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.

Correlating cryo-super resolution radial fluctuations and dual-axis cryo-scanning transmission electron tomography to bridge the light-electron resolution gap.

Visualization of organelles and their interactions with other features in the native cell remains a challenge in modern biology. We have introduced cryo-scanning transmission electron tomography (CSTET), which can access 3D volumes on the scale of 1 micron with a resolution of nanometers, making it ideal for this task. Here we introduce two relevant advances: (a) we demonstrate the utility of multi-color super-resolution radial fluctuation light microscopy under cryogenic conditions (cryo-SRRF), and (b) we extend the use of deconvolution processing for dual-axis CSTET data. We show that cryo-SRRF nanoscopy is able to reach resolutions in the range of 100 nm, using commonly available fluorophores and a conventional widefield microscope for cryo-correlative light-electron microscopy. Such resolution aids in precisely identifying regions of interest before tomographic acquisition and enhances precision in localizing features of interest within the 3D reconstruction. Dual-axis CSTET tilt series data and application of entropy regularized deconvolution during post-processing results in close-to-isotropic resolution in the reconstruction without averaging. The integration of cryo-SRRF with deconvolved dual-axis CSTET provides a versatile workflow for studying unique objects in a cell.

Microbial predators form a new supergroup of eukaryotes.

Molecular phylogenetics of microbial eukaryotes has reshaped the tree of life by establishing broad taxonomic divisions, termed supergroups, that supersede the traditional kingdoms of animals, fungi and plants, and encompass a much greater breadth of eukaryotic diversity1. The vast majority of newly discovered species fall into a small number of known supergroups. Recently, however, a handful of species with no clear relationship to other supergroups have been described2-4, raising questions about the nature and degree of undiscovered diversity, and exposing the limitations of strictly molecular-based exploration. Here we report ten previously undescribed strains of microbial predators isolated through culture that collectively form a diverse new supergroup of eukaryotes, termed Provora. The Provora supergroup is genetically, morphologically and behaviourally distinct from other eukaryotes, and comprises two divergent clades of predators-Nebulidia and Nibbleridia-that are superficially similar to each other, but differ fundamentally in ultrastructure, behaviour and gene content. These predators are globally distributed in marine and freshwater environments, but are numerically rare and have consequently been overlooked by molecular-diversity surveys. In the age of high-throughput analyses, investigation of eukaryotic diversity through culture remains indispensable for the discovery of rare but ecologically and evolutionarily important eukaryotes.

Guidelines for a Morphometric Analysis of Prokaryotic and Eukaryotic Cells by Scanning Electron Microscopy.

The invention of a scanning electron microscopy (SEM) pushed the imaging methods and allowed for the observation of cell details with a high resolution. Currently, SEM appears as an extremely useful tool to analyse the morphology of biological samples. The aim of this paper is to provide a set of guidelines for using SEM to analyse morphology of prokaryotic and eukaryotic cells, taking as model cases Escherichia coli bacteria and B-35 rat neuroblastoma cells. Herein, we discuss the necessity of a careful sample preparation and provide an optimised protocol that allows to observe the details of cell ultrastructure (≥ 50 nm) with a minimum processing effort. Highlighting the versatility of morphometric descriptors, we present the most informative parameters and couple them with molecular processes. In this way, we indicate the wide range of information that can be collected through SEM imaging of biological materials that makes SEM a convenient screening method to detect cell pathology.

Multi-membrane search algorithm.

This research proposes a new multi-membrane search algorithm (MSA) based on cell biological behavior. Cell secretion protein behavior and cell division and fusion strategy are the main inspirations for the algorithm. In order to verify the performance of the algorithm, we used 19 benchmark functions to compare the MSA test results with MVO, GWO, MFO and ALO. The number of iterations of each algorithm on each benchmark function is 100, the population number is 10, and the running is repeated 50 times, and the average and standard deviation of the results are recorded. Tests show that the MSA is competitive in unimodal benchmark functions and multi-modal benchmark functions, and the results in composite benchmark functions are all superior to MVO, MFO, ALO, and GWO algorithms. This paper also uses MSA to solve two classic engineering problems: welded beam design and pressure vessel design. The result of welded beam design is 1.7252, and the result of pressure vessel design is 5887.7052, which is better than other comparison algorithms. Statistical experiments show that MSA is a high-performance algorithm that is competitive in unimodal and multimodal functions, and its performance in compound functions is significantly better than MVO, MFO, ALO, and GWO algorithms.

