DOI: 10.5061/dryad.3r2280gpg
title: Sister chromatid cohesion is mediated by individual cohesin complexes

description: Eukaryotic genomes are organized by loop extrusion and sister chromatid
 cohesion, both mediated by the multimeric cohesin protein complex.
 Understanding how cohesin holds sister DNAs together, and how loss of
 cohesion causes age-related infertility in females, requires knowledge as
 to cohesin’s stoichiometry in vivo. Using quantitative super-resolution
 imaging, we identify two discrete populations of chromatin-bound cohesin
 in post-replicative human cells. While most complexes appear dimeric,
 cohesin localized to sites of sister chromatid cohesion and associated
 with Sororin is exclusively monomeric. The monomeric stoichiometry of
 Sororin:cohesin complexes demonstrates that sister chromatid cohesion is
 conferred by individual cohesin rings, a key prediction of the proposal
 that cohesion arises from their co-entrapment of sister DNAs.
description: Below is the information on how samples were stained and images
 acquired, sorted by microscopy technique.
 <em><strong>Immunofluorescence
 staining</strong></em> Cells were grown to
 80% confluency on 22x22 mm #1.5H high-precision coverslips (thickness
 0.170 ± 0.005 mm, Marienfeld Superior). Cells were washed in PBS,
 pre-extracted in ice-cold PBS 0.2% Triton X-100 (Sigma-Aldrich) for 1.5
 min on ice and fixed in 4% formaldehyde for 15 min. For non-pre-extracted
 samples, cells were fixed and subsequently permeabilized in PBS 0.2%
 Triton X-100 for 5 min. Primary and secondary antibodies were diluted in
 antibody diluent (DMEM medium containing 10% FBS, 0.05% sodium azide,
 sterile filtered 0.2 mm). Samples were incubated in primary antibody for
 1.5 h and for 1 h in secondary antibody solution supplemented with
 4’,6’-diamidino-2-phenylindole-dyhydrochloride (DAPI, 0.5 mg/ml). In
 between primary and secondary antibody staining, samples were washed 3x in
 PBS 0.2% Tween. After secondary antibody incubation, samples were washed
 3x again, samples were post-fixed in 4% formaldehyde for 15 min and
 samples were washed in PBS and distilled water. For QIBC, samples were
 mounted in Mowiol-based mounting medium (Mowiol 488
 (Calbiochem)/glycerol/Tris-HCl, pH 8.5) and in non-hardening Slowfade
 Diamond (Thermo Fisher Scientific, S36963) for Spinning Disk and 3D-SIM
 imaging. For EdU staining, cells were incubated with 10 mM EdU 20 min
 before pre-extraction and EdU detection was performed before primary
 antibody incubation according to the manufacturer’s instructions (Thermo
 Fisher Scientific). For detection of endogenously tagged proteins with
 chemical dyes, samples were incubated for 1.5 h with Halo- or 3 h with
 SNAP-ligands, washed three times in fresh media, and incubated for 30 min
 in the incubator in fresh media for exit wash of unbound dye. For
 experiments with two Halo dyes, cells were incubated with both dyes for
 1.5 h, then washed and chased with a third dye as indicated. For
 calibration purposes, U2OS cells were incubated with 100 nm 4-colour
 Microspheres (fluorescent blue, green, orange, dark red; Thermo Fisher
 Scientific) for 3 days at a density of 30 ml bead slurry / 2x105 cells.
 For G2 analysis, cells with highest mean intensities for Cyclin A (fixed
 cells) or sororin (pre-extracted cells) were chosen.
 <em><strong>DNA
 FISH</strong></em> Methanol-acetic
 acid–fixed cells were prepared and hybridized as described in (Brown et
 al., 2006). Hybridized slides were examined using a fluorescence
 microscope (Olympus BX60).  Hybridization efficiency was assessed by
 scoring FITC signals in ten metaphase spreads and only those
 hybridizations with greater than 80% efficiency were scored.  200 FITC
 signals were scored as either single or split dots for each
 hybridization.  The percentage of split dot values for each cosmid plotted
 represent the average of two independent biological
 experiments. 
