Biology subjects

Keys, H. R.

Publications and source records attributed to Keys, H. R..

13 recordsLinked to original sources

bioRxiv2026

Systematic functional genetic analysis of cell-substrate adhesion

Cellular adhesion is critical for tissue organization and integrity, but the full complement of proteins required for proper adhesion remains unresolved. Here, we define the requirements for cell-substrate adhesion in cultured human cells using orthogonal, large-scale functional genetic approaches. Using mechanical assays to test the maintenance ("shake-off") or formation of adhesion and parallel large-scale assays of cell morphology, we identify dozens of gene targets with roles in adhesion. Our analyses reveal dynamic requirements for adhesion across timepoints and cell lines. We additionally conduct targeted downstream mechanistic analyses to resolve the molecular basis for altered adhesion. Collectively, we identify established and uncharacterized regulators of adhesion, including genes involved in mitosis, focal adhesions, actin regulation, and membrane trafficking. Unexpectedly, we find that cells that fail cytokinesis display impaired adhesion, with strongly altered actin organization and nuclear dynamics. Together, this work provides a comprehensive view of the genetic requirements for cell-substrate adhesion.

cell biology↗

bioRxiv2025

Lysosomal RNA profiling reveals targeting of specific types of RNAs for degradation

Autophagy targets a wide variety of substrates for degradation within lysosomes1. While lysosomes are known to possess RNase activity2, the role of lysosomal RNA degradation in post-transcriptional gene regulation is not well understood. Here, we define RNASET2, PLD3, and both endogenous and exogenous RNase A family members as lysosomal RNases. Cells lacking these RNases accumulated large amounts of lysosomal RNA. Although all types of RNA can be found within lysosomes, SRP RNAs, Y RNAs, 5' TOP mRNAs, long-lived mRNAs, and mRNAs encoding membrane and secreted proteins were specifically enriched. All types of RNA depend on autophagy for lysosomal targeting, but the lysosomally-enriched RNAs are more sensitive to loss of autophagy, implying that selective mechanisms mediate their lysosomal entry. RNA stability measurements revealed that lysosomally-degraded transcripts also had autophagy-dependent changes in stability. In exploring how specific RNAs are targeted for lysosomal degradation, we found that the Alu domain of SRP RNAs is sufficient for targeting these RNAs to lysosomes in fashion that depends on its interactions with the SRP9 and SRP14 proteins. For mRNAs, 5' TOP motifs are sufficient to increase their targeting to lysosomes for degradation in a LARP1-dependent manner. Altogether, our results establish lysosomes as selective modulators of cellular RNA content.

molecular biology↗

bioRxiv2025

Functional genetics with a hypomorphic CENP-C mutant reveals a regulatory system for chromosome congression

Chromosome congression is a key process that acts to align chromosomes at the spindle equator via kinetochore-microtubule interactions, with defects in chromosome alignment leading to chromosomal instability. However, defining the mechanisms that underlie chromosome congression is limited due to the multiple factors that act in parallel to regulate chromosome movement. Here, we conducted a genome-wide Cas9-based functional genetics screen using a hypomorphic CENP-C mutant that affects its kinetochore interactions. Our analysis identified KIF18A, whose knockout resulted in synthetic lethality with the CENP-C mutant. Further analysis revealed that the synthetic defect was due to a reduction in CENP-E function in the CENP-C mutant. Our work suggests that KIF18A promotes chromosome alignment in cooperation with CENP-E downstream of CENP-C during early prometaphase. Thus, our analysis enables us to dissect parallel molecular mechanisms for chromosome congression and identify sensitivities and biomarkers that might guide anti-KIF18A chemotherapeutics.

cell biology↗

bioRxiv2025

Polyamines buffer labile iron to suppress ferroptosis

Polyamines are essential and evolutionarily conserved metabolites present at millimolar concentrations in mammalian cells. Cells tightly regulate polyamine homeostasis through complex feedback mechanisms, yet the precise role necessitating this regulation remains unclear. Here, we show that polyamines contribute to endogenous buffering of redox-active iron, providing a molecular link between polyamine metabolism and ferroptosis. Using a genome-wide CRISPR screen, we identified a synthetic lethal dependency between polyamine depletion and the key ferroptosis suppressor, GPX4. Mechanistically, we show that polyamine deficiency triggers a redistribution of cellular iron, increasing the labile iron pool and upregulating ferritin. To directly visualize this iron buffering in living cells, we developed a genetically encoded fluorescent reporter for redox-active iron. Live-cell analysis revealed a striking inverse correlation between intracellular polyamine levels and redox-active iron at single-cell resolution. These findings reposition polyamines as key regulators of iron homeostasis, with implications for ferroptosis-linked disease states and cellular redox balance.