Rapid tool for cell nanoarchitecture integrity assessment.

With the rapid increase and accessibility of high-resolution imaging technologies of cells, the interpretation of results relies more and more on the assumption that the three-dimensional integrity of the surrounding cellular landscape is not compromised by the experimental setup. However, the only available technology for directly probing the structural integrity of whole-cell preparations at the nanoscale is electron cryo-tomography, which is time-consuming, costly, and complex. We devised an accessible, inexpensive and reliable screening assay to quickly report on the compatibility of experimental protocols with preserving the structural integrity of whole-cell preparations at the nanoscale. Our Rapid Cell Integrity Assessment (RCIA) assay is executed at room temperature and relies solely on light microscopy imaging. Using cellular electron cryo-tomography as a benchmark, we verify that RCIA accurately unveils the adverse impact of reagents and/or protocols such as those used for virus inactivation or to arrest dynamic processes on the cellular nanoarchitecture.

Ribosome Biogenesis and Cancer: Overview on Ribosomal Proteins.

Cytosolic ribosomes (cytoribosomes) are macromolecular ribonucleoprotein complexes that are assembled from ribosomal RNA and ribosomal proteins, which are essential for protein biosynthesis. Mitochondrial ribosomes (mitoribosomes) perform translation of the proteins essential for the oxidative phosphorylation system. The biogenesis of cytoribosomes and mitoribosomes includes ribosomal RNA processing, modification and binding to ribosomal proteins and is assisted by numerous biogenesis factors. This is a major energy-consuming process in the cell and, therefore, is highly coordinated and sensitive to several cellular stressors. In mitochondria, the regulation of mitoribosome biogenesis is essential for cellular respiration, a process linked to cell growth and proliferation. This review briefly overviews the key stages of cytosolic and mitochondrial ribosome biogenesis; summarizes the main steps of ribosome biogenesis alterations occurring during tumorigenesis, highlighting the changes in the expression level of cytosolic ribosomal proteins (CRPs) and mitochondrial ribosomal proteins (MRPs) in different types of tumors; focuses on the currently available information regarding the extra-ribosomal functions of CRPs and MRPs correlated to cancer; and discusses the role of CRPs and MRPs as biomarkers and/or molecular targets in cancer treatment.

Maturing secretory granules: Where secretory and endocytic pathways converge.

Secretory granules (SGs) are specialized organelles responsible for the storage and regulated release of various biologically active molecules from the endocrine and exocrine systems. Thus, proper SG biogenesis is critical to normal animal physiology. Biogenesis of SGs starts at the trans-Golgi network (TGN), where immature SGs (iSGs) bud off and undergo maturation before fusing with the plasma membrane (PM). How iSGs mature is unclear, but emerging studies have suggested an important role for the endocytic pathway. The requirement for endocytic machinery in SG maturation blurs the line between SGs and another class of secretory organelles called lysosome-related organelles (LROs). Therefore, it is important to re-evaluate the differences and similarities between SGs and LROs.

Remodelling of membrane tubules by the actin cytoskeleton.

Inside living cells, the remodelling of membrane tubules by actomyosin networks is crucial for processes such as intracellular trafficking or organelle reshaping. In this review, we first present various in vivo situations in which actin affects membrane tubule remodelling, then we recall some results on force production by actin dynamics and on membrane tubules physics. Finally, we show that our knowledge of the underlying mechanisms by which actomyosin dynamics affect tubule morphology has recently been moved forward. This is thanks to in vitro experiments that mimic cellular membranes and actin dynamics and allow deciphering the physics of tubule remodelling in biochemically controlled conditions, and shed new light on tubule shape regulation.

