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

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

[Unconventional protein secretion - new perspectives in protein trafficking]. / Sécrétion non conventionnelle - Nouvelles perspectives dans le trafic des protéines.

The characterization of the structural and functional organization of eukaryotic cells has revealed the membrane compartments and machinery required for vesicular protein transport. Most proteins essential for intercellular communication contain an N-terminal signal sequence enabling them to be incorporated into the biosynthetic or conventional secretory pathway, in which proteins are sequentially transported through the endoplasmic reticulum (ER) and the Golgi apparatus. However, major research studies have shown the existence of alternative secretory routes that are independent of the ER-Golgi and designated as unconventional secretory pathways. These pathways involve a large number of players that may divert specific compartments from their primary function in favor of secretory roles. The comprehensive description of these processes is therefore of utmost importance to unveil how proteins secreted through these alternative pathways control cell homeostasis or contribute to disease development.

Title: Sécrétion non conventionnelle - Nouvelles perspectives dans le trafic des protéines. Abstract: L'étude de l'organisation structurale et fonctionnelle des cellules eucaryotes a révélé les compartiments membranaires ainsi que la machinerie nécessaires au trafic vésiculaire des protéines. La plupart des protéines essentielles à la communication intercellulaire contiennent une séquence signal leur permettant d'être incorporées dans la voie de sécrétion conventionnelle, par laquelle les protéines sont transportées séquentiellement dans le réticulum endoplasmique (RE) puis l'appareil de Golgi. Cependant, les cellules eucaryotes sont également dotées de voies de sécrétion alternatives ou voies de sécrétion non conventionnelles, qui mettent en jeu de nombreux acteurs susceptibles de détourner certains compartiments de leurs fonctions principales au profit de fonctions sécrétoires.

SnapShot: Eukaryotic ribosome biogenesis II.

Ribosome production is essential for cell growth. Approximately 200 assembly factors drive this complicated pathway that starts in the nucleolus and ends in the cytoplasm. A large number of structural snapshots of the pre-60S pathway have revealed the principles behind large subunit synthesis. To view this SnapShot, open or download the PDF.

Protein thermal sensing regulates physiological amyloid aggregation.

To survive, cells must respond to changing environmental conditions. One way that eukaryotic cells react to harsh stimuli is by forming physiological, RNA-seeded subnuclear condensates, termed amyloid bodies (A-bodies). The molecular constituents of A-bodies induced by different stressors vary significantly, suggesting this pathway can tailor the cellular response by selectively aggregating a subset of proteins under a given condition. Here, we identify critical structural elements that regulate heat shock-specific amyloid aggregation. Our data demonstrates that manipulating structural pockets in constituent proteins can either induce or restrict their A-body targeting at elevated temperatures. We propose a model where selective aggregation within A-bodies is mediated by the thermal stability of a protein, with temperature-sensitive structural regions acting as an intrinsic form of post-translational regulation. This system would provide cells with a rapid and stress-specific response mechanism, to tightly control physiological amyloid aggregation or other cellular stress response pathways.

Intracellular signaling in proto-eukaryotes evolves to alleviate regulatory conflicts of endosymbiosis.

The complex eukaryotic cell resulted from a merger between simpler prokaryotic cells, yet the role of the mitochondrial endosymbiosis with respect to other eukaryotic innovations has remained under dispute. To investigate how the regulatory challenges associated with the endosymbiotic state impacted genome and network evolution during eukaryogenesis, we study a constructive computational model where two simple cells are forced into an obligate endosymbiosis. Across multiple in silico evolutionary replicates, we observe the emergence of different mechanisms for the coordination of host and symbiont cell cycles, stabilizing the endosymbiotic relationship. In most cases, coordination is implicit, without signaling between host and symbiont. Signaling only evolves when there is leakage of regulatory products between host and symbiont. In the fittest evolutionary replicate, the host has taken full control of the symbiont cell cycle through signaling, mimicking the regulatory dominance of the nucleus over the mitochondrion that evolved during eukaryogenesis.

A programmable reaction-diffusion system for spatiotemporal cell signaling circuit design.

Cells self-organize molecules in space and time to generate complex behaviors, but we lack synthetic strategies for engineering spatiotemporal signaling. We present a programmable reaction-diffusion platform for designing protein oscillations, patterns, and circuits in mammalian cells using two bacterial proteins, MinD and MinE (MinDE). MinDE circuits act like "single-cell radios," emitting frequency-barcoded fluorescence signals that can be spectrally isolated and analyzed using digital signal processing tools. We define how to genetically program these signals and connect their spatiotemporal dynamics to cell biology using engineerable protein-protein interactions. This enabled us to construct sensitive reporter circuits that broadcast endogenous cell signaling dynamics on a frequency-barcoded imaging channel and to build control signal circuits that synthetically pattern activities in the cell, such as protein condensate assembly and actin filamentation. Our work establishes a paradigm for visualizing, probing, and engineering cellular activities at length and timescales critical for biological function.

