Crossing boundaries of light microscopy resolution discerns novel assemblies in the nucleolus.

The nucleolus is the largest membraneless organelle and nuclear body in mammalian cells. It is primarily involved in the biogenesis of ribosomes, essential macromolecular machines responsible for synthesizing all proteins required by the cell. The assembly of ribosomes is evolutionarily conserved and accounts for the most energy-consuming cellular process needed for cell growth, proliferation, and homeostasis. Despite the significance of this process, the substructural mechanistic principles of the nucleolar function in preribosome biogenesis have only recently begun to emerge. Here, we provide a new perspective using advanced super-resolution microscopy and single-molecule MINFLUX nanoscopy on the mechanistic principles governing ribosomal RNA-seeded nucleolar formation and the resulting tripartite suborganization of the nucleolus driven, in part, by liquid-liquid phase separation. With recent advances in the cryogenic electron microscopy (cryoEM) structural analysis of ribosome biogenesis intermediates, we highlight the current understanding of the step-wise assembly of preribosomal subunits in the nucleolus. Finally, we address how novel anticancer drug candidates target early steps in ribosome biogenesis to exploit these essential dependencies for growth arrest and tumor control.

DNAJC9 prevents CENP-A mislocalization and chromosomal instability by maintaining the fidelity of histone supply chains.

The centromeric histone H3 variant CENP-A is overexpressed in many cancers. The mislocalization of CENP-A to noncentromeric regions contributes to chromosomal instability (CIN), a hallmark of cancer. However, pathways that promote or prevent CENP-A mislocalization remain poorly defined. Here, we performed a genome-wide RNAi screen for regulators of CENP-A localization which identified DNAJC9, a J-domain protein implicated in histone H3-H4 protein folding, as a factor restricting CENP-A mislocalization. Cells lacking DNAJC9 exhibit mislocalization of CENP-A throughout the genome, and CIN phenotypes. Global interactome analysis showed that DNAJC9 depletion promotes the interaction of CENP-A with the DNA-replication-associated histone chaperone MCM2. CENP-A mislocalization upon DNAJC9 depletion was dependent on MCM2, defining MCM2 as a driver of CENP-A deposition at ectopic sites when H3-H4 supply chains are disrupted. Cells depleted for histone H3.3, also exhibit CENP-A mislocalization. In summary, we have defined novel factors that prevent mislocalization of CENP-A, and demonstrated that the integrity of H3-H4 supply chains regulated by histone chaperones such as DNAJC9 restrict CENP-A mislocalization and CIN.

Listeria monocytogenes ST37 Distribution in the Moscow Region and Properties of Clinical and Foodborne Isolates.

Listerias of the phylogenetic lineage II (PLII) are common in the European environment and are hypovirulent. Despite this, they caused more than a third of the sporadic cases of listeriosis and multi-country foodborne outbreaks. L. monocytogenes ST37 is one of them. During the COVID-19 pandemic, ST37 appeared in clinical cases and ranked second in occurrence among food isolates in the Moscow region. The aim of this study was to describe the genomic features of ST37 isolates from different sources. All clinical cases of ST37 were in the cohort of male patients (age, 48-81 years) with meningitis-septicemia manifestation and COVID-19 or Influenza in the anamnesis. The core genomes of the fish isolates were closely related. The clinical and meat isolates revealed a large diversity. Prophages (2-4/genome) were the source of the unique genes. Two clinical isolates displayed pseudolysogeny, and excided prophages were A006-like. In the absence of plasmids, the assortment of virulence factors and resistance determinants in the chromosome corresponded to the hypovirulent characteristics. However, all clinical isolates caused severe disease, with deaths in four cases. Thus, these studies allow us to speculate that a previous viral infection increases human susceptibility to listeriosis.

Bayesian analysis dissects kinetic modulation during non-stationary gene expression.