 <em><strong>RASER-FISH</strong></em> RASER-FISH allows DNA sequence detection in intact 3D cell nuclei as it maintains fine-scale chromatin structure by replacement of heat denaturation with exonuclease III digestion after UV-generation of DNA nicks. RASER-FISH was carried out as described in (Brown et al., 2022; Ochs et al., 2019). In brief, U2OS cells were seeded on 22x22 mm #1.5H high-precision coverslips (thickness 0.170 ± 0.005 mm) and labelled for 18 h with 10 mM BrdU/BrdC mix (3:1). Cells were either pre-extracted and fixed as described above or fixed in 4% formaldehyde and permeabilized in PBS 0.2% Triton-X-100 for 5 min. Immunofluorescence staining for sororin or Cyclin A was carried out as described above. After incubation with 0.5 mg/ml DAPI for 15 min for UV sensitization, cells were treated with UV light (254 nm) for 15 min followed by incubation with 5 U/ml exonuclease III (NEB) at 37°C for 15 min. Biotin labelled probes were denatured in hybridization mix at 90°C for 10 min, pre-annealed with human Cot-1 DNA (Invitrogen) at 37°C for 15 min and hybridized to samples overnight at 39°C. Coverslips were washed twice in 1x SSC buffer at 37°C for 30 min, once in 1x SSC at room temperature, once in PBS, and biotin was detected by incubation with streptavidin-Alexa488 (1:500, Thermo Fisher). Samples were post-fixed, rinsed in PBS and MilliQ and mounted in Slowfade Diamond. For RAD21 degradation, cells harboring RAD21-Halo were incubated with 2.5 mM HaloPROTAC3 (Promega) for the last 8 h of BrdU/BrdC incubation. For dye mixing experiments, dye mixing was carried out as described above for the last 2 h of BrdU/BrdC incubation. The probes for all FISH assays in this study were obtained from a chromosome 16p specific cosmid library based on genome assembly NCBI36 (Stallings et al., 1990) and have been characterized in (Daniels et al., 2001) for mapping of the terminal 2 Mb of chromosome 16 p-arm. The GenBank accession number for the entire map is AE005175 and the specific probes used in this study have EMBL IDs HS306A4 (AL008727) and HS443D9 (Z92845). Probes were biotin-labelled. <em><strong>Quantitative image-based cytometry (QIBC)</strong></em> QIBC (Ochs et al., 2016; Toledo et al., 2013) was performed on a motorized Olympus IX83 inverted wide-field microscope equipped with Semrock DAPI/FITC/Cy3/Cy5 Quad LED filter set, Hamamatsu Orca Fusion B CMOS camera and Lumencor SPECTRA X light source. Automated unbiased image acquisition was carried out with the proprietary ScanR acquisition software. Identical exposure times were used for all samples within one experiment and settings were chosen for maximum dynamic range under non-saturating conditions. Most data was obtained with the Olympus UPLXAO 20x, NA 0.8 air objective. Depending on cell confluency and type of image analysis, 49-100 images were acquired, aiming for at least 2000 cells per analysis after gating. After acquisition, images were analyzed using the ScanR analysis software. DAPI signal was used for segmentation of nuclei based on intensity threshold. This nucleus mask was then used to quantify pixel intensities in the different channel for each individual nucleus. After segmentation and pixel quantification, measured parameters were extracted (mean and total intensities, area, circularity, well). For automated vermicelli quantification, edge detection was used, and 4 arbitrary classes were defined based on increasing edges (none, mild, moderate, and severe chromatin compaction). All data were exported to Tibco Spotfire, which was used to quantify average/median values in cell populations and to generate color-coded scatter plots in a flow-cytometry fashion, or to GraphPad Prism to generate stacked bar charts. <em><strong>Spinning disk microscopy</strong></em> Representative pseudo-confocal images were acquired with an UltraView Vox spinning-disk microscope (Perkin Elmer) and Volocity software (version 6.3.1) with a 60x, 1.42 NA Plan-Apochromat oil-immersion objective. Images were captured with a Hamamatsu EMCCD 16-bit camera at a spatial resolution of 121x250 nm. Single z-slices of whole nuclei are shown, brightness and contrast were linearly adjusted for optimal display. Color-channels were false-colored. <em><strong>Structured illumination microscopy (SIM)</strong></em> 3D-SIM images were acquired with a DeltaVision OMX SR system (GE Healthcare) equipped with a 60x, 1.5 NA UPLAPO60XOHR oil immersion objective (Olympus), pco.edge 4.2 sCMOs cameras (PCO), and 405 nm, 488 nm, 568 nm and 640 nm lasers. 3D image stacks were acquired over the whole nuclear volume in z with 15 raw images per plane (3 angles, 5 phases). Spherical aberration was minimized using immersion oil with refractive index 1.514 and an objective collar setting of 0.140 for image acquisition. Raw data was computationally reconstructed with SoftWoRx 7.2.0 (GE Healthcare) using channel-specific optical transfer functions (OTFs) recorded using immersion oil with RI 1.516 and Wiener filter setting 0.0030. All SIM data were routinely and meticulously quality-controlled for effective resolution and absence of artifacts using SIMcheck (Ball et al., 2015). Multichannel acquisitions were aligned in 3D with Chromagnon software (Matsuda et al., 2020) using 3D-SIM acquisitions of multicolor EdU-labelled C127 cells as colocalization reference. Images were thresholded based on the MCNR function of SIMcheck, which generates a metric of local stripe modulation contrast in different regions of the raw data and directly correlates this with the level of high-frequency information content in the reconstructed data. Only immunofluorescent signals with underlying MCNR values that exceeded a stringent quality threshold were considered for further analysis, while localizations with low underlying MCNR values were discarded. Thereby, any SIM signal, which falls below reconstruction confidence and is considered to be a labelling/imaging artifact, is excluded from further data interpretation (Ball et al., 2015). For representative images, single z-slices, cropped regions of whole nuclei or partial z-stacks are shown as indicated in figure legends. Brightness was linearly adjusted for optimal presentation. Channels were false-colored. <em><strong>HILO photobleaching</strong></em> Cells were seeded and labelled on 35 mm diameter, No. 1.5 MatTek dishes, pre-extracted and fixed as described above, and imaged in PBS. For photobleaching, a custom-built total internal reflection fluorescence (TIRF) microscope (described in detail in (Szczurek et al., 2023)) was used with a 100x 1.4 NA oil objective (Olympus) and with an iChrome MLE MultiLaser engine (Topica Photonics). Photobleaching was performed using highly inclined laminar optical sheet illumination (HILO). Emission was projected onto the central region of an iXon 897 EMCCD camera (Andor, 512x512 pixels). Pixel size in acquired images was 96 nm. Sample position and focus were controlled with a motorized stage and z-motor (ASI). For photobleaching samples were exposed to 30 % (RAD21-Halo) and 10-20% (sororin-SNAP) 561 nm laser and imaged using 100 ms camera interval for up to 1.5 min. Obtained images were analyzed with the Fiji plugin “Time Series Analyser” (https://imagej.nih.gov/ij/plugins/time-series.html) and background subtraction over time was performed using parallel background measurements.