COQ4 is required for the oxidative decarboxylation of the C1 carbon of coenzyme Q in eukaryotic cells.

Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum. Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role but also via the oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis and shed light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.

Membrane fusion and fission during eukaryogenesis.

All eukaryotes can be traced back to a single shared ancestral lineage that emerged from interactions between different prokaryotic cells. Current models of eukaryogenesis describe various selective forces and evolutionary mechanisms that contributed to the formation of eukaryotic cells. Central to this process were significant changes in cellular structure, resulting in the configuration of a new cell type characterized by internal membrane compartments. Additionally, eukaryogenesis results in a life cycle that relies on cell-cell fusion. We discuss the potential roles of proteins involved in remodeling cellular membranes, highlighting two critical stages in the evolution of eukaryotes: the internalization of symbiotic partners and a scenario wherein the emergence of sexual reproduction is linked to a polyploid ancestor generated by cell-cell fusion.

New Perspectives on Crosstalks Between Bacterial Regulatory RNAs from Outer Membrane Vesicles and Eukaryotic Cells.

Regulatory small RNAs (sRNAs) help the bacteria to survive harsh environmental conditions by posttranscriptional regulation of genes involved in various biological pathways including stress responses, homeostasis, and virulence. These sRNAs can be found carried by different membrane-bound vesicles like extracellular vesicles (EVs), membrane vesicles (MVs), or outer membrane vesicles (OMVs). OMVs provide myriad functions in bacterial cells including carrying a cargo of proteins, lipids, and nucleic acids including sRNAs. A few interesting studies have shown that these sRNAs can be transported to the host cell by membrane vesicles and can regulate the host immune system. Although there is evidence that sRNAs can be exported to host cells and sometimes can even cross the blood-brain barrier, the exact mechanism is still unknown. In this review, we investigated the new techniques implemented in various studies, to elucidate the crosstalks between bacterial cells and human immune systems by membrane vesicles carrying bacterial regulatory sRNAs.

mRNA reading frame maintenance during eukaryotic ribosome translocation.

One of the most critical steps of protein synthesis is coupled translocation of messenger RNA (mRNA) and transfer RNAs (tRNAs) required to advance the mRNA reading frame by one codon. In eukaryotes, translocation is accelerated and its fidelity is maintained by elongation factor 2 (eEF2)1,2. At present, only a few snapshots of eukaryotic ribosome translocation have been reported3-5. Here we report ten high-resolution cryogenic-electron microscopy (cryo-EM) structures of the elongating eukaryotic ribosome bound to the full translocation module consisting of mRNA, peptidyl-tRNA and deacylated tRNA, seven of which also contained ribosome-bound, naturally modified eEF2. This study recapitulates mRNA-tRNA2-growing peptide module progression through the ribosome, from the earliest states of eEF2 translocase accommodation until the very late stages of the process, and shows an intricate network of interactions preventing the slippage of the translational reading frame. We demonstrate how the accuracy of eukaryotic translocation relies on eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs. Our findings shed light on the mechanism of translation arrest by the anti-fungal eEF2-binding inhibitor, sordarin. We also propose that the sterically constrained environment imposed by diphthamide, a conserved eukaryotic posttranslational modification in eEF2, not only stabilizes correct Watson-Crick codon-anticodon interactions but may also uncover erroneous peptidyl-tRNA, and therefore contribute to higher accuracy of protein synthesis in eukaryotes.

Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues.

Microtubules, composed of α/ß-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit dynamic instability as tubulin heterodimer subunits undergo repetitive polymerization and depolymerization. Precise control of microtubule stability and dynamics, achieved through tubulin post-translational modifications and microtubule-associated proteins, is essential for various cellular functions. Dysfunctions in microtubules are strongly implicated in pathogenesis, including neurodegenerative disorders. Ongoing research focuses on microtubule-targeting therapeutic agents that modulate stability, offering potential treatment options for these diseases and cancers. Consequently, understanding the dynamic state of microtubules is crucial for assessing disease progression and therapeutic effects. Traditionally, microtubule dynamics have been assessed in vitro or in cultured cells through rough fractionation or immunoassay, using antibodies targeting post-translational modifications of tubulin. However, accurately analyzing tubulin status in tissues using such procedures poses challenges. In this study, we developed a simple and innovative microtubule fractionation method to separate stable microtubules, labile microtubules, and free tubulin in mouse tissues. The procedure involved homogenizing dissected mouse tissues in a microtubule-stabilizing buffer at a 19:1 volume ratio. The homogenates were then fractionated through a two-step ultracentrifugation process following initial slow centrifugation (2,400 × g) to remove debris. The first ultracentrifugation step (100,000 × g) precipitated stable microtubules, while the resulting supernatant was subjected to a second ultracentrifugation step (500,000 × g) to fractionate labile microtubules and soluble tubulin dimers. This method determined the proportions of tubulin constituting stable or labile microtubules in the mouse brain. Additionally, distinct tissue variations in microtubule stability were observed that correlated with the proliferative capacity of constituent cells. These findings highlight the significant potential of this novel method for analyzing microtubule stability in physiological and pathological conditions.