cell biology↗

bioRxiv2024

A genetically encoded fluorescent reporter for polyamines

Polyamines are abundant and evolutionarily conserved metabolites that are essential for life. Dietary polyamine supplementation extends life-span and health-span. Dysregulation of polyamine homeostasis is linked to Parkinsons disease and cancer, driving interest in therapeutically targeting this pathway. However, measuring cellular polyamine levels, which vary across cell types and states, remains challenging. We introduce a first-in-class genetically encoded polyamine reporter for real-time measurement of polyamine concentrations in single living cells. This reporter utilizes the polyamine-responsive ribosomal frameshift motif from the OAZ1 gene. We demonstrate broad applicability of this approach and reveal dynamic changes in polyamine levels in response to genetic and pharmacological perturbations. Using this reporter, we conducted a genome-wide CRISPR screen and uncovered an unexpected link between mitochondrial respiration and polyamine import, which are both risk factors for Parkinsons disease. By offering a new lens to examine polyamine biology, this reporter may advance our understanding of these ubiquitous metabolites and accelerate therapy development.

cell biology↗

bioRxiv2024

Multi-species genome-wide CRISPR screens identify GPX4 as a conserved suppressor of cold-induced cell death

Cells must adapt to environmental changes to maintain homeostasis. One of the most striking environmental adaptations is entry into hibernation during which core body temperature can decrease from 37C to as low at 4C. How mammalian cells, which evolved to optimally function within a narrow range of temperatures, adapt to this profound decrease in temperature remains poorly understood. In this study, we conducted the first genome-scale CRISPR-Cas9 screen in cells derived from Syrian hamster, a facultative hibernator, as well as human cells to investigate the genetic basis of cold tolerance in a hibernator and a non-hibernator in an unbiased manner. Both screens independently revealed glutathione peroxidase 4 (GPX4), a selenium-containing enzyme, and associated proteins as critical for cold tolerance. We utilized genetic and pharmacological approaches to demonstrate that GPX4 is active in the cold and its catalytic activity is required for cold tolerance. Furthermore, we show that the role of GPX4 as a suppressor of cold-induced cell death extends across hibernating species, including 13-lined ground squirrels and greater horseshoe bats, highlighting the evolutionary conservation of this mechanism of cold tolerance. This study identifies GPX4 as a central modulator of mammalian cold tolerance and advances our understanding of the evolved mechanisms by which cells mitigate cold-associated damage - one of the most common challenges faced by cells and organisms in nature.

cell biology↗

bioRxiv2024

Functional genetics reveals modulators of anti-microtubule drug sensitivity

Microtubules play essential roles in diverse cellular processes and are important pharmacological targets for treating human disease. Here, we sought to identify cellular factors that modulate the sensitivity of cells to anti-microtubule drugs. We conducted a genome-wide CRISPR/Cas9-based functional genetics screen in human cells treated with the microtubule-destabilizing drug nocodazole or the microtubule-stabilizing drug taxol. We further conducted a focused secondary screen to test drug sensitivity for [~]1400 gene targets across two distinct human cell lines and to additionally test sensitivity to the Kif11-inhibitor, STLC. These screens defined gene targets whose loss enhances or suppresses sensitivity to anti-microtubule drugs. In addition to gene targets whose loss sensitized cells to multiple compounds, we observed cases of differential sensitivity to specific compounds and differing requirements between cell lines. Our downstream molecular analysis further revealed additional roles for established microtubule-associated proteins and identified new players in microtubule function.

cell biology↗

bioRxiv2021

Coupled protein quality control during nonsense mediated mRNA decay

Translation of mRNAs containing premature termination codons (PTCs) can result in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance path-way responsible for detecting and degrading PTC containing transcripts. While the molecular mechanisms governing mRNA degradation have been extensively studied, the fate of the nascent protein product remains largely uncharacterized. Here, we use a fluorescent reporter system in mammalian cells to reveal a selective degradation pathway specifically targeting the protein product of an NMD mRNA. We show that this process is post-translational, and dependent on an intact ubiquitin proteasome system. To systematically uncover factors involved in NMD-linked protein quality control, we conducted genome-wide flow cytometry-based screens. Our screens recovered known NMD factors, and suggested a lack of dependence on the canonical ribosome-quality control (RQC) pathway. Finally, one of the strongest hits in our screens was the E3 ubiquitin ligase CNOT4, a member of the CCR4-NOT complex, which is involved in initiating mRNA degradation. We show that CNOT4 is involved in NMD coupled protein degradation, and its role depends on a functional RING ubiquitin ligase domain. Our results demonstrate the existence of a targeted pathway for nascent protein degradation from PTC containing mRNAs, and provide a framework for identifying and characterizing factors involved in this process.

molecular biology↗

bioRxiv2021

Co-deletion of ATAD1 with PTEN primes cells for BIM-mediated apoptosis

PTEN is a potent tumor suppressor gene that is frequently mutated or deleted in human cancers. Such deletions often include portions of the 10q23 locus beyond the bounds of PTEN itself, in many cases resulting in the disruption of additional genes. Coincidental loss of PTEN-adjacent genes might impose vulnerabilities that could either affect patient outcome basally or be exploited therapeutically. Here we describe how the loss of ATAD1, which is adjacent to and frequently co-deleted with PTEN, predisposes cancer cells to apoptosis and correlates with improved survival in cancer patients. ATAD1 directly and specifically extracts the pro-apoptotic BIM protein from mitochondria to inactivate it. Cells lacking ATAD1 are hypersensitive to clinically used proteasome inhibitors, which increase BIM and trigger apoptosis. Thus, we demonstrate that mitochondrial protein quality control interfaces with cell death in a clinically actionable manner.