Dynamic interplay between cell membrane tension and clathrin-mediated endocytosis.

Deformability of the plasma membrane, the outermost surface of metazoan cells, allows cells to be dynamic, mobile and flexible. Factors that affect this deformability, such as tension on the membrane, can regulate a myriad of cellular functions, including membrane resealing, cell motility, polarisation, shape maintenance, membrane area control and endocytic vesicle trafficking. This review focuses on mechanoregulation of clathrin-mediated endocytosis (CME). We first delineate the origins of cell membrane tension and the factors that yield to its spatial and temporal fluctuations within cells. We then review the recent literature demonstrating that tension on the membrane is a fast-acting and reversible regulator of CME. Finally, we discuss tension-based regulation of endocytic clathrin coat formation during physiological processes.

Redox-mediated regulation of low complexity domain self-association.

Eukaryotic cells express thousands of protein domains long believed to function in the absence of molecular order. These intrinsically disordered protein (IDP) domains are typified by gibberish-like repeats of only a limited number of amino acids that we refer to as domains of low sequence complexity. A decade ago, it was observed that these low complexity (LC) domains can undergo phase transition out of aqueous solution to form either liquid-like droplets or hydrogels. The self-associative interactions responsible for phase transition involve the formation of specific cross-ß structures that are unusual in being labile to dissociation. Here we give evidence that the LC domains of two RNA binding proteins, ataxin-2 and TDP43, form cross-ß interactions that specify biologically relevant redox sensors.

Centrosome structure and biogenesis: Variations on a theme?

Centrosomes are composed of two orthogonally arranged centrioles surrounded by an electron-dense matrix called the pericentriolar material (PCM). Centrioles are cylinders with diameters of ~250 nm, are several hundred nanometres in length and consist of 9-fold symmetrically arranged microtubules (MT). In dividing animal cells, centrosomes act as the principal MT-organising centres and they also organise actin, which tunes cytoplasmic MT nucleation. In some specialised cells, the centrosome acquires additional critical structures and converts into the base of a cilium with diverse functions including signalling and motility. These structures are found in most eukaryotes and are essential for development and homoeostasis at both cellular and organism levels. The ultrastructure of centrosomes and their derived organelles have been known for more than half a century. However, recent advances in a number of techniques have revealed the high-resolution structures (at Å-to-nm scale resolution) of centrioles and have begun to uncover the molecular principles underlying their properties, including: protein components; structural elements; and biogenesis in various model organisms. This review covers advances in our understanding of the features and processes that are critical for the biogenesis of the evolutionarily conserved structures of the centrosomes. Furthermore, it discusses how variations of these aspects can generate diversity in centrosome structure and function among different species and even between cell types within a multicellular organism.

SubLocEP: a novel ensemble predictor of subcellular localization of eukaryotic mRNA based on machine learning.

MOTIVATION: mRNA location corresponds to the location of protein translation and contributes to precise spatial and temporal management of the protein function. However, current assignment of subcellular localization of eukaryotic mRNA reveals important limitations: (1) turning multiple classifications into multiple dichotomies makes the training process tedious; (2) the majority of the models trained by classical algorithm are based on the extraction of single sequence information; (3) the existing state-of-the-art models have not reached an ideal level in terms of prediction and generalization ability. To achieve better assignment of subcellular localization of eukaryotic mRNA, a better and more comprehensive model must be developed. RESULTS: In this paper, SubLocEP is proposed as a two-layer integrated prediction model for accurate prediction of the location of sequence samples. Unlike the existing models based on limited features, SubLocEP comprehensively considers additional feature attributes and is combined with LightGBM to generated single feature classifiers. The initial integration model (single-layer model) is generated according to the categories of a feature. Subsequently, two single-layer integration models are weighted (sequence-based: physicochemical properties = 3:2) to produce the final two-layer model. The performance of SubLocEP on independent datasets is sufficient to indicate that SubLocEP is an accurate and stable prediction model with strong generalization ability. Additionally, an online tool has been developed that contains experimental data and can maximize the user convenience for estimation of subcellular localization of eukaryotic mRNA.

