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Progression through the G1 phase of the cell cycle is the most highly regulated step in cellular division. We employed a chemogenetic 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 proliferation 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 antagonizing G1 progression in a diversity of cell linages, including CML, breast cancer and immortalized cell lines.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Conflicts of interest: The author(s) declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility., (© 2023. Springer Nature Limited.)

CRISPR screens are used extensively to systematically interrogate the phenotype-to-genotype problem. In contrast to early CRISPR screens, which defined core cell fitness genes, most current efforts now aim to identify context-specific phenotypes that differentiate a cell line, genetic background, or condition of interest, such as a drug treatment. While CRISPR-related technologies have shown great promise and a fast pace of innovation, a better understanding of standards and methods for quality assessment of CRISPR screen results is crucial to guide technology development and application. Specifically, many commonly used metrics for quantifying screen quality do not accurately measure the reproducibility of context-specific hits. We highlight the importance of reporting reproducibility statistics that directly relate to the purpose of the screen and suggest the use of metrics that are sensitive to context-specific signal. A record of this paper's transparent peer review process is included in the supplemental information., Competing Interests: Declaration of interests Co-author Brenda Andrews is on the advisory board of Cell Systems., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Objective: Current surgical techniques use common surgical instruments for sterile shunt assembly. This study investigated the impact of using these techniques and surgical instruments on the mechanical integrity of the ventriculoperitoneal shunt system, specifically shunt catheters., Methods: The authors conducted failure testing on 85 rifampin-coated catheters and 85 barium-impregnated catheters using 5 different surgical instruments and 2 different surgical techniques. In technique A, the distal end of the catheter was pushed onto the shunt valve inlet connector with the surgical instrument. In technique B, the catheter was pulled over the inlet connector. One hundred sixty catheters underwent 10-repetition-maximum testing, in which the catheter either failed before completion of 10 consecutive assembly/disassembly repetitions or the catheter completed 10 consecutive repetitions. The authors also conducted 100-repetition-maximum tests on 5 barium-impregnated and 5 rifampin-coated catheters using technique A., Results: Catheter failure rates were significantly different among the different instruments used in assembly (p ≤ 0.001). Post hoc analysis showed that using mosquito forceps with shods resulted in a significantly lower catheter failure rate than the other instruments (p < 0.0005). The catheter failure rate of technique A was significantly lower than that of technique B (5% vs 81%, p < 0.001). There was no statistical difference between the failure rates of the barium and rifampin catheters (42% vs 44%, p = 0.9), but the barium catheters outperformed the rifampin catheters in the 100-repetition-maximum trials (p = 0.02). Instrument type (p = 0.0232) and catheter type (p = 0.0096) were both significant factors in determining the number of assembly/disassembly repetitions needed to cause catheter failure. It took an average of 2.79 repetitions of assembly/disassembly to cause catheter failure. DeBakey forceps had significantly lower mean repetitions to failure (mean 1.38) than the Gerald forceps without teeth (mean 2.73, p = 0.05) and bayonet (mean 3.25, p = 0.02)., Conclusions: This study was the first of its kind to analyze how physical manipulation affects the mechanical integrity of ventriculoperitoneal shunt components. The authors demonstrated a significantly lower shunt catheter failure rate when mosquito forceps with shods and technique A were used in assembly. Moreover, the authors established that use of DeBakey forceps resulted in failure with fewer assembly/disassembly repetitions than use of the Gerald and bayonet forceps, suggesting that DeBakey forceps are the most damaging instrument.

A network of transcription factors (TFs) coordinates transcription with cell cycle events in eukaryotes. Most TFs in the network are phosphorylated by cyclin-dependent kinase (CDK), which limits their activities during the cell cycle. Here, we investigate the physiological consequences of disrupting CDK regulation of the paralogous repressors Yhp1 and Yox1 in yeast. Blocking Yhp1/Yox1 phosphorylation increases their levels and decreases expression of essential cell cycle regulatory genes which, unexpectedly, increases cellular fitness in optimal growth conditions. Using synthetic genetic interaction screens, we find that Yhp1/Yox1 mutations improve the fitness of mutants with mitotic defects, including condensin mutants. Blocking Yhp1/Yox1 phosphorylation simultaneously accelerates the G1/S transition and delays mitotic exit, without decreasing proliferation rate. This mitotic delay partially reverses the chromosome segregation defect of condensin mutants, potentially explaining their increased fitness when combined with Yhp1/Yox1 phosphomutants. These findings reveal how altering expression of cell cycle genes leads to a redistribution of cell cycle timing and confers a fitness advantage to cells., Competing Interests: The authors have declared that no competing interests exist.