cancer biology↗

bioRxiv2021

A genome-wide screen in the mouse liver reveals sex-specific and cell non-autonomous regulation of cell fitness

A complete understanding of the genetic determinants underlying mammalian physiology and disease is limited by the availability of tools for high-throughput genetic dissection in the living organism. Genome-wide screening using CRISPR-Cas9 has emerged as a powerful method for uncovering the genetic regulation of cellular processes, but the need to stably deliver single guide RNAs to millions of cells has largely restricted its implementation to ex vivo systems. There thus remains a pressing need for accessible high-throughput functional genomics in a living organism. Here, we establish genome-wide enrichment and depletion screening in the liver of a single mouse and use this approach to uncover the complete regulation of cellular fitness in a living organism. We uncover regulation of cell fitness not previously identified in cell culture screens, underscoring the power of bringing high-throughput functional genomics into the organism. The approach we developed is accessible, robust, scalable, and adaptable to diverse phenotypes and CRISPR applications. We have hereby established a foundation for high-throughput functional genomics in a living mammal, enabling unprecedented insight into physiology and disease.

genomics↗

bioRxiv2020

CRISPR screens in physiologic medium reveal conditionally essential genes in human cells

Forward genetic screens across hundreds of diverse cancer cell lines have started to define the genetic dependencies of proliferating human cells and how these vary by genotype and lineage. Most screens, however, have been carried out in culture media that poorly resemble metabolite availability in human blood. To explore how medium composition influences gene essentiality, we performed CRISPR-based screens of human cancer cell lines cultured in traditional versus human plasma-like medium (HPLM). Sets of medium-dependent fitness genes span several cellular processes and can vary with both natural cell-intrinsic diversity and the specific combination of basal and serum components that comprise typical culture media. Notably, we traced the causes for each of three conditional growth phenotypes to the availability of metabolites uniquely defined in HPLM versus traditional media. Our findings reveal the profound impact of medium composition on gene essentiality in human cells, and also suggest general strategies for using genetic screens in HPLM to uncover new cancer vulnerabilities and gene-nutrient interactions.

cancer biology↗

bioRxiv2018

Inhibition of mitochondrial ferredoxin 1 (FDX1) prevents adaptation to proteotoxic stress

The mechanisms used by cancer cells to resist the severe disruption in protein homeostasis caused by proteasome inhibitors remain obscure. Here, we show this resistance correlates with a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS). Employing small molecule screens, we identified a striking overlap between compounds that preferentially impede the growth of proteasome inhibitor-resistant cancer cells and those that block the growth of high OXPHOS cells. Elesclomol potently exhibits both characteristics. Using genome-wide CRISPR/Cas9-based screening, in vitro validation and NMR spectroscopy we identify mitochondrial protein ferredoxin 1 (FDX1), a critical component of mitochondrial iron-sulfur (Fe-S) cluster biosynthesis, as the primary target of elesclomol. In a mouse model of multiple myeloma, inhibition of FDX1 with elesclomol significantly attenuated the emergence of proteasome inhibitor-resistance and markedly prolonged survival. Our work reveals that the mitochondrial Fe-S cluster pathway is a targetable vulnerability in cancers that are resistant to increased proteotoxic burden.

cancer biology↗

bioRxiv2017

Juxtacap Nucleotide Sequence Modulates eIF4E Binding and Translation

mRNA translation is an energetically costly activity required for almost all biological processes. The multiprotein complex eIF4F, which bridges the 5 cap and the polyA tail through eIF4E and eIF4G, respectively, is necessary for efficient translation initiation of most mRNAs and is an important target of translational control. Previous work suggests that cap-proximal nucleotides can modulate eIF4E binding to mRNAs, but the effect of specific cap-proximal nucleotide sequences on eIF4E recruitment and the ultimate consequences for translation remain unknown. Using RNA Bind-n-Seq on a model 5 UTR, we systematically identify eIF4E-intrinsic cap-proximal nucleotide binding preferences. mRNAs with highly-bound motifs are translated well in a cell-free system, whereas those with low-eIF4E-binding motifs are not. However, eIF4E juxtacap motif preferences do not dictate the ribosome occupancy of endogenous mRNAs in cells, suggesting that the effect of juxtacap sequence on eIF4E binding and translation is mRNA-context-dependent. Accordingly, a single downstream point mutation that disrupts a predicted base pair with a preferred juxtacap nucleotide increases translation. The juxtacap sequence is a previously unappreciated determinant of eIF4E recruitment to mRNAs, and we propose that differences in mRNA 5 end accessibility defined by the juxtacap sequence are important for establishing translational efficiency.

biochemistry↗