Integrating images from multiple microscopy screens reveals diverse patterns of change in the subcellular localization of proteins

Abstract

The evaluation of protein localization changes on a systematic level is a powerful tool for understanding how cells respond to environmental, chemical, or genetic perturbations. To date, work in understanding these proteomic responses through high-throughput imaging has catalogued localization changes independently for each perturbation. To distinguish changes that are targeted responses to the specific perturbation or more generalized programs, we developed a scalable approach to visualize the localization behavior of proteins across multiple experiments as a quantitative pattern. By applying this approach to 24 experimental screens consisting of nearly 400,000 images, we differentiated specific responses from more generalized ones, discovered nuance in the localization behavior of stress-responsive proteins, and formed hypotheses by clustering proteins that have similar patterns. Previous approaches aim to capture all localization changes for a single screen as accurately as possible, whereas our work aims to integrate large amounts of imaging data to find unexpected new cell biology.

YeastRGB: comparing the abundance and localization of yeast proteins across cells and libraries

“…Indeed, storage of the raw data for our proteome-wide screens decreased up to 50 folds, from 0.63TB (C’ GFP), 1.38TB (C’ SWAT) and 1.97TB (N’ SWAT) to 17GB, 28GB, and 39GB respectively (values before compression). Moreover, it will be simpler to apply classification algorithms on the segmented cells than it would be on raw, unsegmented images (6,24–27), e.g. for the automated identification of protein localization.…”

Yeast Rgd3 is a phospho-regulated F-BAR-containing RhoGAP involved in the regulation of Rho3 distribution and cell morphology

“…We next explored whether Rgd3 polarization is dependent on the actin cytoskeleton. Rgd3 is highly polarized in cells induced to shmoo by α-factor treatment, a phenomenon first observed in high-throughput studies (Kraus et al, 2017;Lu et al, 2018). Treatment of shmooing Rgd3-mNG cells with latrunculin B, to depolymerize actin filaments, resulted in complete depolarization of Rgd3-mNG within 40 seconds ( Figure 6A).…”

Organellar Maps Through Proteomic Profiling – A Conceptual Guide

“…A , Relative abundance profiles; subunits in light gray, means in dashed black. SILAC quantification produces a tight profile cluster with finely nuanced resolution of small differences in the low abundance fractions ( 1 – 3 ). The LFQ profiles show substantially more scatter.…”

“…This spatial dimension of the proteome is carefully regulated, highly dynamic, and allows much faster responses to perturbations than alteration of gene expression. Many, perhaps most, cell biological processes involve proteins transitioning between cellular locations; evidence suggests that spatial regulation of the proteome is as important and extensive as regulation of protein abundance ( 1 ). Supporting this notion, an increasing number of diseases are associated with disturbances in protein localization ( 2 – 4 ).…”