Objective: To determine the utility of blood symmetric dimethylarginine (SDMA) concentration measurement as a diagnostic tool for chronic kidney disease (CKD) in tigers (Panthera tigris) by comparing results for SDMA with those for traditional renal biomarkers and investigating correlations between these biomarkers and histopathologic kidney changes in tigers with CKD., Sample: Blood, urine, and kidney samples from 35 tigers with CKD from 2 sanctuaries., Procedures: Blood (serum or plasma) and urine samples were collected antemortem. Necropsy, including gross and histologic assessment, was performed for tigers that died or were euthanized for quality-of-life reasons. Results for CKD biomarkers in blood (BUN, creatinine, phosphorus, and SDMA concentrations) and urine (protein concentration, urine protein-to-creatinine ratio, and urine specific gravity) were evaluated for correlation with histologic kidney damage scored with an objective grading scale defined by percentage of inflammation, fibrosis, and tubular atrophy., Results: Symmetric dimethylarginine had the strongest significant correlation (ρ = 0.667) with histologic kidney damage score, followed by urine specific gravity (ρ = -0.639), blood creatinine concentration (ρ = 0.624), and BUN (ρ = 0.588). No significant correlation with kidney score was identified for blood phosphorus concentration, urine protein concentration, or the urine protein-to-creatinine ratio., Clinical Relevance: We recommend SDMA be prioritized as a renal biomarker in tigers, with SDMA results considered in addition to those of other traditional renal biomarkers when assessing kidney function in tigers. Additionally, the grading scale we developed could be replicated across patients and pathologists for more consistent postmortem assessment of CKD in tigers.

Morphological profiling is an omics-based approach for predicting intracellular targets of chemical compounds in which the dose-dependent morphological changes induced by the compound are systematically compared to the morphological changes in gene-deleted cells. In this study, we developed a reliable high-throughput (HT) platform for yeast morphological profiling using drug-hypersensitive strains to minimize compound use, HT microscopy to speed up data generation and analysis, and a generalized linear model to predict targets with high reliability. We first conducted a proof-of-concept study using six compounds with known targets: bortezomib, hydroxyurea, methyl methanesulfonate, benomyl, tunicamycin, and echinocandin B. Then we applied our platform to predict the mechanism of action of a novel diferulate-derived compound, poacidiene. Morphological profiling of poacidiene implied that it affects the DNA damage response, which genetic analysis confirmed. Furthermore, we found that poacidiene inhibits the growth of phytopathogenic fungi, implying applications as an effective antifungal agent. Thus, our platform is a new whole-cell target prediction tool for drug discovery., (© 2022. The Author(s).)

Single-cell image analysis provides a powerful approach for studying cell-to-cell heterogeneity, which is an important attribute of isogenic cell populations, from microbial cultures to individual cells in multicellular organisms. This phenotypic variability must be explained at a mechanistic level if biologists are to fully understand cellular function and address the genotype-to-phenotype relationship. Variability in single-cell phenotypes is obscured by bulk readouts or averaging of phenotypes from individual cells in a sample; thus, single-cell image analysis enables a higher resolution view of cellular function. Here, we consider examples of both small- and large-scale studies carried out with isogenic cell populations assessed by fluorescence microscopy, and we illustrate the advantages, challenges, and the promise of quantitative single-cell image analysis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which > 5,600 yeast genes are engineered for transcriptional inducibility with single-gene precision at their native loci and without plasmids. Each strain contains SGA screening markers and a unique barcode, enabling high-throughput genetics. We characterized YETI using growth phenotyping and BAR-seq screens, and we used a YETI allele to identify the regulon of Rof1, showing that it acts to repress transcription. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from eukaryotic and prokaryotic systems shows that native expression is a variable that can bias promoter-perturbing screens, including CRISPRi. We engineered a second expression system, Z 3 EB42, that gives lower expression than Z 3 EV, a feature enabling conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

