Self-assembly of the RZZ complex into filaments drives kinetochore expansion in the absence of microtubule attachment

Cláudia Pereira; Rita M. Reis; José B. Gama; Dhanya K. Cheerambathur; Ana X. Carvalho; Reto Gassmann

Volume and intensity of kinetochore fluorescence for CENP-E, CENP-F, Spindly, and GFP were measured using the 3D Objects Counter tool. To each image stack (0.2µm z-steps) encompassing one cell, the Subtract Background function was applied (rolling ball radius of 5 pixels), and a mask was generated based on an empirically determined threshold that maximized the number of detected kinetochores, while minimizing the detection of false objects. The mask was re-directed to the original, unprocessed image stack to determine the number of voxels and the integrated intensity for each object, which could consist of one or several closely apposed kinetochores. Object values were then summed to give the total number of voxels and the total integrated intensity for kinetochores in the image stack. To determine background intensity, the number of voxels and integrated intensity were measured for five separate regions in the image stack that did not contain kinetochores. Values for the five regions were averaged, normalized to the total number of kinetochore voxels, and subtracted from the total integrated kinetochore intensity. The total number of kinetochores in the image stack was determined separately using the CENP-C signal after maximum intensity projection of the image stack, followed by thresholding, automatic particle counting, and verification by visual inspection. Finally, the total number of kinetochore voxels and the total integrated kinetochore intensity were divided by the total number of kinetochores to yield the average number of voxels and integrated intensity per kinetochore per cell. Average kinetochore volumes and integrated intensities were determined for 20 cells in two independent experiments. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/282707 doi: bioRxiv preprint