Brown Nat Protocols Fig 2

Figure 2 | Comparison of hybridization strategies. a, Hybridization efficiencies plotted as the percentage of signal scored (mean with SD) in C127 cells for probes pEx and pCx36 (red and green respectively). As a lack of hybridization signals was occasionally noted in RASER-FISH samples, owing to incomplete BrdU labelling, we assessed hybridization efficiency as follows: on an allelic basis, the presence of a single signal (either green or red) deemed that allele as scorable. At that same allele, the presence (or absence) of the neighbouring signal was recorded. Hybridization frequencies were expressed as number of alleles with both red and green signals / number of all scorable alleles ×100. The hybridization frequencies shown are calculated from 501 (4 min), 715 (4 min+dry), 755 (RASER) and 218 (30 min+dry) alleles. b, Comparison of single-strandedness and nuclear integrity. Single-stranded DNA detected by antibody in example HeLa and C127 cells after simple immunofluorescence (IF control) or the three FISH methods, 4 min, 4 min+dry, RASER. Upper panels show the ssDNA antibody labelling signal (green/white). Scale bar, 5 µm. Lower panels show an expanded view of the nuclear periphery of the same cells with the ssDNA signal (green) against a DAPI counterstain (purple). The disruption to nuclear integrity in the heat-denatured samples is evident. Scale bar, 1 µm. c, Comparison of access to blocks of DNA repeats. Example C127 nuclei hybridized by the three FISH methods with a probe to gamma satellite DNA repeats (green) against a DAPI counterstain, all imaged with the same settings. RASER-FISH provides the most comprehensive labelling. Scale bar, 5 µm.</p

Degradation of cyclin Cln3 by exceeding CENs: Mad3 physical and functional interactions with SCF.

(A) Analysis of Cln3 stability by promoter shut-off experiments in the presence (orange circles) or absence (gray circles) of two YCp–CENGALp vectors in wild-type cells grown under permissive conditions. After tetracycline addition, cells were collected at the indicated times, and obtained Cln3–6FLAG levels are plotted relative to an unspecific cross-reacting band (asterisk) used as loading control. (B) Analysis of Cln3 stability in Mad3-deficient cells as in (A). (C) Analysis of mCitrine–Cln3–11A stability by time-lapse microscopy in the presence (orange circles) or absence (gray circles) of three YCp vectors. Nuclear levels of mCitrine–Cln3–11A in cells were determined at the indicated times after cycloheximide addition, and mean values (N = 100) are plotted. (D) Analysis of mCitrine–Cln3–11A accumulation in the nucleus in the presence (orange circles) or absence (gray circles) of three YCp vectors. Nuclear levels of mCitrine–Cln3–11A were determined in G1 daughter cells with 50–60 μm3 of volume. Individual data (N = 90) and median values are plotted. (E) Cell extracts (input) and GST PDs of cdc4ts grr1 cells expressing Cln3–13myc and GST fusions to Cdc4ΔFbox, Mad3, or Mad3ΔGLEBS were analyzed by immunoblotting with either αmyc (top panels) or αGST (bottom panel) antibodies. (F) Cell extracts (input) and GST PDs of cells expressing Mad3–3HA or Mad3 ΔGLEBS–3HA and either GST or GST–Cdc4 were analyzed by immunoblotting with either αHA (top panels) or αGST (bottom panel) antibodies. (G) Cells with the indicated genotypes carrying three YCp vectors were analyzed as in Fig 1B to determine cell size at budding as a function of copy number. Individual budding volumes (small dots) were binned, and mean values (large circles, N = 50) and a regression line are plotted. Correlation analysis and pairwise comparisons were performed with nonparametric tests as described in Materials and methods. Underlying data can be found in S1 Data. GST, glutathione S-transferase; PD, pulldown; YCp, yeast centromeric plasmid.</p

Exceeding CENs require centromeric Mad3/Bub3 signaling proteins to modulate cell size.

(A) Cells with the indicated genotypes carrying three YCp vectors were analyzed as in Fig 1B to determine cell size at budding as a function of copy number. Individual budding volumes (small dots) were binned, and mean values (large circles, N = 50) and a regression line are plotted. (B) Newborn daughter cells with the indicated genotypes were analyzed to determine cell size at budding. Mad3 overexpression (oMAD3) was attained by inducing a GAL1p–MAD3 construct with 1 mM estradiol in newborn cells expressing the Gal4–hER–VP16 transactivator. Individual data (N > 300) and median values are plotted. Correlation analysis and pairwise comparisons were performed with nonparametric tests as described in Materials and methods. Underlying data can be found in S1 Data. CEN, centromere; YCp, yeast centromeric plasmid.</p

Brown Nat Protocols Fig 3b

Figure 3 | Comparison of chromatin structure and TAD shapes. Super-resolution 3D-SIM imaging of human RPE-1 cells after a control IF treatment or indicated FISH protocol using a KIF23 TAD-specific probe. b, Exemplary TAD FISH signals selected from several cells highlight more defined and distinct edges of the TAD signal after RASER detection compared to 4 min, 4 min+dry (bottom row of middle section) and 30 min+dry heat denaturation. Images show false colour representations of maximum intensity projections. Scale bar, 1 µm