Labelling of nascent stem loops with fluorescent proteins has fostered the visualization of transcription in living cells. Quantitative analysis of recorded fluorescence traces can shed light on kinetic transcription parameters and regulatory mechanisms. However, existing methods typically focus on steady state dynamics. Here, we combine a stochastic process transcription model with a hierarchical Bayesian method to infer global as well locally shared parameters for groups of cells and recover unobserved quantities such as initiation times and polymerase loading of the gene. We apply our approach to the cyclic response of the yeast CUP1 locus to heavy metal stress. Within the previously described slow cycle of transcriptional activity on the scale of minutes, we discover fast time-modulated bursting on the scale of seconds. Model comparison suggests that slow oscillations of transcriptional output are regulated by the amplitude of the bursts. Several polymerases may initiate during a burst.

Architectural basis for cylindrical self-assembly governing Plk4-mediated centriole duplication in human cells.

Proper organization of intracellular assemblies is fundamental for efficient promotion of biochemical processes and optimal assembly functionality. Although advances in imaging technologies have shed light on how the centrosome is organized, how its constituent proteins are coherently architected to elicit downstream events remains poorly understood. Using multidisciplinary approaches, we showed that two long coiled-coil proteins, Cep63 and Cep152, form a heterotetrameric building block that undergoes a stepwise formation into higher molecular weight complexes, ultimately generating a cylindrical architecture around a centriole. Mutants defective in Cep63•Cep152 heterotetramer formation displayed crippled pericentriolar Cep152 organization, polo-like kinase 4 (Plk4) relocalization to the procentriole assembly site, and Plk4-mediated centriole duplication. Given that the organization of pericentriolar materials (PCM) is evolutionarily conserved, this work could serve as a model for investigating the structure and function of PCM in other species, while offering a new direction in probing the organizational defects of PCM-related human diseases.

The histone H3/H4 chaperone CHAF1B prevents the mislocalization of CENP-A for chromosomal stability.

Restricting the localization of the evolutionarily conserved centromeric histone H3 variant CENP-A to centromeres prevents chromosomal instability (CIN). The mislocalization of CENP-A to non-centromeric regions contributes to CIN in yeasts, flies and human cells. Even though overexpression and mislocalization of CENP-A have been reported in cancers, the mechanisms responsible for its mislocalization remain poorly understood. Here, we used an imaging-based high-throughput RNAi screen to identify factors that prevent mislocalization of overexpressed YFP-tagged CENP-A (YFP-CENP-A) in HeLa cells. Among the top five candidates in the screen - the depletion of which showed increased nuclear YFP-CENP-A fluorescence - were the histone chaperones CHAF1B (or p60) and CHAF1A (or p150). Follow-up validation and characterization experiments showed that CHAF1B-depleted cells exhibited CENP-A mislocalization, CIN phenotypes and increased enrichment of CENP-A in chromatin fractions. The depletion of DAXX, a histone H3.3 chaperone, suppressed CENP-A mislocalization and CIN in CHAF1B-depleted cells. We propose that in CHAF1B-depleted cells, DAXX promotes mislocalization of the overexpressed CENP-A to non-centromeric regions, resulting in CIN. In summary, we identified regulators of CENP-A localization and defined a role for CHAF1B in preventing DAXX-dependent CENP-A mislocalization and CIN.

Impact of Saccharomyces cerevisiae on the Field of Single-Molecule Biophysics.

Cellular functions depend on the dynamic assembly of protein regulator complexes at specific cellular locations. Single Molecule Tracking (SMT) is a method of choice for the biochemical characterization of protein dynamics in vitro and in vivo. SMT follows individual molecules in live cells and provides direct information about their behavior. SMT was successfully applied to mammalian models. However, mammalian cells provide a complex environment where protein mobility depends on numerous factors that are difficult to control experimentally. Therefore, yeast cells, which are unicellular and well-studied with a small and completely sequenced genome, provide an attractive alternative for SMT. The simplicity of organization, ease of genetic manipulation, and tolerance to gene fusions all make yeast a great model for quantifying the kinetics of major enzymes, membrane proteins, and nuclear and cellular bodies. However, very few researchers apply SMT techniques to yeast. Our goal is to promote SMT in yeast to a wider research community. Our review serves a dual purpose. We explain how SMT is conducted in yeast cells, and we discuss the latest insights from yeast SMT while putting them in perspective with SMT of higher eukaryotes.

