Proteome-scale movements and compartment connectivity during the eukaryotic cell cycle.

Cell cycle progression relies on coordinated changes in the composition and subcellular localization of the proteome. By applying two distinct convolutional neural networks on images of millions of live yeast cells, we resolved proteome-level dynamics in both concentration and localization during the cell cycle, with resolution of ∼20 subcellular localization classes. We show that a quarter of the proteome displays cell cycle periodicity, with proteins tending to be controlled either at the level of localization or concentration, but not both. Distinct levels of protein regulation are preferentially utilized for different aspects of the cell cycle, with changes in protein concentration being mostly involved in cell cycle control and changes in protein localization in the biophysical implementation of the cell cycle program. We present a resource for exploring global proteome dynamics during the cell cycle, which will aid in understanding a fundamental biological process at a systems level.

K29-linked free polyubiquitin chains affect ribosome biogenesis and direct ribosomal proteins to the intranuclear quality control compartment.

Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs) Ubp2 and Ubp14, and E3 ligases Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the intranuclear quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with ribosomopathies.

Expanding TheCellVision.org: a central repository for visualizing and mining high-content cell imaging projects.

We previously constructed TheCellVision.org, a central repository for visualizing and mining data from yeast high-content imaging projects. At its inception, TheCellVision.org housed two high-content screening (HCS) projects providing genome-scale protein abundance and localization information for the budding yeast Saccharomyces cerevisiae, as well as a comprehensive analysis of the morphology of its endocytic compartments upon systematic genetic perturbation of each yeast gene. Here, we report on the expansion of TheCellVision.org by the addition of two new HCS projects and the incorporation of new global functionalities. Specifically, TheCellVision.org now hosts images from the Cell Cycle Omics project, which describes genome-scale cell cycle-resolved dynamics in protein localization, protein concentration, gene expression, and translational efficiency in budding yeast. Moreover, it hosts PIFiA, a computational tool for image-based predictions of protein functional annotations. Across all its projects, TheCellVision.org now houses >800,000 microscopy images along with computational tools for exploring both the images and their associated datasets. Together with the newly added global functionalities, which include the ability to query genes in any of the hosted projects using either yeast or human gene names, TheCellVision.org provides an expanding resource for single-cell eukaryotic biology.

The PRC2.1 Subcomplex Opposes G1 Progression through Regulation of CCND1 and CCND2.

Progression through the G1 phase of the cell cycle is the most highly regulated step in cellular division. We employed a chemogenomics approach to discover novel cellular networks that regulate cell cycle progression. This approach uncovered functional clusters of genes that altered sensitivity of cells to inhibitors of the G1/S transition. Mutation of components of the Polycomb Repressor Complex 2 rescued growth inhibition caused by the CDK4/6 inhibitor palbociclib, but not to inhibitors of S phase or mitosis. In addition to its core catalytic subunits, mutation of the PRC2.1 accessory protein MTF2, but not the PRC2.2 protein JARID2, rendered cells resistant to palbociclib treatment. We found that PRC2.1 (MTF2), but not PRC2.2 (JARID2), was critical for promoting H3K27me3 deposition at CpG islands genome-wide and in promoters. This included the CpG islands in the promoter of the CDK4/6 cyclins CCND1 and CCND2, and loss of MTF2 lead to upregulation of both CCND1 and CCND2. Our results demonstrate a role for PRC2.1, but not PRC2.2, in promoting G1 progression.

The TSC22D, WNK, and NRBP gene families exhibit functional buffering and evolved with Metazoa for cell volume regulation.

The ability to sense and respond to osmotic fluctuations is critical for the maintenance of cellular integrity. We used gene co-essentiality analysis to identify an unappreciated relationship between TSC22D2, WNK1, and NRBP1 in regulating cell volume homeostasis. All of these genes have paralogs and are functionally buffered for osmo-sensing and cell volume control. Within seconds of hyperosmotic stress, TSC22D, WNK, and NRBP family members physically associate into biomolecular condensates, a process that is dependent on intrinsically disordered regions (IDRs). A close examination of these protein families across metazoans revealed that TSC22D genes evolved alongside a domain in NRBPs that specifically binds to TSC22D proteins, which we have termed NbrT (NRBP binding region with TSC22D), and this co-evolution is accompanied by rapid IDR length expansion in WNK-family kinases. Our study reveals that TSC22D, WNK, and NRBP genes evolved in metazoans to co-regulate rapid cell volume changes in response to osmolarity.