description: # **Sister chromatid cohesion is mediated by individual cohesin
 complexes** \[Access this dataset on Dyrad] (DOI:10.5061/dryad.3r2280gpg)
 Eukaryotic genomes are organized by loop extrusion and sister chromatid
 cohesion, both mediated by the multimeric cohesin protein complex.
 Understanding how cohesin holds sister DNAs together, and how loss of
 cohesion causes age-related infertility in females, requires knowledge as
 to cohesin’s stoichiometry *in vivo*. Using quantitative super-resolution
 imaging, we identify two discrete populations of chromatin bound cohesin
 in post-replicative human cells. While most complexes appear dimeric,
 cohesin localized to sites of sister chromatid cohesion and associated
 with sororin is exclusively monomeric. The monomeric stoichiometry of
 sororin:cohesin complexes demonstrates that sister chromatid cohesion is
 conferred by individual cohesin rings, a key prediction of the proposal
 that cohesion arises from their co-entrapment of sister DNAs. ###
 Description of the data and file structure Imaging data is structured by
 figures and figure subpanels. **Fig.1 Chromatin bound cohesin exists in a
 monomeric and a dimeric state** **(A)** Schematic of 3D-SIM imaging
 approach of endogenously tagged RAD21 in G2 phase and on chromatin.
 **(B)** Representative widefield and super-resolution 3D-SIM images of
 chromatin bound RAD21-Halo. Single nucleus z-slice shown or copped region.
 False coloring by RAD21-Halo signal intensity shows different cohesin
 populations. **(C)** Frequency distribution of chromatin bound RAD21-Halo
 signal volumes with fitted non-linear regressions, n&gt; 60.000,
 RAD21-Halo signals from 2 independent biological experiments. **(D)**
 Sub-labeling of chromatin bound RAD21-Halo with 50 pM JFX554 dye to
 achieve single molecule labeling, n&gt; 900 RAD21-Halo signals. **(E)**
 Photobleaching of sub-labelled RAD21-Halo, example single-step bleach
 curve is shown corrected for background signal. Quantification in **Fig.
 S1C**. **(F)** Frequency distribution of single RAD21-Halo molecule
 volumes obtained from **(D)** and **(E)** was overlaid with frequency
 distribution of chromatin bound cohesin volumes as shown in **(C)**.
 Quantification of absolute distributions of chromatin bound cohesin
 yielded the following frequencies: 22% monomer, 61% dimer, 17% trimer.
 Quantification is based on non-linear regression analysis. Experiments
 were carried out in two biological replicates (**B-G**). File titles
 contain information on the experimental day, treatment, staining,
 replicate, image number, and processing steps as: DATE_TREATMENT_STAINING
 (488)_STAINING (568)_STAINING (647)_REPLICATE_IMAGE NUMBER_PROCESSING
 STEPS.tif. An exception is Fig.1E, where the file is labelled as: EXPOSURE
 TIME_LASER POWER_STAINING_IMAGE NUMBER.tif. **Fig.3 Cohesin complexes
 associated with sororin specifically mark sites of sister chromatid
 cohesion** **(A)** Schematic depiction of RASER-FISH approach for imaging
 of sites of sister chromatid cohesion. Two possible outcomes are shown;
 the cohesed sister chromatids are visible as one FISH signal or
 non-cohesed sister chromatids are visible as “split dots” due to their
 spatial separation. This figure was made with Biorender.com. **(B)**
 Frequencies of “split dots” (separated sister chromatids) measured by FISH
 and conventional microscopy in G2 cells. Probe locations are midpoints of
 cosmids in kb from chromosome 16 p telomere. TA; human lymphoblastoid cell
 line established from a normal individual, MEJY JY5.4; mouse
 erythroleukaemia hybrid cell line containing a normal human chromosome 16.
 N&gt; 200 alleles analyzed per cosmid, averaged from 2 independent
 biological repeats. **(C)** RASER-FISH outcomes of two probes, marking
 sites of sister chromatid cohesion are shown, ether in wildtype conditions
 or conditions of cohesin loss induced by PROTAC3-mediated degradation of
 RAD21 (8h treatment). G2 cells were identified by sororin mean intensity
 (wild type) or Cyclin A mean intensity (no cohesin). Example images of G2
 cells shown (left) and quantification shown (right). Experiments were
 carried out in biological duplicates. N=57 HS443D9 WT, n=43 HSD443D9 no
 cohesin, n=28 HS306A4 WT and n=31 HS306A4 no cohesin. Kruskal-Wallis test
 yielded p=0.0095, mean and standard deviation (SD) are plotted. **(D)**
 Co-localization of sororin to cohesed (top) and non-cohesed (bottom) sites
 and absolute quantification of sororin occupancy of the respective sites.
