Dataset DOI: 10.5061/dryad.9p8cz8ww4

Description of the data and file structure

Chromatin & HP1a condensate fusion and heat-reversibility movies shown in Figures 2, 3, & 4 of the corresponding manuscript. Additional 3-dimensional images of mutant HP1a-chromatin mixtures are also available here as representative image-sets of the single slices shown in Figure 5. All movies were collected on a Zeiss 880 confocal and are uploaded here as .czi files, which can be opened and viewed in FIJI/ ImageJ. Axial-scans of 3-dimensional z-stacks were created using the reslice function in FIJI (see below).

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The following data were collected and analyzed by LD Brennan for the manuscript: "In vitro reconstitution of heterochromatin compartments reveals the spontaneous formation of tunable liquid-liquid interfaces."

Figure 2: Chromatin + HP1a condensate heating experiments

File names: "240108_me3-cy5_wt-cy3_25uM-HP1a_22-37_002part-1", "240108_me3-cy5_wt-cy3_25uM-HP1a_22-37_002", "230802_27to37realtime_1uM-chromatin_25uM-HP1a_005"

These movies of chromatin and HP1a co-condensates were collected using a Zeiss 880 Airyscan microscope with a 63x/1.4 oil immersion objective. Time-lapse image stacks were collected every 5 seconds at a single imaging plane and have a pixel size of 0.132μm X 0.132μm. Channel 1 corresponds with the Cy3 channel, channel 2 with Alexa-488, and channel 3 with Cy5. Heating starts at Frame 11 in file "230802_27to37realtime_1uM-chromatin_25uM-HP1a_005" and "240108_me3-cy5_wt-cy3_25uM-HP1a_22-37_002part-1" which then continues, with heating, in "240108_me3-cy5_wt-cy3_25uM-HP1a_22-37_002".

Files can be opened in FIJI using the BioFormats extension. The Cy3 and Cy5 channels were split into individual files in FIJI, saved as a Tif series, and then analyzed using the following custom Python scripts: make_segments, make_group_segments, and calculate_region_correlations. Data were plotted using the seaborn boxplot functionality of Python.

Figure 3: chromatin condensate material properties

240115_1uM-wt-Chromatin-fusions-63x_DIC_002_crop029.nd2

240115_1uM-wt-Chromatin-fusions-63x_DIC_002_crop030.nd2

240115_1uM-me3-Chromatin-fusions-63x_DIC_005_crop025.nd2

240115_1uM-me3-Chromatin-fusions-63x_DIC_005_crop022-2for.nd2

Figure 4: chromatin + HP1a condensate material properties

240115_1uM-wt-25uM-HP1a-Chromatin-fusions-63x_DIC_009_crop041.nd2

240115_1uM-wt-25uM-HP1a-Chromatin-fusions-63x_DIC_009_crop042.nd2

240115_1uM-me3-25uM-HP1a-Chromatin-fusions-63x_DIC_008_crop019.nd2

240115_1uM-me3-25uM-HP1a-Chromatin-fusions-63x_DIC_008_crop024.nd2

These movies are representative examples of chromatin condensates fusions captured on a Nikon Ti2 with a 63x oil objective lens at 0.02 sec per frame with brightfield illumination and have a pixel size of 0.108 μm X 0.108 μm. These files can be opened in FIJI and were manually analyzed using the line tool to measure the long-axis of the droplets undergoing fusion: pre-fusion, mid-fusion, and post-fusion. See methods.

Figure 5: HP1a mutants

“AADA-topdown” shows an overlay of both chromatin types, shown as a z-stack along the X-Y plane.

“AADA-reslice” shows the same droplet as in the top-down view, shown along the X-Z planes.

“KED-topdown” shows an overlay of both chromatin types, shown as a z-stack along the X-Y plane.

“KED-reslice” shows the same droplet as in the top-down view, shown along the X-Z planes.

These movies of chromatin and HP1a co-condensates were collected using a Zeiss 880 Airyscan microscope with a 63x/1.4 oil immersion objective. Z-stacks were collected at an interval of 0.42 μm and have a pixel size of 0.132 μm X 0.132 μm. In all movies, Me-chromatin is red, and U-chromatin is cyan. HP1a is not shown. Scale bars are 0.5 μm.

Files and variables

File: 240108_me3-cy5_wt-cy3_25uM-HP1a_22-37_002.czi

Description: heating and cooling of the chromatin+ HP1a mixtures images in part1, quantitation shown in Figure 2

File: 240108_me3-cy5_wt-cy3_25uM-HP1a_22-37_002part-1.czi

Description: pre-heating of chromatin +HP1a mixtures, quantitation shown in Figure 2

File: 230802_27to37-realtime_1uM-chromatin_25uM-HP1a_005.czi

Description: duplicate heating experiments of chromatin + HP1a mixtures, stilled shown in Figure 2

