Supporting data pertaining to two distinct nuclear basket remodeling events during budding yeast meiosis. (A) Montage of a cell with Seh1-GFP, a Y-complex nucleoporin, and Htb1-mCherry, a histone, progressing through meiosis (UB24613). The onset of anaphase II was defined by the presence of four Htb1-mCherry lobes. (B and C) Montages of cells containing FKBP12-Mlp1-GFP and Seh1-FRB, treated with either (B) DMSO or (C) 10 μM rapamycin after 4 h in SPM (UB29337). (D) Montages of cells with different fluorescently tagged basket nucleoporins—Nup60-GFP (UB30174), Nup2-GFP (UB30168), and Nup1-GFP (UB30166)—and the inducible Mlp1 tether (FKBP12-Mlp1 and Seh1-FRB) treated with 10 μM rapamycin after 4 h in SPM. For B–D, the transmembrane nucleoporin Pom34-mCherry was used to monitor the core of the NPC, with the GUNC indicated by a white box. The onset of anaphase II was defined as the first time point with GUNC formation. All cells were fpr1Δ to facilitate rapamycin access to the tether. (E) A schematic depicting RITE (Verzijlbergen et al., 2010), a technique that facilitates differentiation between pre-existing and newly synthesized protein pools. Nup2 was tagged with a RITE cassette (Nup2-RITE), allowing for genetically encoded tag switching upon induction of Cre recombinase. After tag exchange, any GFP-tagged Nup2 represents a protein that was present prior to the conversion event. Tag exchange was induced with over 95% efficiency prior to meiotic induction, as assayed by loss of hygromycin resistance. Tetrad end-point analysis of Nup2-GFP signal intensity allowed individual cells to be scored for successful tag exchange by microscopy. (F and G) Montages of cells containing Nup2-RITE, a basket nucleoporin, and Spc42-mCherry, a spindle pole body (SPB) component, progressing through meiosis. (F) No Cre-EBD present, such that GFP-tagged protein represents either pre-existing or newly synthesized Nup2 (UB34454). (G) Cre-EBD present and induced, such that GFP-tagged protein represents only pre-existing Nup2 (UB34452). The onset of anaphase I was defined as the Nup2-GFP signal exhibiting distortion from a spherical shape consistent with chromosome segregation. Note that GFP intensity was not scaled identically in F and G, and that the selected RFP imaging settings did not allow for robust detection of newly synthesized Nup2-mCherry. Scale bars, 2 μm.
Figure S3.

Supporting data pertaining to two distinct nuclear basket remodeling events during budding yeast meiosis. (A) Montage of a cell with Seh1-GFP, a Y-complex nucleoporin, and Htb1-mCherry, a histone, progressing through meiosis (UB24613). The onset of anaphase II was defined by the presence of four Htb1-mCherry lobes. (B and C) Montages of cells containing FKBP12-Mlp1-GFP and Seh1-FRB, treated with either (B) DMSO or (C) 10 μM rapamycin after 4 h in SPM (UB29337). (D) Montages of cells with different fluorescently tagged basket nucleoporins—Nup60-GFP (UB30174), Nup2-GFP (UB30168), and Nup1-GFP (UB30166)—and the inducible Mlp1 tether (FKBP12-Mlp1 and Seh1-FRB) treated with 10 μM rapamycin after 4 h in SPM. For B–D, the transmembrane nucleoporin Pom34-mCherry was used to monitor the core of the NPC, with the GUNC indicated by a white box. The onset of anaphase II was defined as the first time point with GUNC formation. All cells were fpr1Δ to facilitate rapamycin access to the tether. (E) A schematic depicting RITE (Verzijlbergen et al., 2010), a technique that facilitates differentiation between pre-existing and newly synthesized protein pools. Nup2 was tagged with a RITE cassette (Nup2-RITE), allowing for genetically encoded tag switching upon induction of Cre recombinase. After tag exchange, any GFP-tagged Nup2 represents a protein that was present prior to the conversion event. Tag exchange was induced with over 95% efficiency prior to meiotic induction, as assayed by loss of hygromycin resistance. Tetrad end-point analysis of Nup2-GFP signal intensity allowed individual cells to be scored for successful tag exchange by microscopy. (F and G) Montages of cells containing Nup2-RITE, a basket nucleoporin, and Spc42-mCherry, a spindle pole body (SPB) component, progressing through meiosis. (F) No Cre-EBD present, such that GFP-tagged protein represents either pre-existing or newly synthesized Nup2 (UB34454). (G) Cre-EBD present and induced, such that GFP-tagged protein represents only pre-existing Nup2 (UB34452). The onset of anaphase I was defined as the Nup2-GFP signal exhibiting distortion from a spherical shape consistent with chromosome segregation. Note that GFP intensity was not scaled identically in F and G, and that the selected RFP imaging settings did not allow for robust detection of newly synthesized Nup2-mCherry. Scale bars, 2 μm.

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