 Samples were prepared under pre-extraction conditions to visualize
 sororin, which maintained cohesion. Experiments were carried out in
 biological duplicates. N=48 HS443D9 (top), n=28 HSD443D9 (bottom), n=31
 HS306A4 (top) and n=20 HS306A4 (bottom). Kruskal-Wallis test yielded
 p=0.0075 (top) and p=0.0086 (bottom), mean and SD are plotted. **(E)**
 Distance analysis between sister chromatids (distance between intensity
 maxima) at sites occupied by or void of sororin. Experiments were carried
 out in biological duplicates and n=49 for sororin positive sites and n=55
 for sororin negative sites. Mann-Whitney U test yielded p&lt;0.000001,
 mean and SD are plotted. Graphs are artificially jittered in x to show
 distributions. File titles contain information on the experimental day,
 treatment, staining, replicate, image number, and processing steps as:
 DATE_TREATMENT_STAINING (488)_STAINING (568)_STAINING
 (647)_REPLICATE_IMAGE NUMBER_PROCESSING STEPS.tif. in Fig. 3C & Fig.
 S5C, sub-folders indicate probes used, treatment conditions and
 replicates. Note: this folder also contains the imaging files for Fig.
 S5C, as the same images underlie the quantifications in Fig.3C and Fig.
 S5C. **Fig.4 Sororin is a monomer and associated with &lt;1/3 of chromatin
 bound cohesin** **(A)** Schematic depiction of 3D-SIM imaging approach of
 endogenously tagged RAD21 together with sororin in G2 phase and on
 chromatin. **(B)** Representative widefield and 3D-SIM images of chromatin
 bound sororin. Single nucleus z-slice shown or cropped region shown. False
 coloring by sororin signal intensity shows uniform population. **(C)**
 Frequency distribution of chromatin bound sororin signal intensities with
 fitted non-linear regression (Gaussian distribution); SumInt; Sum
 Intensity; A.U., arbitrary units, n&gt; 140.000 sororin signals from two
 biologically independent experiments. **(D)** Frequency distribution of
 signal intensities of chromatin bound sororin in **Fig. 4C** is overlaid
 with non-specific antibody conjugate intensities observed on imaging
 slides outside nuclei as shown in **(E)**. N&gt;3700 antibody conjugates
 and data are from two biologically independent experiments. **(E)**
 Example image showing analysis of single sororin intensities. On imaging
 slides, primary and secondary antibody conjugates, which occur through
 non-specific binding, were segmented, and their frequency distribution
 analyzed. Arrows indicate non-specific antibody conjugates on slide (blue)
 and specific sororin signals (green). Antibody specificity in cells has
 been controlled for in **Fig. S5A**. Quantification of absolute
 distributions of chromatin bound sororin from **Fig. 4C** yielded the
 following frequencies: 74.4% (monomer), 15.75% (dimer) and 9.85%
 (multimer). Quantification is based on non-linear regression analysis.
 **(F)** Example image of RAD21-Halo and sororin colocalization in G2 phase
 cells. Maximum projections of 5 z-slices are shown, as entire nucleus and
 cropped region. Overlaps are indicated in white circles and numbered lines
 mark the normalized intensity profiles show measurements. **(G)**
 Colocalization analysis of RAD21 at sites of sister chromatid cohesion
 marked by sororin. Bar charts consist of dots, each representing an
 analyzed RAD21 signal. Colocalization was analyzed in two biologically
 independent experiments with &gt;38.000 RAD21 molecules analyzed per
 repeat. Absolute numbers for RAD21 colocalization with sororin are 28.47%
 (repeat 1) and 28.56% (repeat 2). Two-tailed student t-test determined
 p&lt;0.0001, means are shown. File titles contain information on the
 experimental day, treatment, staining, replicate, image number, and
 processing steps as: DATE_TREATMENT_STAINING (488)_STAINING (568)_STAINING
 (647)_REPLICATE_IMAGE NUMBER_PROCESSING STEPS.tif. An exception is Fig.
 4F, where the word MAX indicates that a z projection is is shown. **Fig.5
 Sister chromatid cohesion is maintained by individual cohesin rings**
 **(A)** Schematic depiction of labeling approach. U2OS RAD21-Halo cells
 were incubated with two different HaloTag ligands, each labeling ~50% of
 RAD21-Halo (see **Fig. S7A-C** for controls) The table shows expected
 frequencies of observing one Halo dye at a sororin site, or the other, or
 both at the same time, for monomeric, dimeric and multimeric cohesin.
 **(B)** Example 3D-SIM image of G2 cell after incubation with JFX554 and
 JFX650 against RAD21-Halo (each labeling ~50%) and immunostaining with
 sororin antibody. A single z-slice of a nucleus is shown and
 quantification in **(D)** and **(H)**. **(C)** Examples of classes I-III
 (sororin-containing signals) detected in G2 cells. Single z-slices of
 3D-SIM example crops are shown. **(D)** Quantification of **(C)**.
 Three-way colocalization was measured in two biologically independent
 experiments, with 1200 sororin sites analyzed per repeat. Means are 8.21%
 (sororin without RAD21-Halo), 45.00% (sororin with RAD21-Halo JFX554),
 44.46% (sororin with RAD21-Halo JFX650) and 2.33% (sororin with RAD21-Halo
 JFX554 and RAD21-Halo JFX650). Dots represent individual cells.
