Misseg

Code written for ImageJ, R and IGOR Pro to analyse data associated with manuscript #p055p032

Endomembranes promote chromosome missegregation by ensheathing misaligned chromosomes

Nuria Ferrandiz, Laura Downie, Georgina P. Starling, Stephen J Royle

J Cell Biol, (2022) 221: e202203021 doi: 10.1083/jcb.202203021

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Misseg

The main program in this repo is Misseg/Misseg.ipf. Full details of how to use it with IGOR Pro can be found in Misseg/README.md

The other directories are:

• ERRemoval
• kinetochoreColoc
• Progression
• SankeyFate

ER Removal

CongressionAnalysis - Quantifying the congression of a misaligned chromosome to the plate.

DNA channel is processed and segmented in ImageJ manually. User defines the segments belonging to chromosome and plate using Magic Wand tool and adding to ROI manager. ROIs are converted to csv using a python script and files are saved into a folder for chromosome and a folder for plate, within a folder for the movie.

ER Removal - Quantifying the clearance of ER in mitotic cells.

Using the same ImageJ scripts, the ER channel was processed and segmented using ER_Removal.ijm or the two subscripts (divided to allow the slowest step to be completed and different segmentation approaches tried with the other). Resulting data are processed in Igor using MissegERRemoval.ipf

Kinetochore coloc

A series of scripts to examine co-localisation of kinetochore signals in 3D image stacks.

• kinetochoreColoc.ijm loads dv files segments them and uses 3DOC to find the positions of two signals in channels 3 and 4. This is a substitute for Imaris (since we were having problems with the bridge to MATLAB and we did not have a current version)
• kinetochoreSegmentation.ijm is a tool to check how the segmentation (thresholding) performed in the previous script (it is not needed for analysis per se)
• dv2RGB.ijm will convert a folder of dv files to RGB tiffs for classification in Igor
• kinetochoreColoc.ipf is a menu-based workflow to classify the kinetochores from kinetochoreColoc.ijm and then analyse their proximity. There is an option to verify the classification done by a user and even to add additional kinetochores that have been missed by the automated procedure.

The classifier above allows a user to scroll through a Z-stack and classify kinetochores as Aligned, Misaligned (called Free in the paper) or Misaligned - ER (called Ensheathed in the paper).

Mad2

These experiments were analysed in Igor using Mad2/ChromosomeCongression.ipf by processing csv outputs generated in Fiji using Mad2/ChromosomeCongressionMagicWand.ijm

The method for chromosome and plate objects segmentation is as described for ER Clearance above.

MnQuant

ImageJ scripts were used to generate the orthogonal views shown in the figure as well as do the quantification of micronuclei. Outputs from the quantification scripts are in Data/ for LBR and BAF. For space reasons, TIFFs and Masks are not included. The processMNQuant.R script will read these files and generate the plots in Output/Plots/

Sankey Fate

An R-based script to generate Sankey diagrams from manual fate mapping.

Notes

Mitotic progression figures were generated using MitoticTiming