We present FLEX (Functional evaluation of experimental perturbations), a pipeline that leverages several functional annotation resources to establish reference standards for benchmarking human genome-wide CRISPR screen data and methods for analyzing them. FLEX provides a quantitative measurement of the functional information captured by a given gene-pair dataset and a means to explore the diversity of functions captured by the input dataset. We apply FLEX to analyze data from the diverse cell line screens generated by the DepMap project. We identify a predominant mitochondria-associated signal within co-essentiality networks derived from these data and explore the basis of this signal. Our analysis and time-resolved CRISPR screens in a single cell line suggest that the variable phenotypes associated with mitochondria genes across cells may reflect screen dynamics and protein stability effects rather than genetic dependencies. We characterize this functional bias and demonstrate its relevance for interpreting differential hits in any CRISPR screening context. More generally, we demonstrate the utility of the FLEX pipeline for performing robust comparative evaluations of CRISPR screens or methods for processing them., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: Currently, bevacizumab (BEV), an antiangiogenic agent, is used as an adjunctive therapy to re-irradiation and surgery in patients with recurrent high-grade gliomas (rHGG). BEV has shown to decrease enhancement on MRI, but it is often unclear if these changes are due to tumor response to BEV or treatment-induced changes in the blood brain barrier. Preliminary studies show that amino acid PET can aid in distinguishing these changes on MRI., Methods: The authors performed a systematic review of PubMed and Embase through July 2020 with the search terms 'bevacizumab' or 'Avastin' and 'recurrent glioma' and 'PET,' yielding 38 papers, with 14 meeting inclusion criteria., Results: Thirteen out of fourteen studies included in this review used static PET and three studies used dynamic PET to evaluate the use of BEV in rHGG. Six studies used the amino acid tracer [18F]FET, four studies used [11C]MET, and four studies used [18F]FDOPA., Conclusion: [18F]FET, [11C]MET, and [18F]FDOPA PET in combination with MRI have shown promising results for improving accuracy in diagnosing tumor recurrence, detecting early treatment failure, and distinguishing between tumor progression and treatment-induced changes in patients with rHGG treated with BEV., (© The Author(s) 2021. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

The purpose of this study is to determine the tibiofemoral forces during functional electrical stimulation (FES) rowing in individuals with spinal cord injury (SCI). We analysed the motion of five participants with SCI during FES rowing, with simultaneous measurements of (i) three-dimensional marker trajectories, (ii) foot reaction forces (FRFs), (iii) ergometer handle forces, and (iv) timestamps for electrical stimulation of the quadriceps and hamstrings muscles. We created full-body musculoskeletal models in OpenSim to determine subject-specific tibiofemoral forces during FES rowing. The peak magnitudes of tibiofemoral forces averaged over five participants with SCI were 2.43 ± 0.39 BW and 2.25 ± 0.71 BW for the left and right legs, respectively. The peak magnitudes of FRFs were 0.19 ± 0.04 BW in each leg. The peak magnitude of handle forces was 0.47 ± 0.19 BW. Peak tibiofemoral force was associated with peak FRF (magnitudes, R 2 = 0.56, p = 0.013) and peak handle force (magnitudes, R 2 = 0.54, p = 0.016). The ratios of peak magnitude of tibiofemoral force to peak magnitude of FRF were 12.9 ± 1.9 (left) and 11.6 ± 2.4 (right), and to peak magnitude of handle force were 5.7 ± 2.3 (left) and 4.9 ± 0.9 (right). This work lays the foundation for developing a direct exercise intensity metric for bone mechanical stimulus at the knee during rehabilitation exercises. Clinical Significance: Knowledge of tibiofemoral forces from exercises such as FES rowing may provide clinicians the ability to personalize rehabilitation protocols to ensure that an SCI patient is receiving the minimum dose of mechanical stimulus necessary to maintain bone health.

Systematic complex genetic interaction studies have provided insight into high-order functional redundancies and genetic network wiring of the cell. Here, we describe a method for screening and quantifying trigenic interactions from ordered arrays of yeast strains grown on agar plates as individual colonies. The protocol instructs users on the trigenic synthetic genetic array analysis technique, τ-SGA, for high-throughput screens. The steps describe construction of the double-mutant query strains and the corresponding single-mutant control query strains, which are screened in parallel in two replicates. The screening experimental set-up consists of sequential replica-pinning steps that enable automated mating, meiotic recombination and successive haploid selection steps for the generation of triple mutants, which are scored for colony size as a proxy for fitness, which enables the calculation of trigenic interactions. The procedure described here was used to conduct 422 trigenic interaction screens, which generated ~460,000 yeast triple mutants for trigenic interaction analysis. Users should be familiar with robotic equipment required for high-throughput genetic interaction screens and be proficient at the command line to execute the scoring pipeline. Large-scale screen computational analysis is achieved by using MATLAB pipelines that score raw colony size data to produce τ-SGA interaction scores. Additional recommendations are included for optimizing experimental design and analysis of smaller-scale trigenic interaction screens by using a web-based analysis system, SGAtools. This protocol provides a resource for those who would like to gain a deeper, more practical understanding of trigenic interaction screening and quantification methodology.