 Statistical testing by two-tailed student t-test yielded p&lt;0.000001.
 **(E)** Examples of classes IV-VI (cohesin at sororin absent sites)
 detected in G2 cells. Single z-slices of 3D-SIM example crops are shown.
 **(F)** Cohesin at sites without sororin was analyzed in two biologically
 independent experiments, with 1200 cohesin signals analyzed per repeat.
 Means are 26.67% (2 x RAD21-Halo JFX554), 24.79% (2 x RAD21-Halo JFX650)
 and 48.54% (RAD21-Halo JFX554 and RAD21-Halo JFX650). Dots represent
 individual cells. Statistical testing by two-tailed student t-test yielded
 p&lt;0.000001. **(G)** RASER-FISH example of probe HS443D9 co-localizing
 with RAD21-Halo in a dye mixing experiment (left) and quantification
 (right). N=34 probe signals from two biologically independent experiments.
 Mann-Whitney U test yielded p=0.33. File titles contain information on the
 experimental day, treatment, staining, replicate, image number, and
 processing steps as: DATE_TREATMENT_STAINING (488)_STAINING (568)_STAINING
 (647)_REPLICATE_IMAGE NUMBER_PROCESSING STEPS.tif. **Fig. S1 Molecular
 counting of chromatin bound RAD21-Halo** **(A)** Representative widefield
 and super-resolution 3D-SIM images of chromatin bound RAD21-Halo as shown
 in **Fig. 1B** with added orthogonal views for illustration of 3D
 resolution increase by SIM. Single nucleus z-slice shown or copped region.
 **(B)** HILO-photobleaching of sub-labeled chromatin bound RAD21-Halo.
 Example cell nucleus with sparse RAD21-Halo signals is shown as well as
 crops at consecutive time points to visualize single step bleach reactions
 of events (highlighted with magenta arrows). **(C)** Quantification of
 single step-wise photobleaching of 10 example signals of chromatin bound
 RAD21-Halo after sub-labeling. Data is background corrected using regions
 of identical sizes. Bleaching occurs at different timepoints due to
 Gaussian nature of HILO laser beam. **(D)** Quantification of steps in
 bleach events observed in RAD21-Halo after sub-labeling and on chromatin.
 The experiment was carried out in two independent biological repeats with
 &gt;90 bleach reactions analyzed per repeat. Dots indicate independent
 technical repeats per biological repeat. Mann-Whitney U test yielded
 p=0.0022. Files are labelled as: EXPOSURE TIME_LASER POWER_STAINING_IMAGE
 NUMBER.tif. **Fig. S2 Generation of cell line for endogenously tagged
 cohesin complex** **(A)** Sequence alignment for SMC3 hinge region from H.
 sapiens to *S. cerevisiae* colored by amino acid conservation (60%
 threshold). The loop targeted for endogenous tagging surrounding amino
 acid residue E602 is highlighted in magenta. **(B) *M. musculus* cohesin
 hinge with loop targeted for endogenous tagging of SMC3 highlighted in
 magenta (PDB: 2WD5) (*55*)**(C) PCR showing homozygous tagging of
 SMC3-E602 with Spot-tag in two independent clones. **(D)** Western blot of
 SMC3-E602-Spot clones 1 and 2 compared to parental cell lines to show
 homozygous tagging. Membrane was immunostained for Spot, RAD21, SMC1 and
 Lamin-B1. Halo-tag was detected in-gel with JF549 and SMC3 was detected on
 overnight midi-gel to visualize 1.4 kDa size difference of Spot-tag
 insertion. **(E)** IP of SMC3-E602-Spot clones 1 and 2 by a-SMC1 antibody,
 immunostained for SMC1, SMC3 and RAD21. Input is 1% total sample. **(F)**
 Scatter plots showing the cell cycle distribution pattern of chromatin
 bound SMC3 detected in the endogenously tagged SMC3-E602-Spot cell line
 with Spot antibody (left) versus the cell cycle distribution pattern of
 chromatin bound SMC3 detected with SMC3 antibody in RAD21-Halo cells. m;
 mouse, rb; rabbit, ab; antibody. N=9700, the experiment was repeated twice
 biologically independently. **(G)** Quantification of metaphase spreads in
 RAD21-Halo parental cells and SMC3-E602-Spot clones 1 and 2. Spreads were
 counted as fully cohered, and railroad as well as prematurely separated
 chromosomes were counted as loss of cohesin phenotype. Two biologically
 independent experiments were carried out, with &gt;20 metaphase spreads
 analyzed per cell line and repeat. Mean and SD are shown. **(H)** Western
 blot showing siRNA depletion efficiency of PDS5A and B in RAD21-Halo U2OS
 cells. Vermicelli were induced by combined siRNA depletion of PDS5A and B.
