresses sister separation caused by Sgo1 depletion. Cycling HeLa cells carrying inducible myc-tagged versions of Scc3-SA2 were treated with (or without) doxycycline at 2 μg/ml for 72 h prior to and during transfection to induce expression, as indicated. Cells were harvested 18 h and 24 h post transfection, chromosomes were spread on glass slides and Giemsa-stained. As in Figure 7, the percentage of mitotic cells were calculated out of 200 cells, and mitotic chromosome spreads were then further classified into one of five categories (n = 200 mitotic spreads for each time point). The frequency of each category of mitotic cell (see examples in Figures 3B, 7B, and 7D) is given as a percentage of total cell numbers, such that the sum of each column represents the mitotic index.
(C) Nonphosphorylatable Scc3-SA2-mediated suppression is still observed when combined with nocodazole arrest. The indicated treatments were repeated as outlined in (B) in the presence of nocodazole which was added to cultures 4 h posttransfection, i.e., 14 and 20 h prior to harvesting of cells. Samples were processed as in (B), with the addition of a sixth mitotic category.
(D) Live cell analysis of Sgo1 depletion of nonphosphorylatable Scc3-SA2 (SA2–12xA) expressing cells. To follow chromosome behaviour, cells stably expressing EGFP-H2B and inducibly expressing SA2–12xA were used. Expression of SA2–12xA was induced 72 h prior to transfection as in (B). Cells were transfected with Sgo1 siRNA as in Figure 1A and examined by time-lapse fluorescence microscopy. Significant number of cells exit mitosis by expressing nonphosphorylatable Scc3-SA2.
(E) Nonphosphorylatable SA2 (SA2–12xA) is found at centromeres even in the absence of Sgo1. HeLa cells containing either the myc-tagged wild-type (a) or SA2–12xA (b) inducible transgene were either uninduced or induced as in (B) 72 h before transfection. Transfection of Sgo1 siRNA was performed prior to the second thymidine block as in Figure 1A. At 8.5 h after the release from early S phase, mitotic cells were spun down on glass slides and analysed for cohesin localisation by immunofluorescence microscopy using antibodies to the myc epitope (shown in green). Cells were costained with CREST antiserum to label kinetochores (shown in red) DNA was counterstained with DAPI (shown in blue).
(F) Quantification of SA2-myc staining. Samples similar to those described in (E) were stained with myc and P-H3 antibodies (the latter to identify cells from prophase to metaphase). Approximately 200 P-H3-positive cells were assessed for SA2-myc staining, and the percentage of cells that were both P-H3- and SA2-myc-positive was plotted. We believe that the apparent drop in the number of SA2–12xA-myc positive cells observed with depletion of Sgo1 relative to mock transfection is a statistical artefact caused by the mitotic arrest and accumulation of those cells that did not express SA2–12xA-myc (∼30%) but that were depleted of Sgo1.