Results for “cell-cycle proteins”

DOE OSTI2024

Parallel measurement of transcriptomes and proteomes from same single cells using nanodroplet splitting

Single-cell multiomics provides comprehensive insights into gene regulatory networks, cellular diversity, and temporal dynamics. Here, we introduce nanoSPLITS (nanodroplet SPlitting for Linked-multimodal Investigations of Trace Samples), an integrated platform that enables global profiling of the transcriptome and proteome from same single cells via RNA sequencing and mass spectrometry-based proteomics, respectively. Benchmarking of nanoSPLITS demonstrates high measurement precision with deep proteomic and transcriptomic profiling of single-cells. We apply nanoSPLITS to cyclin-dependent kinase 1 inhibited cells and found phospho-signaling events could be quantified alongside global protein and mRNA measurements, providing insights into cell cycle regulation. We extend nanoSPLITS to primary cells isolated from human pancreatic islets, introducing an efficient approach for facile identification of unknown cell types and their protein markers by mapping transcriptomic data to existing large-scale single-cell RNA sequencing reference databases. Accordingly, we establish nanoSPLITS as a multiomic technology incorporating global proteomics and anticipate the approach will be critical to furthering our understanding of biological systems.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Conformational and oligomeric states of SPOP from small-angle X-ray scattering and molecular dynamics simulations

Speckle-type POZ protein (SPOP) is a substrate adaptor in the ubiquitin proteasome system, and plays important roles in cell-cycle control, development, and cancer pathogenesis. SPOP forms linear higher-order oligomers following an isodesmic self-association model. Oligomerization is essential for SPOP’s multivalent interactions with substrates, which facilitate phase separation and localization to biomolecular condensates. Structural characterization of SPOP in its oligomeric state and in solution is, however, challenging due to the inherent conformational and compositional heterogeneity of the oligomeric species. Here, we develop an approach to simultaneously and self-consistently characterize the conformational ensemble and the distribution of oligomeric states of SPOP by combining small-angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations. We build initial conformational ensembles of SPOP oligomers using coarse-grained molecular dynamics simulations, and use a Bayesian/maximum entropy approach to refine the ensembles, along with the distribution of oligomeric states, against a concentration series of SAXS experiments. Our results suggest that SPOP oligomers behave as rigid, helical structures in solution, and that a flexible linker region allows SPOP’s substrate-binding domains to extend away from the core of the oligomers. Additionally, our results are in good agreement with previous characterization of the isodesmic self-association of SPOP. In the future, the approach presented here can be extended to other systems to simultaneously characterize structural heterogeneity and self-assembly.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Phosphoproteome Response to Dithiothreitol Reveals Unique Versus Shared Features of Saccharomyces cerevisiae Stress Responses

To cope with sudden changes in the external environment, the budding yeast Saccharomyces cerevisiae orchestrates a multifaceted response that spans many levels of physiology. Several studies have interrogated the transcriptome response to endoplasmic reticulum (ER) stress and the role of regulators such as the Ire1 kinase and Hac1 transcription factors. However, less is known about responses to ER stress at other levels of physiology. Here, we used quantitative phosphoproteomics and computational network inference to uncover the yeast phosphoproteome response to the reducing agent dithiothreitol (DTT) and the upstream signaling network that controls it. We profiled wild-type cells and mutants lacking IRE1 or MAPK kinases MKK1 and MKK2, before and at various times after DTT treatment. In addition to revealing downstream targets of these kinases, our inference approach predicted new regulators in the DTT response, including cell-cycle regulator Cdc28 and osmotic-response kinase Rck2, which we validated computationally. Our results also revealed similarities and surprising differences in responses to different stress conditions, especially in the response of protein kinase A targets. Furthermore, these results have implications for the breadth of signaling programs that can give rise to common stress response signatures.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Aneuploidy induces premature aging in yeast due to defects in Ribosome Quality Control [RNASeqData]