 **(I)** Example images of RAD21-Halo and SMC3-E602-Spot clones 1 and 2
 after control or PDS5A/B siRNA treatment. RAD21-Halo was visualized in all
 cell lines by JFX554 dye and DNA by DAPI staining. Images were acquired
 with a spinning disk microscope and false-colored. **(J)** Vermicelli were
 categorized as wild type, mild, moderate and severe chromatin compaction
 phenotypes by automated ScanR analysis. Mean and SD are shown. Two
 biologically independent experiments were carried out, with &gt;1000 cells
 analyzed per cell line, condition and repeat. File titles contain
 information on the experimental day, treatment, staining, replicate, image
 number, and processing steps as: DATE_TREATMENT_STAINING (488)_STAINING
 (568)_STAINING (647)_REPLICATE_IMAGE NUMBER_PROCESSING STEPS.tif. An
 exception is Fig. S2F where imaging files from QIBC are labelled as WELL
 NUMBER_POSITION NUMBER_TIMEPOINT_STAINING.tif. In Fig. S2G, sub-folders
 indicate the different cell lines used. **Fig S3 SIMinspector, an image
 analysis tool for 3D super-resolution microscopy** **(A)** Schematic
 depiction of the SIMinspector workflow. 3D-SIM images are acquired and
 processed. Calibration samples are analyzed to determine microscope- and
 sample-specific parameters. Script 1 (SIMinspector_mainobject_Fiji)
 segments the main object based on intensity thresholding. Script 2
 (SIMinspector_subobject_Fiji) segments subobjects by intensity
 thresholding and watershedding and exports intensity, volume, pairwise
 colocalization measurements and center positions. 3-way colocalization is
 determined by 2x two-way colocalization. **(B)** For calibration, U2OS
 cells were incubated with 100 nm 3-colour beads for 48 hours, which led to
 nuclear incorporation. This allowed calibration measurements in same
 cellular environment as actual samples. **(C)** Example 3D-SIM images of
 DAPI stained nucleus with incorporated 3-colour beads (5 slice maximum
 projection). **(D)** Full width at half maximum (FWHM) measurements in
 lateral and axial dimensions for monomeric beads within U2OS cells for all
 3 channels, n=10 single bead signals, shown are median (middle line) and
 interquartile range (IGQ; box), whiskers are 1.5* IQR. **(E)** Measurement
 of two-way colocalization between green and red emission signals (left)
 and between green and far-red emission signals (right) detected from
 three-color 100 nm beads at a varying distance threshold, solid line
 represents sigmoid fit. N=80 beads. **(F)** Three-way colocalization
 analysis of beads within U2OS cells. At 99 nm distance threshold, &gt;95%
 of bead signals from 3 channels colocalize. N=80 beads, experiment was
 carried out twice biologically independently. Kruskal-Wallis test
 determined p&lt;0.0001, mean and SD are shown. File titles contain
 information on the experimental day, treatment, staining, replicate, image
 number, and processing steps as: DATE_TREATMENT_STAINING (488)_STAINING
 (568)_STAINING (647)_REPLICATE_IMAGE NUMBER_PROCESSING STEPS.tif. An
 exception is Fig. S3C, where the word MAX indicates that a z projection is
 is shown. **Fig. S4 Chromatin bound RAD21 marks cohesin complexes**
 **(A**) Schematic depiction of 3D-SIM imaging approach of endogenously
 tagged cohesin complexes in G2 phase and on chromatin. RAD21 is
 Halo-tagged and SMC3 Spot-tagged at the cohesin hinge. **(B)**
 Chromatin-bound cohesin visualized by 3D-SIM microscopy, nucleus shown as
 single z-slice, colocalization shown in cropped regions of single z-slices
 and intensity line profiles. RAD21-Halo was visualized by incubation with
 JFX554 dye and SMC3-E602-Spot with Spot antibody. Images were false
 colored. **(C)** Quantification of colocalization between RAD21-Halo and
 SMC3-E602-Spot as detected in **(B)**. 2 biologically independent repeats
 are shown in overlay, single spots represent single RAD21-Halo signals
 analyzed. N&gt;90.000 analyzed RAD21 signals per repeat, the experiment
 was repeated twice in biologically independent manner. Statistical testing
 by two-tailed student t-test yielded p=0.0004. File titles contain
 information on the experimental day, treatment, staining, replicate, image
 number, and processing steps as: DATE_TREATMENT_STAINING (488)_STAINING
 (568)_STAINING (647)_REPLICATE_IMAGE NUMBER_PROCESSING STEPS.tif. **Fig S6
 Sororin is a monomer and its recruitment to chromatin coupled to DNA
 replication** **(A)** Cell cycle analysis of sororin binding to chromatin
 by immunofluorescence. Cell cycle stages were identified based on EdU
 (5-ethynyl-2’-deoxyurdine, thymidine analogue, S phase marker), MCM2
 antibody (marker for pre-replicative chromatin) and DAPI (DNA marker).
 Example images for each cell cycle stage were acquired on a Spinning Disk
 microscope and quantification shown in **(B)**. Cells were false-colored.
 **(B)** QIBC of chromatin bound sororin. Scatter plots shows single cells
 as dots over cell cycle by plotting of EdU over DAPI nuclear intensities.