File: 240115_1uM-wt-25uM-HP1a-Chromatin-fusions-63x_DIC_009_crop041.nd2

Description: Example of brightfield condensate fusions analyzed in Figure 4

File: 240115_1uM-wt-25uM-HP1a-Chromatin-fusions-63x_DIC_009_crop042.nd2

Description: Example of brightfield condensate fusions analyzed in Figure 4

File: 240115_1uM-wt-Chromatin-fusions-63x_DIC_002_crop029.nd2

Description: Example of brightfield condensate fusions analyzed in Figure 3

File: 240115_1uM-wt-Chromatin-fusions-63x_DIC_002_crop030.nd2

Description: Example of brightfield condensate fusions analyzed in Figure 3

File: 240115_1uM-me3-Chromatin-fusions-63x_DIC_005_crop022-2for.nd2

Description: Example of brightfield condensate fusions analyzed in Figure 3

File: 240115_1uM-me3-Chromatin-fusions-63x_DIC_005_crop025.nd2

Description: Example of brightfield condensate fusions analyzed in Figure 3

File: 240115_1uM-me3-25uM-HP1a-Chromatin-fusions-63x_DIC_008_crop019.nd2

Description: Example of brightfield condensate fusions analyzed in Figure 4

File: 240115_1uM-me3-25uM-HP1a-Chromatin-fusions-63x_DIC_008_crop024.nd2

Description: Example of brightfield condensate fusions analyzed in Figure 4

File: AADA-topdown.m4v

Description: shows an overlay of both chromatin types, shown as a z-stack along the X-Y plane.

File: AADA-reslice.m4v

Description: shows the same droplet as in the top-down view, shown along the X-Z planes, analyzed in Figure 5.

File: KED-topDown.m4v

Description: shows an overlay of both chromatin types, shown as a z-stack along the X-Y plane.

File: KED-reslice.m4v

Description: shows the same droplet as in the topdown view, shown along the X-Z planes, analyzed in Figure 5.

Code/software

Python analysis code and the plotted datasets can be found in the PythonNotebooks.zip file. 

This zip file is organized into folders for each figure that include the raw, tabular data or Python "pickled" data, along with the relevant Python notebook used to visualize said data. For ease of use, we suggest that users open the Python notebooks first, as they point to the relevant tabular data file and provide commented roadmaps for how to interact with the raw data (such as variable names and units).

Tabular datasets:

Folder Figure 1&5: The tabular data was generated from segmenting single z-plane imaging of HP1-chromatin mixtures in Arivis and thus contains columns that are labelled such that the segment identity ("name" and "ID") can be traced and cross-correlated with the "parent droplet" ("parent ID") that may contain multiple segment types depending on HP1a content. The units for each column are included in the column name (or key if imported into Python) except for the pixel intensity values (arbitrary units), which are denoted as follows: 

• 'Intensity #1' is Me-chromatin
• 'Intensity #2 ' is HP1a
• 'Intensity #3' is U-chromatin

Folder Figure 2: The raw data is stored as a .pickle binary file, which is accessible only through the associated Python notebook. This notebook also provides the method used for analyzing the pearson-correlation coefficent for each chromatin type over the duration of the heating experiments and can be re-run in this notebook. The columns in the binary file are labeled as such: frame_ch1, frame_ch2, region_label, correlation, and time which correlates to: the file name for each frame of the movie separated by channel 1 (me-chromatin) or channel 2 (wt-chromatin) respectively, "region_label" is an arbitrary index of segmented regions identified in the initial analysis, "correlation" is the pearson-correlation coefficent for the pixel intensities in each frame, and "time" in frame-count in the movies for which the frame rate is 5 seconds/per frame.

Folder Figure 3&4: This folder contains a tabular dataset that is for the droplet fusion analysis: "231020_DropletFusions," used to plot the fusion times shown inFigures 3 and 4. This file has several sheets that correspond to raw data (230919 me3, 230713 wt, 230811 me3 hp1a, 230811 wt hp1a, 240115 wt, 240115 me3, 240115 wt hp1a, and 240115 me3 hp1a) of fusion times measured as delineated in the methods section of the manuscript. And the following sheets: "comparison", which is the statistical tests comparing fusions of the two chromatin types with and without HP1a. "pooled-0-HP1a", pooled-25-HP1a", and "pooled", which each contain the combined fusion times from each experimental sheet for without HP1a, with HP1a, and both experimental conditions, respectively.

This folder also includes several .pickle binary files, which contain the raw data of droplet counts, areas, and HP1a intensities, which were used to plot how the addition of increasing HP1a concentrations changes droplet morphology and HP1a partitioning into the different chromatin condensates. These files can be accessed by loading them as indicated in the Python notebooks. This files labeled "...AllDroplet data..." contain the following "columns" of data: 'label', 'area', 'area (micron)', 'axis_major_length', 'axis_minor_length', 'eccentricity', 'intensity_max', 'intensity_mean', 'intensity_min', 'perimeter', 'centroid-0', 'centroid-1', 'chromatin_intensity', 'background_intensity', 'partition_intensity', 'fraction_area', 'Frame', 'date', 'Chromatin Type', 'HP1a Conc', 'partition_avgBackground', 'intensity_correct', 'partition_correct', 'bleedthrough' where size/ length measurements are provided in pixels unless otherwise specified in column key and intensity is in arbitrary units summed for each droplet labeled during the segmentation analysis. These datasets were used to generate the binary files labeled "... HP1a-partitioning," which can be recapitulated using the Python notebook in this folder. Or, this data can be directly imported and plotted in the notebook as well as is contains the following: 'Chromatin Type', 'HP1a Conc' = HP1a concentration, 'mean' = mean intensity of the HP1a channel in the me-3 chromatin droplet, 'std' = standard deviation of the intensity of the HP1a channel in the me-3 chromatin droplet, 'unmod HP1 mean' = mean intensity of the HP1a channel in the unmod-chromatin droplet, 'unmod HP1 std' = standard deviation of the intensity of the HP1a channel in the unmod-chromatin droplet, 'HP1 partition' = ratio of HP1 in the me3-chromatin to the unmod-chromatin, 'partition std' = the standard deviation of HP1 partitioning.