Premature aging is a hallmark of Down syndrome, caused by trisomy of human chromosome 21; but the reason is unclear and difficult to study in humans. We used an aneuploid model in wild yeast to show that chromosome amplification disrupts nutrient-induced cell-cycle arrest, quiescence entry, and healthy aging, across genetic backgrounds and amplified chromosomes. We discovered that these defects are due in part to aneuploidy-induced dysfunction in Ribosome Quality Control (RQC). Aneuploids entering quiescence display aberrant ribosome profiles, accumulate RQC intermediates, and harbor an increased load of protein aggregates. Although they have normal proteasome capacity, aneuploids show signs of ubiquitin dysregulation, which impacts cyclin abundance to disrupt arrest. Remarkably, inducing ribosome stalling in euploids produces similar aberrations, while up-regulating limiting RQC subunits or proteins in ubiquitin metabolism alleviates many of the aneuploid defects. Our results raise major implications for other aneuploidy disorders including Down syndrome.

aneuploidy↗

DOE OSTI2024

Aneuploidy induces premature aging in yeast due to defects in Ribosome Quality Control [MoBYSeqData]

Premature aging is a hallmark of Down syndrome, caused by trisomy of human chromosome 21; but the reason is unclear and difficult to study in humans. We used an aneuploid model in wild yeast to show that chromosome amplification disrupts nutrient-induced cell-cycle arrest, quiescence entry, and healthy aging, across genetic backgrounds and amplified chromosomes. We discovered that these defects are due in part to aneuploidy-induced dysfunction in Ribosome Quality Control (RQC). Aneuploids entering quiescence display aberrant ribosome profiles, accumulate RQC intermediates, and harbor an increased load of protein aggregates. Although they have normal proteasome capacity, aneuploids show signs of ubiquitin dysregulation, which impacts cyclin abundance to disrupt arrest. Remarkably, inducing ribosome stalling in euploids produces similar aberrations, while up-regulating limiting RQC subunits or proteins in ubiquitin metabolism alleviates many of the aneuploid defects. Our results raise major implications for other aneuploidy disorders including Down syndrome.

aneuploidy↗

DOE OSTI2024

Premature aging in aneuploid yeast is influenced by aneuploidy-induced defects in Ribosome Quality Control [RiboSeq]

Premature aging is a hallmark of Down syndrome, caused by trisomy of human chromosome 21; but the reason is unclear and difficult to study in humans. We used an aneuploid model in wild yeast to show that chromosome amplification disrupts nutrient-induced cell-cycle arrest, quiescence entry, and healthy aging, across genetic backgrounds and amplified chromosomes. We discovered that these defects are due in part to aneuploidy-induced dysfunction in Ribosome Quality Control (RQC). Aneuploids entering quiescence display aberrant ribosome profiles, accumulate RQC intermediates, and harbor an increased load of protein aggregates. Although they have normal proteasome capacity, aneuploids show signs of ubiquitin dysregulation, which impacts cyclin abundance to disrupt arrest. Remarkably, inducing ribosome stalling in euploids produces similar aberrations, while up-regulating limiting RQC subunits or proteins in ubiquitin metabolism alleviates many of the aneuploid defects. Our results raise major implications for other aneuploidy disorders including Down syndrome.

aneuploidy↗

DOE OSTI2025

Chromosome duplication causes premature aging via defects in ribosome quality control

Down syndrome, caused by an extra copy of Chromosome 21, causes lifelong problems. One of the most common phenotypes among people with Down syndrome is premature aging, including early tissue decline, neurodegeneration, and shortened life span. Yet the reasons for premature systemic aging are a mystery and difficult to study in humans. Here we show that chromosome amplification in wild yeast also produces premature aging and shortens life span. Chromosome duplication disrupts nutrient-induced cell-cycle arrest, entry into quiescence, and cellular health during chronological aging, across genetic background and independent of which chromosome is amplified. Using a genomic screen, we discovered that these defects are due in part to aneuploidy-induced dysfunction in Ribosome Quality Control (RQC). We show that aneuploids entering quiescence display aberrant ribosome profiles, accumulate RQC intermediates, and harbor an increased load of protein aggregates compared to euploid cells. Although they maintain proteasome activity, aneuploids also show signs of ubiquitin dysregulation and sequestration into foci. Remarkably, inducing ribosome stalling in euploids produces similar aging phenotypes, while up-regulating limiting RQC subunits or poly-ubiquitin alleviates many of the aneuploid defects. We propose that the increased translational load caused by having too many mRNAs accelerates a decline in translational fidelity, contributing to premature aging.