Brown Nat Protocols Fig 2b

2b, Comparison of single-strandedness and nuclear integrity. Single-stranded DNA detected by antibody in example HeLa and C127 cells after simple immunofluorescence (IF control) or the three FISH methods, 4 min, 4 min+dry, RASER. Upper panels show the ssDNA antibody labelling signal (green/white). Scale bar, 5 µm. Lower panels show an expanded view of the nuclear periphery of the same cells with the ssDNA signal (green) against a DAPI counterstain (purple). The disruption to nuclear integrity in the heat-denatured samples is evident. Scale bar, 1 µm

Brown Nat Protocols Fig 2c

2c, Comparison of access to blocks of DNA repeats. Example C127 nuclei hybridized by the three FISH methods with a probe to gamma satellite DNA repeats (green) against a DAPI counterstain, all imaged with the same settings. RASER-FISH provides the most comprehensive labelling. Scale bar, 5 µm.</p

Brown Nat Protocols Fig 2a

Figure 2 | Comparison of hybridization strategies. a, Hybridization efficiencies plotted as the percentage of signal scored (mean with SD) in C127 cells for probes pEx and pCx34 (red and green respectively). As a lack of hybridization signals was occasionally noted in RASER-FISH samples, owing to incomplete BrdU labelling, we assessed hybridization efficiency as follows: on an allelic basis, the presence of a single signal (either green or red) deemed that allele as scorable. At that same allele, the presence (or absence) of the neighbouring signal was recorded. Hybridization frequencies were expressed as number of alleles with both red and green signals / number of all scorable alleles ×100. The hybridization frequencies shown are calculated from 501 (4 min), 715 (4 min+dry), 755 (RASER) and 218 (30 min+dry) alleles

Brown Nat Protocols Fig 6

Figure 6 | RASER-FISH hybridization examples with inset magnifications. a, Deconvolved widefield image of plasmids pCx (green) and pEx (red) from the α-globin gene region36 hybridized to C127 nucleus detected with DAPI. b, Fosmids recognising NKX2 (green) and Pax2 (red) hybridized to mouse ES cells39 with nuclei detected with DAPI. c, BAC RP24-217l105 (red) hybridized to C127 nucleus with chromatin stained with SYTOX Green (grey), imaged by 3D-SIM. Orthogonal (top) and lateral (bottom) cross sections are shown. d, 3D-SIM image of a C127 nucleus hybridized with 6 pools of oligonucleotide probes directly labelled with Atto 565, Abberior Star Red and Oregon Green and covering 1030 kb of the α-globin gene region37. Maximum projection with the nuclear boundary defined by DAPI outlined (DAPI not shown). e, 3D-STED image (maximum intensity projection) of a mouse erythroblast nucleus hybridized with 3 pools of oligonucleotide probes directly labelled with Atto 565, Abberior Star Red and Oregon Green and covering 78.5 kb of the α-globin gene region36. (f) RNA-DNA RASER-FISH image of a mouse erythroblast showing the α-globin genes detected with plasmid pA36 (red) against nascent α-globin transcripts54 (green) imaged by widefield deconvolution (maximum intensity projection). g-i, Immuno-RASER-FISH examples imaged by widefield deconvolution. g, Antibody detection of HP1α (red) combined with a plasmid probe pCx (green) (Brown 2018) detected in a DAPI-stained C127 cell nucleus (maximum intensity projection covering the central region). h, Immuno-RASER-FISH image of an antibody to fibrillarin (detecting nucleoli) (red) combined with a plasmid probe pCx36 (green) detected in a DAPI-stained C127 cell nucleus. i, Immuno-RASER-FISH image of an antibody to 53BP1 (red) combined with a BAC RP11-347N18 probe partly covering the KIF23 TAD38 (green) detected in a DAPI-stained U2OS cell nucleus (maximum intensity projection covering the central region). Scale bars are 5 µm and 1 µm (insets).</p

Centromeric signaling proteins boost G1 cyclin degradation and modulate cell size in budding yeast

<div><p>Cell size scales with ploidy in a great range of eukaryotes, but the underlying mechanisms remain unknown. Using various orthogonal single-cell approaches, we show that cell size increases linearly with centromere (CEN) copy number in budding yeast. This effect is due to a G1 delay mediated by increased degradation of Cln3, the most upstream G1 cyclin acting at Start, and specific centromeric signaling proteins, namely Mad3 and Bub3. Mad3 binds both Cln3 and Cdc4, the adaptor component of the Skp1/Cul1/F-box (SCF) complex that targets Cln3 for degradation, these interactions being essential for the CEN-dosage dependent effects on cell size. Our results reveal a pathway that modulates cell size as a function of CEN number, and we speculate that, in cooperation with other CEN-independent mechanisms, it could assist the cell to attain efficient mass/ploidy ratios.</p></div

CEN signaling proteins cooperate with SCF–Cdc4 to enhance degradation of the yeast G1 cyclin and modulate cell size in budding yeast.

When present in excess, centromeres accelerate Cln3 degradation in the nucleus with the essential participation of Mad3, a centromeric signaling protein that interacts with Cdc4 and requires SCF function to modulate cell size as a function of centromere number. CEN, centromere.</p