MYC amplifies gene expression through global changes in transcription factor dynamics.

The MYC oncogene has been studied for decades, yet there is still intense debate over how this transcription factor controls gene expression. Here, we seek to answer these questions with an in vivo readout of discrete events of gene expression in single cells. We engineered an optogenetic variant of MYC (Pi-MYC) and combined this tool with single-molecule RNA and protein imaging techniques to investigate the role of MYC in modulating transcriptional bursting and transcription factor binding dynamics in human cells. We find that the immediate consequence of MYC overexpression is an increase in the duration rather than in the frequency of bursts, a functional role that is different from the majority of human transcription factors. We further propose that the mechanism by which MYC exerts global effects on the active period of genes is by altering the binding dynamics of transcription factors involved in RNA polymerase II complex assembly and productive elongation.

The role of protease-activated receptor 1 signaling in CD8 T cell effector functions.

CD8 T cells are essential for adaptive immunity against viral infections. Protease activated receptor 1 (PAR1) is expressed by CD8 T cells; however, its role in T cell effector function is not well defined. Here we show that in human CD8 T cells, PAR1 stimulation accelerates calcium mobilization. Furthermore, PAR1 is involved in cytotoxic T cell function by facilitating granule trafficking via actin polymerization and repositioning of the microtubule organizing center (MTOC) toward the immunological synapse. In vivo, PAR1-/- mice have reduced cytokine-producing T cells in response to a lymphocytic choriomeningitis virus (LCMV) infection and fail to efficiently control the virus. Specific deletion of PAR1 in LCMV GP33-specific CD8 T cells results in reduced expansion and diminished effector function. These data demonstrate that PAR1 plays a role in T cell activation and function, and this pathway could represent a new therapeutic strategy to modulate CD8 T cell effector function.

Towards a 'Spot On' Understanding of Transcription in the Nucleus.

Regulation of transcription by RNA Polymerase II (RNAPII) is a rapidly evolving area of research. Technological developments in microscopy have revealed insight into the dynamics, structure, and localization of transcription components within single cells. A frequent observation in many studies is the appearance of 'spots' in cell nuclei associated with the transcription process. In this review we highlight studies that characterize the temporal and spatial characteristics of these spots, examine possible pitfalls in interpreting these kind of imaging data, and outline directions where single-cell imaging may advance in ways to further our understanding of transcription regulation.

CENP-A overexpression promotes aneuploidy with karyotypic heterogeneity.

Chromosomal instability (CIN) is a hallmark of many cancers. Restricting the localization of centromeric histone H3 variant CENP-A to centromeres prevents CIN. CENP-A overexpression (OE) and mislocalization have been observed in cancers and correlate with poor prognosis; however, the molecular consequences of CENP-A OE on CIN and aneuploidy have not been defined. Here, we show that CENP-A OE leads to its mislocalization and CIN with lagging chromosomes and micronuclei in pseudodiploid DLD1 cells and xenograft mouse model. CIN is due to reduced localization of proteins to the kinetochore, resulting in defects in kinetochore integrity and unstable kinetochore-microtubule attachments. CENP-A OE contributes to reduced expression of cell adhesion genes and higher invasion of DLD1 cells. We show that CENP-A OE contributes to aneuploidy with karyotypic heterogeneity in human cells and xenograft mouse model. In summary, our results provide a molecular link between CENP-A OE and aneuploidy, and suggest that karyotypic heterogeneity may contribute to the aggressive phenotype of CENP-A-overexpressing cancers.