 This delineates G1 phase (bottom left cloud with 2N DNA), S phase cells
 (arch) and G2 phase cells (bottom right cloud with 4N DNA). The color code
 shows the mean intensity of chromatin bound sororin signal per cell. 3400
 cells are shown, the experiment was repeated twice in a biologically
 independent manner. **(C)** Western blot of sororin depletion from U2OS
 wild type cells with two independent siRNAs (Sor-1 and Sor-2) over 72 or
 96 h, with immunostaining for sororin and total protein extract. ST,
 single transfection; DT; double transfection; Con, control siRNA; Sor-1,
 siRNA1 against sororin; Sor-2; siRNA2 against sororin. **(D)** PCR showing
 homozygous tagging of sororin with SNAP-tag. **(E)** Western blot showing
 endogenous tagging of sororin with SNAP-tag. Western blot was
 immunostained for Lamin-B1, RAD21, SMC1, SMC3, sororin and sororin-SNAP
 detected in gel with JFX650. Cell lines used were U2OS RAD21-Halo
 (RAD21-Halo), U2OS RAD21-Halo SMC3 E602-Spot (SMC3-E602-Spot), U2OS
 RAD21-Halo, SMC3-E602-Spot, sororin-SNAP (sororin-SNAP). **(F)** QIBC
 analysis of sororin antibody (left) and endogenously tagged sororin-SNAP
 (right) serves as antibody specificity control. 3500 cells are shown per
 condition. The experiment was repeated twice independently. **(G)**
 Example of 1-step photobleaching of chromatin bound sororin-SNAP incubated
 with JF549 by HILO. **(H)** Quantification of photobleaching of chromatin
 bound sororin-SNAP incubated with JF549 dye. The experiment was carried
 out in two independent biological repeats with &gt;80 bleach reactions
 analyzed per repeat. Dots indicate independent technical repeats per
 biological repeat. Mann-Whitney U test determined p=0.002165, mean and SD
 are shown. File titles contain information on the experimental day,
 treatment, staining, replicate, image number, and processing steps as:
 DATE_TREATMENT_STAINING (488)_STAINING (568)_STAINING
 (647)_REPLICATE_IMAGE NUMBER_PROCESSING STEPS.tif. Files is Fig. S6B and
 Fig. S6F are labelled as WELL NUMBER_POSITION
 NUMBER_TIMEPOINT_STAINING.tif. Files in Fig. S6G &H are labelled as:
 EXPOSURE TIME_LASER POWER_STAINING_IMAGE NUMBER.tif. **Fig S7 HaloTag
 dyemixing allows molecular counting** **(A**) Titration of RAD21-Halo dyes
 JFX554 and JFX650 in U2OS RAD21-Halo cells. EC50 was determined to 3.29 nM
 (JFX554) and 1.05 nM (JFX650). **(B)** Labeling approach (top) and
 quantification of signal frequencies (bottom). Samples were incubated with
 varying concentrations of mixed dyes JFX554 and JFX650 for 1.5 h, washed
 and chased with a third dye JF479. Samples were imaged with 3D-SIM and
 single RAD21-Halo signals analyzed. N&gt;28.000 RAD21-Halo analyzed per
 condition. W; wash. Mean and SD are shown. **(C)** To validate labeling
 efficiency of JF479 dye used for chasing in **Fig. S7B**, cells were
 incubated with dye JFX554 for 1 h, chased by JF479 and vice versa.
 RAD21-Halo labelling frequency was obtained from normalized intensity
 distributions for n&gt;4000 cells imaged by QIBC. The experiment was
 repeated twice in a biologically independent manner. W; wash. Mean and SD
 are shown. **(D)** 3D-SIM image crop of 2 sororin signals overlapping with
 two RAD21-Halo molecules, one labelled with JFX554 (magenta) and one
 labelled with JFX 650 (cyan). A single z-slice is shown and intensity
 quantification below. **(E)** Overlay of frequency distribution of
 chromatin bound RAD21 monomer volumes shown in **Fig. 1F** with volumes of
 sororin-associated RAD21. N&gt;33.000 sororin-cohesin complexes and the
 experiment was carried out twice in biologically independent manner. File
 titles contain information on the experimental day, treatment, staining,
 replicate, image number, and processing steps as: DATE_TREATMENT_STAINING
 (488)_STAINING (568)_STAINING (647)_REPLICATE_IMAGE NUMBER_PROCESSING
 STEPS.tif. Files is Fig. S7C are labelled as WELL NUMBER_POSITION
 NUMBER_TIMEPOINT_STAINING.tif, and sub-folders indicate dye chase
 conditions and replicates. Please see the below overview of abbreviations
 for processing steps: ALN: channel aligned file LUT: false-coded by look
 up table MCF_THR: MCNR corrected file PWF: pseudo-widefield image SIR:
 reconstructed SIM image THR: 16-bit conversed and thresholded file For
 information on how these processing steps were executed, please see the
 SIM section in methods here on Dyrad. All images can be opened with
 Fiji/ImageJ. ### Code/Software We have deposited the code for
 SIMinspector, our SIM image processing pipeline, as well as Jupyter
 notebooks for the analysis of ChIP and sister chromatid sensitive-HiC
 (scs-HiC) data. ##### SIMinspector For SIMinspector two files have been
 deposited, called SIMinspector_Mainobject_Fiji and
 SIMinspector_Subobject_Fiji, which are Fiji plug-ins. SIMinspector is a
 custom image analysis tool for the segmentation of sub-cellular features
 and analysis of colocalization in multicolor 3D-super-resolution
 microscopy images. SIMinspector consists of 2 Fiji scripts that are
 explained below and can be executed in the following order: 1.   
 **SIMinspector_Mainobject_Fiji:** This Fiji script segments the main
 object, e.g. cell nucleus, based on intensity thresholding. For this, a
 selected channel (“Channel for mask creation”) is used. Objects are
 detected based on intensity thresholding (“Threshold method”) and all
 objects but the largest are excluded. It produces segmented and cropped 3D
 images in the specified output directory. 2.   