Complex genetic interactions occur when mutant alleles of multiple genes combine to elicit an unexpected phenotype, which could not be predicted given the expectation based on the combination of phenotypes associated with individual mutant alleles. Trigenic Synthetic Genetic Array (τ-SGA) methodology was developed for the systematic analysis of complex interactions involving combinations of three gene perturbations. With a series of replica pinning steps of the τ-SGA procedure, haploid triple mutants are constructed through automated mating and meiotic recombination. For example, a double-mutant query strain carrying two mutant alleles of interest, such as a deletion allele of a nonessential gene and a conditional temperature-sensitive allele of an essential gene, is crossed to an input array of yeast mutants, such as the diagnostic array set of ~1200 mutants, to generate an output array of triple mutants. The colony-size measurements of the resulting triple mutants are used to estimate cellular fitness and quantify trigenic interactions by incorporating corresponding single- and double-mutant fitness estimates. Trigenic interaction networks can be further analyzed for functional modules using various clustering and enrichment analysis tools. Complex genetic interactions are rich in functional information and provide insight into the genotype-to-phenotype relationship, genome size, and speciation.

We describe a protocol for live-cell high-throughput (HTP) screening of yeast mutant strains carrying fluorescent protein markers for subcellular compartments of choice using automated confocal microscopy. This procedure, which combines HTP genetics and microscopy, results in the acquisition of thousands of images that can be analyzed in a systematic and quantitative way to identify morphology defects in the tagged subcellular compartments. This HTP protocol is readily adapted for screening any combination of markers and can be expanded to different growth conditions or higher order mutant genetic backgrounds.

Objective  Primary repair of posteriorly located anterior skull base (ASB) dural defects following cranial trauma is made difficult by narrow operative corridors and adherent dura mater. Inadequate closure may result in continued cerebrospinal fluid (CSF) leak and infectious sequelae. Here, we report surgical outcomes following the use of nonpenetrating titanium microclips as an adjunctive repair technique in traumatic anterior skull base dural defects extending from the olfactory groove to the tuberculum sellae. Methods  All trauma patients who underwent a bifrontal craniotomy from January 2013 to October 2019 were retrospectively reviewed. Patients with ASB defects located at posterior to the olfactory groove were analyzed. Patients with isolated frontal sinus fractures were excluded. All patients presented with CSF leak or radiographic signs of dural compromise. Patients were divided according to posterior extent of injury. Patient characteristics, imaging, surgical technique, and outcomes are reported. Results  A total of 19 patients who underwent a bifrontal craniotomy for repair of posteriorly located ASB dural defects using nonpenetrating titanium microclips were included. Defects were divided by location: olfactory groove (10/19), planum sphenoidale (6/19), and tuberculum sellae (3/19). No patients demonstrated a postoperative CSF leak. No complications related to the microclip technique was observed. Clip artifact did not compromise postoperative imaging interpretation. Conclusion  Primary repair of posteriorly located ASB dural defects is challenging due to narrow working angles and thin dura mater. Use of nonpenetrating titanium microclips for primary repair of posteriorly located dural defects is a reasonable adjunctive repair technique and was associated with no postoperative CSF leaks in this cohort., Competing Interests: Conflict of Interest None declared., (Thieme. All rights reserved.)

Advances in genome engineering and high throughput imaging technologies have enabled genome-scale screens of single cells for a variety of phenotypes, including subcellular morphology and protein localization. We constructed TheCellVision.org, a freely available and web-accessible image visualization and data browsing tool that serves as a central repository for fluorescence microscopy images and associated quantitative data produced by high-content screening experiments. Currently, TheCellVision.org hosts ∼575,590 images and associated analysis results from two published high-content screening (HCS) projects focused on the budding yeast Saccharomyces cerevisiae TheCellVision.org allows users to access, visualize and explore fluorescence microscopy images, and to search, compare, and extract data related to subcellular compartment morphology, protein abundance, and localization. Each dataset can be queried independently or as part of a search across multiple datasets using the advanced search option. The website also hosts computational tools associated with the available datasets, which can be applied to other projects and cell systems, a feature we demonstrate using published images of mammalian cells. Providing access to HCS data through websites such as TheCelllVision.org enables new discovery and independent re-analyses of imaging data., (Copyright © 2020 Masinas et al.)