Consistency and variation of protein subcellular location annotations.

A major challenge for protein databases is reconciling information from diverse sources. This is especially difficult when some information consists of secondary, human-interpreted rather than primary data. For example, the Swiss-Prot database contains curated annotations of subcellular location that are based on predictions from protein sequence, statements in scientific articles, and published experimental evidence. The Human Protein Atlas (HPA) consists of millions of high-resolution microscopic images that show protein spatial distribution on a cellular and subcellular level. These images are manually annotated with protein subcellular locations by trained experts. The image annotations in HPA can capture the variation of subcellular location across different cell lines, tissues, or tissue states. Systematic investigation of the consistency between HPA and Swiss-Prot assignments of subcellular location, which is important for understanding and utilizing protein location data from the two databases, has not been described previously. In this paper, we quantitatively evaluate the consistency of subcellular location annotations between HPA and Swiss-Prot at multiple levels, as well as variation of protein locations across cell lines and tissues. Our results show that annotations of these two databases differ significantly in many cases, leading to proposed procedures for deriving and integrating the protein subcellular location data. We also find that proteins having highly variable locations are more likely to be biomarkers of diseases, providing support for incorporating analysis of subcellular location in protein biomarker identification and screening.

LncLocation: Efficient Subcellular Location Prediction of Long Non-Coding RNA-Based Multi-Source Heterogeneous Feature Fusion.

Recent studies uncover that subcellular location of long non-coding RNAs (lncRNAs) can provide significant information on its function. Due to the lack of experimental data, the number of lncRNAs is very limited, experimentally verified subcellular localization, and the numbers of lncRNAs located in different organelle are wildly imbalanced. The prediction of subcellular location of lncRNAs is actually a multi-classification small sample imbalance problem. The imbalance of data results in the poor recognition effect of machine learning models on small data subsets, which is a puzzling and challenging problem in the existing research. In this study, we integrate multi-source features to construct a sequence-based computational tool, lncLocation, to predict the subcellular location of lncRNAs. Autoencoder is used to enhance part of the features, and the binomial distribution-based filtering method and recursive feature elimination (RFE) are used to filter some of the features. It improves the representation ability of data and reduces the problem of unbalanced multi-classification data. By comprehensive experiments on different feature combinations and machine learning models, we select the optimal features and classifier model scheme to construct a subcellular location prediction tool, lncLocation. LncLocation can obtain an 87.78% accuracy using 5-fold cross validation on the benchmark data, which is higher than the state-of-the-art tools, and the classification performance, especially for small class sets, is improved significantly.

Mitochondria: A worthwhile object for ultrastructural qualitative characterization and quantification of cells at physiological and pathophysiological states using conventional transmission electron microscopy.

Mitochondria are highly dynamic intracellular organelles with ultrastructural heterogeneity reflecting the behaviour and functions of the cells. The ultrastructural remodelling, performed by the counteracting active processes of mitochondrial fusion and fission, enables the organelles to respond to diverse cellular requirements and cues. It is also an important part of mechanisms underlying adaptation of mitochondria to pathophysiological conditions that challenge the cell homeostasis. However, if the stressor is constantly acting, the adaptive capacity of the cell can be exceeded and defective changes in mitochondrial morphology (indicating the insufficient functionality of mitochondria or development of mitochondrial disorders) may appear. Beside qualitative description of mitochondrial ultrastructure, stereological principles concerning the estimation of alterations in mitochondrial volume density or surface density are invaluable approaches for unbiased quantification of cells under physiological or pathophysiological conditions. In order to improve our understanding of cellular functions and dysfunctions, transmission electron microscopy (TEM) still remains a gold standard for qualitative and quantitative ultrastructural examination of mitochondria from various cell types, as well as from those experienced to different stimuli or toxicity-inducing factors. In the current study, general morphological and functional features of mitochondria, and their ultrastructural heterogeneity related to physiological and pathophysiological states of the cells are reviewed. Moreover, stereological approaches for accurate quantification of mitochondrial ultrastructure from electron micrographs taken from TEM are described in detail.