Aggregation and Prion-Inducing Properties of the G-Protein Gamma Subunit Ste18 are Regulated by Membrane Association.

Yeast prions and mnemons are respectively transmissible and non-transmissible self-perpetuating protein assemblies, frequently based on cross-ß ordered detergent-resistant aggregates (amyloids). Prions cause devastating diseases in mammals and control heritable traits in yeast. It was shown that the de novo formation of the prion form [PSI+] of yeast release factor Sup35 is facilitated by aggregates of other proteins. Here we explore the mechanism of the promotion of [PSI+] formation by Ste18, an evolutionarily conserved gamma subunit of a G-protein coupled receptor, a key player in responses to extracellular stimuli. Ste18 forms detergent-resistant aggregates, some of which are colocalized with de novo generated Sup35 aggregates. Membrane association of Ste18 is required for both Ste18 aggregation and [PSI+] induction, while functional interactions involved in signal transduction are not essential for these processes. This emphasizes the significance of a specific location for the nucleation of protein aggregation. In contrast to typical prions, Ste18 aggregates do not show a pattern of heritability. Our finding that Ste18 levels are regulated by the ubiquitin-proteasome system, in conjunction with the previously reported increase in Ste18 levels upon the exposure to mating pheromone, suggests that the concentration-dependent Ste18 aggregation may mediate a mnemon-like response to physiological stimuli.

Blockade of Toll-like receptor 4 (TLR4) reduces oxidative stress and restores phospho-ERK1/2 levels in Leydig cells exposed to high glucose.

AIMS: Hyperglycemia in combination with oxidative stress plays a significant pathophysiological role in diabetic testicular dysfunction, often leading to infertility. Activation of Toll-like receptor 4 (TLR4) has been reported to mediate oxidative stress during diabetes. However, engagement of the TLR4 signaling pathway in diabetic testicular dysfunction has not been previously explored. Herein, we investigated the role of TLR4 in reactive oxygen species (ROS) production and in the phosphorylation status of ERK1/2 in primary Leydig cells exposed to high glucose and in testis isolated from diabetic rats. MAIN METHODS: Testicular levels of TLR4 and phospho-ERK1/2 were determined by Western blotting. ROS production was detected with a fluorescent probe. Additionally, primary Leydig cells were exposed to normal (5.5 mmol/l) or elevated (33 mmol/l) glucose concentrations and treated with or without a TLR4 inhibitor, CLI095 (10-5 mol/l) for 24 h, followed by evaluation of TLR4 and phospho-ERK1/2 expression levels by Western blotting and immunofluorescence staining, respectively. KEY FINDINGS: We show that high glucose induces the expression of TLR4 in Leydig cells. Additionally, we demonstrate that blockade of this receptor in this cell population reduces oxidative stress and restores the levels of phospho-ERK1/2. SIGNIFICANCE: Our findings provide new insight into TLR4 interaction with ROS and MEK/ERK pathway in Leydig cells exposed to high glucose and present a rationale for the development of new therapeutics for diabetic testicular dysfunction.

Toll-like receptor 4 (TLR4) as a possible pathological mechanism in hyperglycemia-associated testicular dysfunction.

Hyperglycemia is a chief factor in diabetes, a complex disease associated with reproductive disorders, mainly testicular dysfunction, which contributes to male infertility. Leydig cells are the predominant cell population in the testis interstitium and, when stimulated, they are capable of initiating immune responses playing crucial roles in the mechanisms related to testis' homeostasis. These cells express TLR4, an innate immune receptor, which is known to be modulated by hyperglycemia in other cell populations and tissue types. Still, whether TLR4 contributes to hyperglycemia-associated testicular dysfunction remains elusive. Activation of TLR4 in response to high glucose levels involves redox imbalance and stimulation of transcriptional factors, especially nuclear factor (NF)-κB, which could be a potential pathological mechanism compromising the testis integrity. Additionally, emerging evidence shows crosstalk between TLR4 and Nrf2, an anti-inflammatory mediator that was previously shown to be reduced in diabetic testis. Therefore, we hypothesize that hyperglycemia-mediated TLR4 activation in testicular cells, especially Leydig cells might be a crucial event triggering oxidative stress and inflammation, which in turn, drives testicular dysfunction.