The Liaison between Metabolism and Oxidative Stress in Human Diseases.

Mitochondria have been the prerequisite to eukaryote complexity since their likely endosymbiotic origin, allowing a remarkable expansion in the number of genes expressed [...].

Chromatinization modulates topoisomerase II processivity.

Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress. While cellular processes constantly create varying torsional stress, how this variation impacts type IIA topoisomerase function remains obscure. Using multiple single-molecule approaches, we examined the torsional dependence of eukaryotic topoisomerase II (topo II) activity on naked DNA and chromatin. We observed that topo II is ~50-fold more processive on buckled DNA than previously estimated. We further discovered that topo II relaxes supercoiled DNA prior to plectoneme formation, but with processivity reduced by ~100-fold. This relaxation decreases with diminishing torsion, consistent with topo II capturing transient DNA loops. Topo II retains high processivity on buckled chromatin (~10,000 turns) and becomes highly processive even on chromatin under low torsional stress (~1000 turns), consistent with chromatin's predisposition to readily form DNA crossings. This work establishes that chromatin is a major stimulant of topo II function.

The Nuclear Cap-Binding Complex, a multitasking binding partner of RNA polymerase II transcripts.

In eukaryotic cells, RNAs transcribed by RNA polymerase-II receive the modification at the 5' end. This structure is called the cap structure. The cap structure has a fundamental role for translation initiation by recruiting eukaryotic translation initiation factor 4F (eIF4F). The other important mediator of the cap structure is a nuclear cap-binding protein complex (CBC). CBC consists of two proteins, which are renamed as NCBP1 and NCBP2 (previously called as CBP80/NCBP and CBP20/NIP1, respectively). This review article discusses the multiple roles CBC mediates and co-ordinates in several gene expression steps in eukaryotes.

A structural biology view on the enzymes involved in eukaryotic mRNA turnover.

The cellular environment contains numerous ribonucleases that are dedicated to process mRNA transcripts that have been targeted for degradation. Here, we review the three dimensional structures of the ribonuclease complexes (Pan2-Pan3, Ccr4-Not, Xrn1, exosome) and the mRNA decapping enzymes (Dcp2, DcpS) that are involved in mRNA turnover. Structures of major parts of these proteins have been experimentally determined. These enzymes and factors do not act in isolation, but are embedded in interaction networks which regulate enzyme activity and ensure that the appropriate substrates are recruited. The structural details of the higher order complexes that form can, in part, be accurately deduced from known structural data of sub-complexes. Interestingly, many of the ribonuclease and decapping enzymes have been observed in structurally different conformations. Together with experimental data, this highlights that structural changes are often important for enzyme function. We conclude that the known structural data of mRNA decay factors provide important functional insights, but that static structural data needs to be complemented with information regarding protein motions to complete the picture of how transcripts are turned over. In addition, we highlight multiple aspects that influence mRNA turnover rates, but that have not been structurally characterized so far.

Towards unraveling the origins of eukaryotic nuclear genome organization.

With 3D genome mapping maturing over the past decade, studies exposed the differences between eukaryotic and prokaryotic genome organization. This raises the question of how the complex eukaryotic genome organization originated. Here, I explore potential pathways to answering this question, guided by our changing understanding of the origins of eukaryotes.

Hiker, a new family of DNA transposons encoding transposases with DD35E motifs, displays a distinct phylogenetic relationship with most known DNA transposon families of IS630-Tc1-mariner (ITm).