Overproduction (op) of proteins triggers cellular defects. One of the consequences of overproduction is the protein burden/cost, which is produced by an overloading of the protein synthesis process. However, the physiology of cells under a protein burden is not well characterized. We performed genetic profiling of protein burden by systematic analysis of genetic interactions between GFP-op, surveying both deletion and temperature-sensitive mutants in budding yeast. We also performed genetic profiling in cells with overproduction of triple-GFP (tGFP), and the nuclear export signal-containing tGFP (NES-tGFP). The mutants specifically interacted with GFP-op were suggestive of unexpected connections between actin-related processes like polarization and the protein burden, which was supported by morphological analysis. The tGFP-op interactions suggested that this protein probe overloads the proteasome, whereas those that interacted with NES-tGFP involved genes encoding components of the nuclear export process, providing a resource for further analysis of the protein burden and nuclear export overload., Competing Interests: RK, KM, SN, HK, KK, SO, YO, BA, CB, HM No competing interests declared, (© 2020, Kintaka et al.)

Essential genes tend to be highly conserved across eukaryotes, but, in some cases, their critical roles can be bypassed through genetic rewiring. From a systematic analysis of 728 different essential yeast genes, we discovered that 124 (17%) were dispensable essential genes. Through whole-genome sequencing and detailed genetic analysis, we investigated the genetic interactions and genome alterations underlying bypass suppression. Dispensable essential genes often had paralogs, were enriched for genes encoding membrane-associated proteins, and were depleted for members of protein complexes. Functionally related genes frequently drove the bypass suppression interactions. These gene properties were predictive of essential gene dispensability and of specific suppressors among hundreds of genes on aneuploid chromosomes. Our findings identify yeast's core essential gene set and reveal that the properties of dispensable essential genes are conserved from yeast to human cells, correlating with human genes that display cell line-specific essentiality in the Cancer Dependency Map (DepMap) project., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Whole-genome duplication has played a central role in the genome evolution of many organisms, including the human genome. Most duplicated genes are eliminated, and factors that influence the retention of persisting duplicates remain poorly understood. We describe a systematic complex genetic interaction analysis with yeast paralogs derived from the whole-genome duplication event. Mapping of digenic interactions for a deletion mutant of each paralog, and of trigenic interactions for the double mutant, provides insight into their roles and a quantitative measure of their functional redundancy. Trigenic interaction analysis distinguishes two classes of paralogs: a more functionally divergent subset and another that retained more functional overlap. Gene feature analysis and modeling suggest that evolutionary trajectories of duplicated genes are dictated by combined functional and structural entanglement factors., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The de novo synthesis of fatty acids has emerged as a therapeutic target for various diseases, including cancer. Because cancer cells are intrinsically buffered to combat metabolic stress, it is important to understand how cells may adapt to the loss of de novo fatty acid biosynthesis. Here, we use pooled genome-wide CRISPR screens to systematically map genetic interactions (GIs) in human HAP1 cells carrying a loss-of-function mutation in fatty acid synthase (FASN), whose product catalyses the formation of long-chain fatty acids. FASN-mutant cells show a strong dependence on lipid uptake that is reflected in negative GIs with genes involved in the LDL receptor pathway, vesicle trafficking and protein glycosylation. Further support for these functional relationships is derived from additional GI screens in query cell lines deficient in other genes involved in lipid metabolism, including LDLR, SREBF1, SREBF2 and ACACA. Our GI profiles also identify a potential role for the previously uncharacterized gene C12orf49 (which we call LUR1) in regulation of exogenous lipid uptake through modulation of SREBF2 signalling in response to lipid starvation. Overall, our data highlight the genetic determinants underlying the cellular adaptation associated with loss of de novo fatty acid synthesis and demonstrate the power of systematic GI mapping for uncovering metabolic buffering mechanisms in human cells.