Single molecule analysis of lamin dynamics.

The nuclear envelope (NE) is an essential cellular structure that contributes to nuclear stability, organization, and function. Mutations in NE-associated proteins result in a myriad of pathologies with widely diverse clinical manifestations, ages of onsets, and affected tissues. Notably, several hundred disease-causing mutations have been mapped to the LMNA gene, which encodes the intermediate filament proteins lamin A and C, two of the major architectural components of the nuclear envelope. However, how NE dysfunction leads to the highly variable pathologies observed in patient cells and tissues remains poorly understood. One model suggests alterations in the dynamic properties of the nuclear lamina and its associated proteins contribute to disease phenotype. Here, we describe the application of single molecule tracking (SMT) methodology to characterize the behavior of nuclear envelope transmembrane proteins and nuclear lamins in their native cellular environment at the single molecule level. As proof-of-concept, we demonstrate by SMT that Halo-tagged lamin B1, Samp1, lamin A, and lamin AΔ50 have distinct binding and kinetic properties, and we identify several disease-relevant mutants which exhibit altered binding dynamics. SMT is also able to separately probe the dynamics of the peripheral and the nucleoplasmic populations of lamin A mutants. We suggest that SMT is a robust and sensitive method to investigate the relationship between pathogenic mutations or cellular processes and protein dynamics at the NE.

Glucose Signaling Is Connected to Chromosome Segregation Through Protein Kinase A Phosphorylation of the Dam1 Kinetochore Subunit in Saccharomycescerevisiae.

The Dam1 complex is an essential component of the outer kinetochore that mediates attachments between spindle microtubules and chromosomes. Dam1p, a subunit of the Dam1 complex, binds to microtubules and is regulated by Aurora B/Ipl1p phosphorylation. We find that overexpression of cAMP-dependent protein kinase (PKA) catalytic subunits (i.e., TPK1, TPK2, TPK3) is lethal in DAM1 mutants and increases the rate of chromosome loss in wild-type cells. Replacing an evolutionarily conserved PKA site (S31) in Dam1p with a nonphosphorylatable alanine suppressed the high-copy PKA dosage lethality in dam1-1 Consistent with Dam1p as a target of PKA, we find that in vitro PKA can directly phosphorylate S31 in Dam1p and we observed phosphorylation of S31 in Dam1p purified from asynchronously growing yeast cells. Cells carrying high-copy TPK2 or a Dam1p phospho-mimetic S31D mutant displayed a reduction in Dam1p localization at the kinetochore, suggesting that PKA phosphorylation plays a role in assembly and/or stability of the Dam1 complex. Furthermore, we observed spindle defects associated with S31 phosphorylation. Finally, we find that phosphorylation of Dam1p on S31 is reduced when glucose is limiting as well as during α-factor arrest, conditions that inhibit PKA activity. These observations suggest that the PKA site of Dam1p participates in regulating kinetochore activity. While PKA is a well-established effector of glucose signaling, our work shows for the first time that glucose-dependent PKA activity has an important function in chromosome segregation.

Single-Molecule Analysis Reveals Linked Cycles of RSC Chromatin Remodeling and Ace1p Transcription Factor Binding in Yeast.