DNA transposons play a crucial role in determining the size and structure of eukaryotic genomes. In this study, a new family of IS630-Tc1-mariner (ITm) DNA transposons, named Hiker (HK), was identified. HK is characterized by a DD35E catalytic domain and is distinct from all previously known families of the ITm group. Phylogenetic analyses showed that DD35E/Hiker forms a monophyletic clade with DD34E/Gambol, indicating that they may represent a separate superfamily of ITm. A total of 178 Hiker species were identified, with 170 found mainly in Actinopterygii, one in Chondrichthyes, six in Anura and one in Mollusca. Gambol (GM), on the other hand, are found in invertebrates, with 18 in Arthropoda and one in Platyhelminthes. Hiker transposons have a total length ranging from 2.14 to 3.67 kb and contain a single open reading frame that encodes a protein of approximately 370 amino acids (range 311-413 aa). They are flanked by short terminal inverted repeats (TIRs) of 16-30 base pairs and two base pair (TA) target-site duplications. In contrast, most transposons of the Gambol family have a total length of 1.35-5.96 kb, encode a transposase protein of approximately 350 amino acids (range 306-374 aa), and are flanked by TIRs that range from 32 to 1097 bp in length. Both Hiker and Gambol transposases have several conserved motifs, including helix-turn-helix (HTH) motifs and a DDE domain. Our study observed multiple amplification waves and repeated horizontal transfer (HT) events of HK transposons in vertebrate genomes, indicating their role in diversifying and shaping the genomes of Actinopterygii, Chondrichthyes, and Anura. Conversely, GM transposons showed few Horizontal transfer events. According to cell-based transposition assays, most HK transposons are likely inactive due to the truncated DNA binding domains of their transposases. We present an updated classification of the ITm group based on these findings, which will enhance the understanding of both the evolution of ITm transposons and that of their hosts.

Comparative Clustering (CompaCt) of eukaryote complexomes identifies novel interactions and sheds light on protein complex evolution.

Complexome profiling allows large-scale, untargeted, and comprehensive characterization of protein complexes in a biological sample using a combined approach of separating intact protein complexes e.g., by native gel electrophoresis, followed by mass spectrometric analysis of the proteins in the resulting fractions. Over the last decade, its application has resulted in a large collection of complexome profiling datasets. While computational methods have been developed for the analysis of individual datasets, methods for large-scale comparative analysis of complexomes from multiple species are lacking. Here, we present Comparative Clustering (CompaCt), that performs fully automated integrative analysis of complexome profiling data from multiple species, enabling systematic characterization and comparison of complexomes. CompaCt implements a novel method for leveraging orthology in comparative analysis to allow systematic identification of conserved as well as taxon-specific elements of the analyzed complexomes. We applied this method to a collection of 53 complexome profiles spanning the major branches of the eukaryotes. We demonstrate the ability of CompaCt to robustly identify the composition of protein complexes, and show that integrated analysis of multiple datasets improves characterization of complexes from specific complexome profiles when compared to separate analyses. We identified novel candidate interactors and complexes in a number of species from previously analyzed datasets, like the emp24, the V-ATPase and mitochondrial ATP synthase complexes. Lastly, we demonstrate the utility of CompaCt for the automated large-scale characterization of the complexome of the mosquito Anopheles stephensi shedding light on the evolution of metazoan protein complexes. CompaCt is available from https://github.com/cmbi/compact-bio.

The minimal intrinsic stochasticity of constitutively expressed eukaryotic genes is sub-Poissonian.

Gene expression inherently gives rise to stochastic variation ("noise") in the production of gene products. Minimizing noise is crucial for ensuring reliable cellular functions. However, noise cannot be suppressed below a certain intrinsic limit. For constitutively expressed genes, this limit is typically assumed to be Poissonian noise, wherein the variance in mRNA numbers is equal to their mean. Here, we demonstrate that several cell division genes in fission yeast exhibit mRNA variances significantly below this limit. The reduced variance can be explained by a gene expression model incorporating multiple transcription and mRNA degradation steps. Notably, in this sub-Poissonian regime, distinct from Poissonian or super-Poissonian regimes, cytoplasmic noise is effectively suppressed through a higher mRNA export rate. Our findings redefine the lower limit of eukaryotic gene expression noise and uncover molecular requirements for achieving ultralow noise, which is expected to be important for vital cellular functions.

Multiple approaches of cellular metabolism define the bacterial ancestry of mitochondria.

We breathe at the molecular level when mitochondria in our cells consume oxygen to extract energy from nutrients. Mitochondria are characteristic cellular organelles that derive from aerobic bacteria and carry out oxidative phosphorylation and other key metabolic pathways in eukaryotic cells. The precise bacterial origin of mitochondria and, consequently, the ancestry of the aerobic metabolism of our cells remain controversial despite the vast genomic information that is now available. Here, we use multiple approaches to define the most likely living relatives of the ancestral bacteria from which mitochondria originated. These bacteria live in marine environments and exhibit the highest frequency of aerobic traits and genes for the metabolism of fundamental lipids that are present in the membranes of eukaryotes, sphingolipids, and cardiolipin.