Objective : To determine the effect of a functional electrical stimulation (FES) rowing program on bone mineral density (BMD) when implemented within two years after SCI. Design : Prospective. Setting : Health Care Facility. Participants : Convenience sample; four adults with recent (<2 years) traumatic, motor complete SCI (C7-T12 AIS A-B). Intervention : A 90-session FES rowing exercise program; participants attended 30-minute FES training sessions approximately three times each week for the duration of their participation. Outcome Measures : BMD in the distal femur and tibia were measured using peripheral Quantitative Computed Tomography (pQCT) at enrollment (T 0 ) and after 30 (T 1 ), 60 (T 2 ), and 90 (T 3 ) sessions. Bone stimulus was calculated for each rower at each time point using the average number of weekly loading cycles, peak foot reaction force, and bone mineral content from the previous time point. A regression analysis was used to determine the relationship between calculated bone stimulus and change in femoral trabecular BMD between time points. Results : Trabecular BMD in the femur and tibia decreased for all participants in T 0-1 , but the rate of loss slowed or reversed between T 1-2 , with little-to-no bone loss for most participants during T 2-3 . The calculated bone stimulus was significantly correlated with change in femoral trabecular BMD (P = 0.016; R 2  = 0.458). Conclusion : Consistent participation in an FES rowing program provides sufficient forces and loading cycles to reduce or reverse expected bone loss at the distal femur and tibia, at least temporarily, in some individuals within two years after SCI. Trial Registration : NCT02008149.

Our ability to understand the genotype-to-phenotype relationship is hindered by the lack of detailed understanding of phenotypes at a single-cell level. To systematically assess cell-to-cell phenotypic variability, we combined automated yeast genetics, high-content screening and neural network-based image analysis of single cells, focussing on genes that influence the architecture of four subcellular compartments of the endocytic pathway as a model system. Our unbiased assessment of the morphology of these compartments-endocytic patch, actin patch, late endosome and vacuole-identified 17 distinct mutant phenotypes associated with ~1,600 genes (~30% of all yeast genes). Approximately half of these mutants exhibited multiple phenotypes, highlighting the extent of morphological pleiotropy. Quantitative analysis also revealed that incomplete penetrance was prevalent, with the majority of mutants exhibiting substantial variability in phenotype at the single-cell level. Our single-cell analysis enabled exploration of factors that contribute to incomplete penetrance and cellular heterogeneity, including replicative age, organelle inheritance and response to stress., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The phenotypic consequence of a given mutation can be influenced by the genetic background. For example, conditional gene essentiality occurs when the loss of function of a gene causes lethality in one genetic background but not another. Between two individual Saccharomyces cerevisiae strains, S288c and Σ1278b, ∼1% of yeast genes were previously identified as "conditional essential." Here, in addition to confirming that some conditional essential genes are modified by a nonchromosomal element, we show that most cases involve a complex set of genomic modifiers. From tetrad analysis of S288C/Σ1278b hybrid strains and whole-genome sequencing of viable hybrid spore progeny, we identified complex sets of multiple genomic regions underlying conditional essentiality. For a smaller subset of genes, including CYS3 and CYS4 , each of which encodes components of the cysteine biosynthesis pathway, we observed a segregation pattern consistent with a single modifier associated with conditional essentiality. In natural yeast isolates, we found that the CYS3 / CYS4 conditional essentiality can be caused by variation in two independent modifiers, MET1 and OPT1 , each with roles associated with cellular cysteine physiology. Interestingly, the OPT1 allelic variation appears to have arisen independently from separate lineages, with rare allele frequencies below 0.5%. Thus, while conditional gene essentiality is usually driven by genetic interactions associated with complex modifier architectures, our analysis also highlights the role of functionally related, genetically independent, and rare variants., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Gram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host cells to promote pathogen fitness by manipulating host cellular processes. Despite their crucial role in promoting virulence, relatively few T3SEs have well-characterized enzymatic activities or host targets. This is in part due to functional redundancy within pathogen T3SE repertoires as well as the promiscuity of individual T3SEs that can have multiple host targets. To overcome these challenges, we generated and characterized a collection of yeast strains stably expressing 75 T3SE constructs from the plant pathogen Pseudomonas syringae This collection is devised to facilitate heterologous genetic screens in yeast, a non-host organism, to identify T3SEs that target conserved eukaryotic processes. Among 75 T3SEs tested, we identified 16 that inhibited yeast growth on rich media and eight that inhibited growth on stress-inducing media. We utilized Pathogenic Genetic Array (PGA) screens to identify potential host targets of P. syringae T3SEs. We focused on the acetyltransferase, HopZ1a, which interacts with plant tubulin and alters microtubule networks. To uncover putative HopZ1a host targets, we identified yeast genes with genetic interaction profiles most similar ( i.e. , congruent) to the PGA profile of HopZ1a and performed a functional enrichment analysis of these HopZ1a-congruent genes. We compared the congruence analyses above to previously described HopZ physical interaction datasets and identified kinesins as potential HopZ1a targets. Finally, we demonstrated that HopZ1a can target kinesins by acetylating the plant kinesins HINKEL and MKRP1, illustrating the utility of our T3SE-expressing yeast library to characterize T3SE functions., (Copyright © 2019 Lee et al.)