It is unknown how the dynamic binding of transcription factors (TFs) is molecularly linked to chromatin remodeling and transcription. Using single-molecule tracking (SMT), we show that the chromatin remodeler RSC speeds up the search process of the TF Ace1p for its response elements (REs) at the CUP1 promoter. We quantified smFISH mRNA data using a gene bursting model and demonstrated that RSC regulates transcription bursts of CUP1 only by modulating TF occupancy but does not affect initiation and elongation rates. We show by SMT that RSC binds to activated promoters transiently, and based on MNase-seq data, that RSC does not affect the nucleosomal occupancy at CUP1. Therefore, transient binding of Ace1p and rapid bursts of transcription at CUP1 may be dependent on short repetitive cycles of nucleosome mobilization. This type of regulation reduces the transcriptional noise and ensures a homogeneous response of the cell population to heavy metal stress.

Associations of Simian Immunodeficiency Virus (SIV)-Specific Follicular CD8+ T Cells with Other Follicular T Cells Suggest Complex Contributions to SIV Viremia Control.

Follicular CD8+ T (fCD8) cells reside within B cell follicles and are thought to be immune-privileged sites of HIV/SIV infection. We have observed comparable levels of fCD8 cells between chronically SIV-infected rhesus macaques with low viral loads (LVL) and high viral loads (HVL), raising the question concerning their contribution to viremia control. In this study, we sought to clarify the role of SIV-specific fCD8 cells in lymph nodes during the course of SIV infection in rhesus macaques. We observed that fCD8 cells, T follicular helper (Tfh) cells, and T follicular regulatory cells (Tfreg) were all elevated in chronic SIV infection. fCD8 cells of LVL animals tended to express more Gag-specific granzyme B and exhibited significantly greater killing than did HVL animals, and their cell frequencies were negatively correlated with viremia, suggesting a role in viremia control. Env- and Gag-specific IL-21+ Tfh of LVL but not HVL macaques negatively correlated with viral load, suggesting better provision of T cell help to fCD8 cells. Tfreg positively correlated with fCD8 cells in LVL animals and negatively correlated with viremia, suggesting a potential benefit of Tfreg via suppression of chronic inflammation. In contrast, in HVL macaques, Tfreg and fCD8 cell frequencies tended to be negatively correlated, and a positive correlation was seen between Tfreg number and viremia, suggesting possible dysfunction and suppression of an effective fCD8 cell immune response. Our data suggest that control of virus-infected cells in B cell follicles not only depends on fCD8 cell cytotoxicity but also on complex fCD8 cell associations with Tfh cells and Tfreg.

Mislocalization of centromeric histone H3 variant CENP-A contributes to chromosomal instability (CIN) in human cells.

Chromosomal instability (CIN) is a hallmark of many cancers and a major contributor to tumorigenesis. Centromere and kinetochore associated proteins such as the evolutionarily conserved centromeric histone H3 variant CENP-A, associate with centromeric DNA for centromere function and chromosomal stability. Stringent regulation of cellular CENP-A levels prevents its mislocalization in yeast and flies to maintain genome stability. CENP-A overexpression and mislocalization are observed in several cancers and reported to be associated with increased invasiveness and poor prognosis. We examined whether there is a direct relationship between mislocalization of overexpressed CENP-A and CIN using HeLa and chromosomally stable diploid RPE1 cell lines as model systems. Our results show that mislocalization of overexpressed CENP-A to chromosome arms leads to chromosome congression defects, lagging chromosomes, micronuclei formation and a delay in mitotic exit. CENP-A overexpressing cells showed altered localization of centromere and kinetochore associated proteins such as CENP-C, CENP-T and Nuf2 leading to weakened native kinetochores as shown by reduced interkinetochore distance and CIN. Importantly, our results show that mislocalization of CENP-A to chromosome arms is one of the major contributors for CIN as depletion of histone chaperone DAXX prevents CENP-A mislocalization and rescues the reduced interkinetochore distance and CIN phenotype in CENP-A overexpressing cells. In summary, our results establish that CENP-A overexpression and mislocalization result in a CIN phenotype in human cells. This study provides insights into how overexpression of CENP-A may contribute to CIN in cancers and underscore the importance of understanding the pathways that prevent CENP-A mislocalization for genome stability.