Imaging of DNA and RNA in Living Eukaryotic Cells to Reveal Spatiotemporal Dynamics of Gene Expression.

This review focuses on imaging DNA and single RNA molecules in living cells to define eukaryotic functional organization and dynamic processes. The latest advances in technologies to visualize individual DNA loci and RNAs in real time are discussed. Single-molecule fluorescence microscopy provides the spatial and temporal resolution to reveal mechanisms regulating fundamental cell functions. Novel insights into the regulation of nuclear architecture, transcription, posttranscriptional RNA processing, and RNA localization provided by multicolor fluorescence microscopy are reviewed. A perspective on the future use of live imaging technologies and overcoming their current limitations is provided.

Isolation of an archaeon at the prokaryote-eukaryote interface.

The origin of eukaryotes remains unclear1-4. Current data suggest that eukaryotes may have emerged from an archaeal lineage known as 'Asgard' archaea5,6. Despite the eukaryote-like genomic features that are found in these archaea, the evolutionary transition from archaea to eukaryotes remains unclear, owing to the lack of cultured representatives and corresponding physiological insights. Here we report the decade-long isolation of an Asgard archaeon related to Lokiarchaeota from deep marine sediment. The archaeon-'Candidatus Prometheoarchaeum syntrophicum' strain MK-D1-is an anaerobic, extremely slow-growing, small coccus (around 550 nm in diameter) that degrades amino acids through syntrophy. Although eukaryote-like intracellular complexes have been proposed for Asgard archaea6, the isolate has no visible organelle-like structure. Instead, Ca. P. syntrophicum is morphologically complex and has unique protrusions that are long and often branching. On the basis of the available data obtained from cultivation and genomics, and reasoned interpretations of the existing literature, we propose a hypothetical model for eukaryogenesis, termed the entangle-engulf-endogenize (also known as E3) model.

High-Aspect-Ratio Nanostructured Surfaces as Biological Metamaterials.

Materials patterned with high-aspect-ratio nanostructures have features on similar length scales to cellular components. These surfaces are an extreme topography on the cellular level and have become useful tools for perturbing and sensing the cellular environment. Motivation comes from the ability of high-aspect-ratio nanostructures to deliver cargoes into cells and tissues, access the intracellular environment, and control cell behavior. These structures directly perturb cells' ability to sense and respond to external forces, influencing cell fate, and enabling new mechanistic studies. Through careful design of their nanoscale structure, these systems act as biological metamaterials, eliciting unusual biological responses. While predominantly used to interface eukaryotic cells, there is growing interest in nonanimal and prokaryotic cell interfacing. Both experimental and theoretical studies have attempted to develop a mechanistic understanding for the observed behaviors, predominantly focusing on the cell-nanostructure interface. This review considers how high-aspect-ratio nanostructured surfaces are used to both stimulate and sense biological systems.

Dealing with several flagella in the same cell.

Flagella are sophisticated organelles found in many eukaryotic microbes where they perform functions related to motility, signal detection, or cell morphogenesis. In many cases, several flagella are present per cell, and these can have a different composition, length, age, or function, raising the question of how this is managed. When the flagella are equivalent and constructed simultaneously such as in Chlamydomonas or Naegleria, we propose an equal access model where molecular components have free access to each organelle. By contrast, Trypanosoma and Leishmania contain temporally distinct organelles and elongate a new flagellum whilst maintaining the existing one. The equal access model could function providing that the mature flagellum is "locked" so that it can no longer be elongated or shortened. Alternatively, access of flagellar components could be restricted at the level of the basal body, the transition zone, or the loading on intraflagellar transport trains. In organisms that contains flagella of different age and composition such as Giardia, a temporal dimension is necessary, with the production of protein components of flagella spreading over one or more cell cycles. In the future, deciphering the molecular mechanisms involved in these processes should reveal new insights in flagellum assembly and function.