Aneuploidy↗

DOE OSTI2025

The genome of the polyextremophilic yeast, Naganishia friedmannii, reveals adaptations involved in stress response pathways, carbohydrate metabolism expansion, and a limited DNA repair repertoire

Here we report the draft genome sequence of Naganishia friedmannii (formerly Cryptococcus friedmannii) isolate, a Basidiomycota yeast commonly found in some of the most extreme environments of the Earth's cryosphere. We isolated N. friedmannii strain Llullensis from soils at 6000 m above sea level on Volcán Llullaillaco, Argentina. The genome was 22.2 Mb with 6251 identified protein coding genes. Proteins known to be associated with thermal, osmotic, and radiation stress were identified in the genome. Comparative analysis with seven other Naganishia genomes revealed unique features underlying its polyextremophilic lifestyle. Naganishia friedmannii showed an expansion of genes involved in breaking down plant-derived carbohydrates, supporting the hypothesis that it survives at high elevations by metabolizing wind-deposited organic matter. Surprisingly, many genes involved in cell-cycle checkpoints and DNA repair were missing, as in several other Naganishia species. This extensive loss may be adaptive in extreme environments prone to abiotic stress, where a high mutation rate could generate advantageous traits, and reduced cell-cycle control may allow for faster reproduction that would be advantageous for rapid growth during brief periods of soil wetting following rare snow events.

Vimercati, Lara↗

DOE OSTI2022

The MuvB complex binds and stabilizes nucleosomes downstream of the transcription start site of cell-cycle dependent genes

The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene expression.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Coupling between DNA replication, segregation, and the onset of constriction in Escherichia coli

Escherichia coli cell cycle features two critical cell-cycle checkpoints: initiation of replication and the onset of constriction. While the initiation of DNA replication has been extensively studied, it is less clear what triggers the onset of constriction and when exactly it occurs during the cell cycle. Here, using high-throughput fluorescence microscopy in microfluidic devices, we determine the timing for the onset of constriction relative to the replication cycle in different growth rates. Our single-cell data and modeling indicate that the initiation of constriction is coupled to replication-related processes in slow growth conditions. Furthermore, our data suggest that this coupling involves the mid-cell chromosome blocking the onset of constriction via some form of nucleoid occlusion occurring independently of SlmA and the Ter linkage proteins. This work highlights the coupling between replication and division cycles and brings up a new nucleoid mediated control mechanism in E. coli.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Productivity and bioproduct formation in phototropic knock/out mutants in micro algae

Phototropin is a blue light receptor, which mediates a variety of blue-light elicited physiological processes in plants and algae. In higher plants these processes include phototropism, chloroplast movement and stomatal opening. In the green alga Chlamydomonas reinhardtii, phototropin plays a vital role in progression of the sexual life cycle and in the control of the eye spot size and light sensitivity. Phototropin is also involved in blue-light mediated changes in the synthesis of chlorophylls, carotenoids, chlorophyll binding proteins. We compared the transcriptome of phototropin knock out (PHOT KO) mutant and wild-type parent to analyze differences in gene expression in high light grown cultures (500 μmol photons m⁻²s⁻¹). Our results indicate the up-regulation of genes involved in photosynthetic electron transport chain, carbon fixation pathway, starch, lipid, and cell cycle control genes. With respect to photosynthetic electron transport genes, genes encoding proteins of the cytochrome b6f and ATP synthase complex were up regulated potentially facilitating proton-coupled electron transfer. In addition genes involved in limiting steps in the Calvin cycle Ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO), Sidoheptulose 1,7 biophosphatase (SBPase), Glyceraldehyde-3-phosphate dehydrogenase (3PGDH) and that mediate cell-cycle control (CDK) were also up regulated along with starch synthase and fatty acid biosynthesis genes involved in starch and lipid synthesis. In addition, transmission electron micrographs show increased accumulation of starch granules in PHOT mutant compared to wild type, which is consistent with the higher expression of starch synthase genes. Collectively, the altered patterns of gene expression in the PHOT mutants were associated with a two-fold increase in growth and biomass accumulation compared to wild type when grown in environmental photobioreactors (Phenometrics) that simulate a pond environment. In conclusion, our studies suggest that phototropin may be a master gene regulator that suppresses rapid cell growth and promotes gametogenesis and sexual recombination in wild type strains.

59 BASIC BIOLOGICAL SCIENCES↗