A semi-automated machine learning-aided approach to quantitative analysis of centrosomes and microtubule organization

Abstract

Microtubules (MTs) perform important cellular functions including migration, intracellular trafficking, and chromosome segregation. The centrosome, comprised of two centrioles surrounded by the pericentriolar material (PCM), is the cell's central MT organizing center. The PCM proteins, including -tubulin and Pericentrin, promote MT nucleation and organization. Centrosomes in cancer cells are commonly numerically amplified. However, the question of how amplification of centrosomes alters the MT organization capacity is not well-studied. We developed a quantitative image-processing and machine learning-aided approach for the automated analysis of MT organization.We designed a convolutional neural network-based approach for detecting centrosomes and an automated pipeline for analyzing MT organization around centrosomes, encapsulated in a semi-automatic graphical tool. Using this tool, we analyzed the spatial distribution of PCM proteins, the growing ends of MTs and the total MT density in breast cancer cells. We find that breast cancer cells with supernumerary centrosomes not only have increased PCM protein but also exhibit expansion in PCM size. Moreover, centrosome amplified cells have a greater MT density and more growing MT ends near centrosomes than unamplified cells. The semi-automated approach developed here enables facile, unbiased and quantitative measurements of centrosome aberrations. We show that these aberrations increase MT nucleation and promote changes to MT density and the spatial distribution of MTs around amplified centrosomes.

The SON RNA splicing factor is required for intracellular trafficking structures that promote centriole assembly and ciliogenesis

“…We examined the distributions of centrosomal and centriolar satellite proteins whose splicing is affected by SON using a semi-automated radial intensity analysis algorithm in control and SON knockdown cells (Figure 5, A and B) (Sankaran et al, 2020). -tubulin, together with tubulin complex members, acts to nucleate microtubules and is concentrated at the centrosome (Stearns et al, 1991;Tovey and Conduit, 2018).…”

“…For radial fluorescence intensity analyses, we utilized the algorithm described in Sankaran et al (Sankaran et al, 2020) using the PCM or microtubule analysis tool. Briefly, maximum intensity projected images were selected for analysis.…”

“…Dysregulation of centrosome number can instigate cellular transformation to a cancerous state (Levine et al, 2017). More than two centrosomes results in chromosomal instability (Ganem et al, 2009;Silkworth et al, 2009;Zhang et al, 2015), altered cell migration (Godinho et al, 2014) and disruptions to the microtubule network (Godinho et al, 2014;Marteil et al, 2018;Sankaran et al, 2020). Differentiated cells, on the other hand, often utilize non-centrosomal microtubule organizing centers (MTOCs) and must attenuate the microtubule nucleating function of the centrosome (Sanchez and Feldman, 2017), suggesting the existence of broad programs to regulate MTOC activity.…”

Trisomy 21 increases microtubules and disrupts centriolar satellite localization

“…Tetracycline-inducible lentiviral stable RPE-1 cell lines were generated according to methods outlined in (Sankaran et al, 2020). Briefly, HEK293T cells were transfected with the tetracycline-inducible mNeon-EB3 construct and lentivirus packaging plasmids using Lipofectamine 2000 (11668-027; Invitrogen).…”