 **SIMinspector_Subobject_Fiji:** This Fiji script segments subobjects from
 2 channels, measures volumes, intensities and centers as well as
 colocalization based on voxel overlap, center-to-center distances and
 center-to-edge distances. To this end, images are made isotropic and
 channels are thresholded (“Segmentation algorithm”). Maxima are detected
 (“Maxima radius”) and made into labels. Maxima-derived labels were used
 for marker-controlled intensity-based watershed filtering. Optional,
 resulting labels can be eroded to optimize segmentation (“Label erosion
 channel”). Afterwards images were resliced and objects below minimum and
 above maximum volumes excluded (“Minimum spot volume in voxels”, “Maximum
 spot volume in voxels”).  Resulting stacks are converted to 16-bit,
 original pixel sizes restored, and statistics of labels computed. Computed
 centers of mass are mapped. Fiji’s plugin DiAna (Gilles et al., 2017) is
 used to calculate voxel overlaps, center-to-center and center-to-edge
 distances for the N nearest neighbors. The script produces 4 .csv files.
 The file ending on “StatisticsOfLabelmap_C1” contains all measurements for
 channel 1, the file ending on “StatisticsOfLabelmap_C2” contains all
 measurements for channel 2 and the files “AdjacencyResults_C1vsC2” contain
 the colocalization results originating from either center of mass or
 geometric center values. The dependencies for these scripts are 3D ImageJ
 Suite(Ollion et al., 2013), Java 8, CLIJ (Haase et al., 2020), CLIJ2,
 clijx-assistant, clijx-assistant extension, IJPB (Legland et al., 2016)
 and DiAna (Gilles et al., 2017). ##### ChIP/scs-HiC We have also deposited
 all code for the alignment of ChIP and sister chromatid sensitive Hi-C,
 which can be found as 6 files: 001_Generate_intervals.ipynb
 002_Generate_stacks_SMC3_w_Sororin.ipynb
 002_Generate_stacks_SMC3_wo_Sororin.ipynb
 003_HiC_aggregate_maps_SMC3_w_Sororin.ipynb
 003_HiC_aggregate_maps_SMC3_wo_Sororin.ipynb
 004_Generate_HiC_line_profiles_SMC3_w_Sororin.ipynb
 004_Generate_HiC_line_profiles_SMC3_wo_Sororin.ipynb All files are Jupyter
 notebooks and are differentiated based on cohesin (SMC3) with or without
 co-localising Sororin, as described in the manuscript Fig.2. Details on
 the processing of ChIP and scs-HiC datasets can be found below. ##### SMC3
 and sororin ChIP-Seq heatmaps ChIP-Seq data for SMC3 and sororin from
 (Ladurner et al., 2016)
 () were mapped
 using bwa (v0.7.17) against human genome assembly GRCh37/hg19. Raw read
 counts were normalized to counts per million (CPM) values and
 log-transformed using a base two logarithm. The peaks were called using
 macs2 (*54*) using a p-value threshold of 1e‑10. For each detected SMC3
 peak the average sororin signal was measured using pybbi package version
 0.3.2 (). SMC3 peaks with a mean
 sororin signal higher than 0.1 CPM were marked as “SMC3 with sororin”
 (20943 peaks). The remaining SMC3 peaks were marked as “SMC3 without
 sororin” (13848 peaks). Heatmap plots centered at SMC3 sites with or
 without sororin were calculated using a custom ipython notebook. Samples
 come from wild type HeLa Kyoto cells synchronized to G2, and from two
 biological replicates. SMC3 and sororin ChIP-Seq data from (Ladurner et
 al., 2016) can be accessed in the European Nucleotide Archive (ENA) with
 the accession number PRJEB12214. ##### Hi-C aggregate maps Scs-Hi-C data
 for wild type HeLa Kyoto cells synchronized in G2 (GSE152373) were
 processed as described in (Mitter et al., 2020). Observed-over-expected
 scsHi-C aggregate maps were calculated using the cooltools package version
 0.5.4 (). 1 Mb ICE-corrected
 snippets around SMC3 sites with or without sororin (“observed”) were
 extracted and each value in the snippets was normalized by the value of
 the scaling at that diagonal (“expected”). The pixel-wise average across
 the snippet was then calculated. The main diagonal and one neighboring
 diagonal were blanked out to avoid Hi-C artefacts. G2 maps were
 constructed from 1.7 billion Hi-C reads with 195 million unique reads from
 11 biological repeats. HeLa scsHi-C data from (Mitter et al., 2020) can be
 access at Gene Expression Omnibus (GEO) with accession number GSE152373.
 ##### Stack-up analysis of line profiles for ChIP-Seq and scsHi-C data
 Stack-ups of line profiles within 1 Mb windows centered at SMC3 sites with
 or without sororin were calculated using a custom iPython notebook. The
 contact density within a sliding diamond of 100 kbs was calculated along
 each region within the set of regions of length m for
 observed-over-expected matrices, resulting in a vector of size n for each
 region. Then, these vectors were stacked into an m × n matrix with m
 denoting the number of regions and n denoting the length of the line
 profile along each region. For display of observed-over-expected values, a
 pseudocount of 0.01 was added before log-transformation. Line profiles
 containing only invalid Hi-C bins were removed from the stack-up. Code
 underlying ChIP and scs-HiC analysis, can be downloaded from Dyrad with
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