The genotype-to-phenotype relationship in health and disease is complex and influenced by both an individual's environment and their unique genome. Personal genetic variants can modulate gene function to generate a phenotype either through a single gene effect or through genetic interactions involving two or more genes. The relevance of genetic interactions to disease phenotypes has been particularly clear in cancer research, where an extreme genetic interaction, synthetic lethality, has been exploited as a therapeutic strategy. The obvious benefits of unmasking genetic background-specific vulnerabilities, coupled with the power of systematic genome editing, have fueled efforts to translate genetic interaction mapping from model organisms to human cells. Here, we review recent developments in genetic interaction mapping, with a focus on CRISPR-based genome editing technologies and cancer., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Systematic experimental approaches have led to construction of comprehensive genetic and protein-protein interaction networks for the budding yeast, Saccharomyces cerevisiae. Genetic interactions capture functional relationships between genes using phenotypic readouts, while protein-protein interactions identify physical connections between gene products. These complementary, and largely non-overlapping, networks provide a global view of the functional architecture of a cell, revealing general organizing principles, many of which appear to be evolutionarily conserved. Here, we focus on insights derived from the integration of large-scale genetic and protein-protein interaction networks, highlighting principles that apply to both unicellular and more complex systems, including human cells. Network integration reveals fundamental connections involving key functional modules of eukaryotic cells, defining a core network of cellular function, which could be elaborated to explore cell-type specificity in metazoans., (Copyright © 2018. Published by Elsevier Ltd.)

The phenotypic consequences of a given mutation can vary across individuals. This so-called background effect is widely observed, from mutant fitness of loss-of-function variants in model organisms to variable disease penetrance and expressivity in humans; however, the underlying genetic basis often remains unclear. Taking insights gained from recent large-scale surveys of genetic interaction and suppression analyses in yeast, we propose that the genetic network context for a given mutation may shape its propensity of exhibiting background-dependent phenotypes. We argue that further efforts in systematically mapping the genetic interaction networks beyond yeast will provide not only key insights into the functional properties of genes, but also a better understanding of the background effects and the (un)predictability of traits in a broader context., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

To systematically explore complex genetic interactions, we constructed ~200,000 yeast triple mutants and scored negative trigenic interactions. We selected double-mutant query genes across a broad spectrum of biological processes, spanning a range of quantitative features of the global digenic interaction network and tested for a genetic interaction with a third mutation. Trigenic interactions often occurred among functionally related genes, and essential genes were hubs on the trigenic network. Despite their functional enrichment, trigenic interactions tended to link genes in distant bioprocesses and displayed a weaker magnitude than digenic interactions. We estimate that the global trigenic interaction network is ~100 times as large as the global digenic network, highlighting the potential for complex genetic interactions to affect the biology of inheritance, including the genotype-to-phenotype relationship., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., Competing Interests: AL, YC, IH, BS, LH, OK, BA, AM No competing interests declared, (© 2018, Lu et al.)

Fluorescent reporter genes have long been used to quantify various cell features such as transcript and protein abundance. Here, we describe a method, reporter synthetic genetic array (R-SGA) analysis, which allows for the simultaneous quantification of any fluorescent protein readout in thousands of yeast strains using an automated pipeline. R-SGA combines a fluorescent reporter system with standard SGA analysis and can be used to examine any array-based strain collection available to the yeast community. This protocol describes the R-SGA methodology for screening different arrays of yeast mutants including the deletion collection, a collection of temperature-sensitive strains for the assessment of essential yeast genes and a collection of inducible overexpression strains. We also present an alternative pipeline for the analysis of R-SGA output strains using flow cytometry of cells in liquid culture. Data normalization for both pipelines is discussed.

Genetic interactions occur when the combination of multiple mutations yields an unexpected phenotype, and they may confound our ability to fully understand the genetic mechanisms underlying complex diseases. Genetic interactions are challenging to study because there are millions of possible different variant combinations within a given genome. Consequently, they have primarily been systematically explored in unicellular model organisms, such as yeast, with a focus on pairwise genetic interactions between loss-of-function alleles. However, there are many different types of genetic interactions, such as those occurring between gain-of-function or heterozygous mutations. Here, we review recent advances made in the systematic analysis of such diverse genetic interactions in yeast, and briefly discuss how similar studies could be undertaken in human cells.

Genes encoding essential components of core cellular processes are typically highly conserved across eukaryotes. However, a small proportion of essential genes are highly taxonomically restricted; there appear to be no similar genes outside the genomes of highly related species. What are the functions of these poorly characterized taxonomically restricted genes (TRGs)? Systematic screens in Saccharomyces cerevisiae and Caenorhabditis elegans previously identified yeast or nematode TRGs that are essential for viability and we find that these genes share many molecular features, despite having no significant sequence similarity. Specifically, we find that those TRGs with essential phenotypes have an expression profile more similar to highly conserved genes, they have more protein-protein interactions and more protein disorder. Surprisingly, many TRGs play central roles in chromosome segregation; a core eukaryotic process. We thus find that genes that appear to be highly evolutionarily restricted do not necessarily play roles in species-specific biological functions but frequently play essential roles in core eukaryotic processes., (Copyright © 2017 Verste et al.)

The capacity to coordinate environmental sensing with initiation of cellular responses underpins microbial survival and is crucial for virulence and stress responses in microbial pathogens. Here we define circuitry that enables the fungal pathogen Candida albicans to couple cell cycle dynamics with responses to cell wall stress induced by echinocandins, a front-line class of antifungal drugs. We discover that the C. albicans transcription factor Cas5 is crucial for proper cell cycle dynamics and responses to echinocandins, which inhibit β-1,3-glucan synthesis. Cas5 has distinct transcriptional targets under basal and stress conditions, is activated by the phosphatase Glc7, and can regulate the expression of target genes in concert with the transcriptional regulators Swi4 and Swi6. Thus, we illuminate a mechanism of transcriptional control that couples cell wall integrity with cell cycle regulation, and uncover circuitry governing antifungal drug resistance.Cas5 is a transcriptional regulator of responses to cell wall stress in the fungal pathogen Candida albicans. Here, Xie et al. show that Cas5 also modulates cell cycle dynamics and responses to antifungal drugs.

The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols. In this study, we reanalyze 17 genome-scale knockout screens in human cell lines from three research groups, using three different genome-scale gRNA libraries. Using the Bayesian Analysis of Gene Essentiality algorithm to identify essential genes, we refine and expand our previously defined set of human core essential genes from 360 to 684 genes. We use this expanded set of reference core essential genes, CEG2, plus empirical data from six CRISPR knockout screens to guide the design of a sequence-optimized gRNA library, the Toronto KnockOut version 3.0 (TKOv3) library. We then demonstrate the high effectiveness of the library relative to reference sets of essential and nonessential genes, as well as other screens using similar approaches. The optimized TKOv3 library, combined with the CEG2 reference set, provide an efficient, highly optimized platform for performing and assessing gene knockout screens in human cell lines., (Copyright © 2017 Hart et al.)

Recent analysis of genome sequences has identified individuals that are healthy despite carrying severe disease-associated mutations. A possible explanation is that these individuals carry a second genomic perturbation that can compensate for the detrimental effects of the disease allele, a phenomenon referred to as suppression. In model organisms, suppression interactions are generally divided into two classes: genomic suppressors which are secondary mutations in the genome that bypass a mutant phenotype, and dosage suppression interactions in which overexpression of a suppressor gene rescues a mutant phenotype. Here, we describe the general properties of genomic and dosage suppression, with an emphasis on the budding yeast. We propose that suppression interactions between genetic variants are likely relevant for determining the penetrance of human traits. Consequently, an understanding of suppression mechanisms may guide the discovery of protective variants in healthy individuals that carry disease alleles, which could direct the rational design of new therapeutics., (© 2017 WILEY Periodicals, Inc.)

To better understand the health implications of personal genomes, we now face a largely unmet challenge to identify functional variants within disease-associated genes. Functional variants can be identified by trans-species complementation, e.g., by failure to rescue a yeast strain bearing a mutation in an orthologous human gene. Although orthologous complementation assays are powerful predictors of pathogenic variation, they are available for only a few percent of human disease genes. Here we systematically examine the question of whether complementation assays based on paralogy relationships can expand the number of human disease genes with functional variant detection assays. We tested over 1,000 paralogous human-yeast gene pairs for complementation, yielding 34 complementation relationships, of which 33 (97%) were novel. We found that paralog-based assays identified disease variants with success on par with that of orthology-based assays. Combining all homology-based assay results, we found that complementation can often identify pathogenic variants outside the homologous sequence region, presumably because of global effects on protein folding or stability. Within our search space, paralogy-based complementation more than doubled the number of human disease genes with a yeast-based complementation assay for disease variation.