Genome-wide mapping of human DNA replication by optical replication mapping supports a stochastic model of eukaryotic replication.

The heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation sites make mapping the location and timing of replication initiation in human cells difficult. To address this challenge, we have developed optical replication mapping (ORM), a high-throughput single-molecule approach, and used it to map early-initiation events in human cells. The single-molecule nature of our data and a total of >2,500-fold coverage of the human genome on 27 million fibers averaging ∼300 kb in length allow us to identify initiation sites and their firing probability with high confidence. We find that the distribution of human replication initiation is consistent with inefficient, stochastic activation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.

Spontaneous electrical low-frequency oscillations: a possible role in Hydra and all living systems.

As one of the first model systems in biology, the basal metazoan Hydra has been revealing fundamental features of living systems since it was first discovered by Antonie van Leeuwenhoek in the early eighteenth century. While it has become well-established within cell and developmental biology, this tiny freshwater polyp is only now being re-introduced to modern neuroscience where it has already produced a curious finding: the presence of low-frequency spontaneous neural oscillations at the same frequency as those found in the default mode network in the human brain. Surprisingly, increasing evidence suggests such spontaneous electrical low-frequency oscillations (SELFOs) are found across the wide diversity of life on Earth, from bacteria to humans. This paper reviews the evidence for SELFOs in diverse phyla, beginning with the importance of their discovery in Hydra, and hypothesizes a potential role as electrical organism organizers, which supports a growing literature on the role of bioelectricity as a 'template' for developmental memory in organism regeneration. This article is part of the theme issue 'Basal cognition: conceptual tools and the view from the single cell'.

What determines eukaryotic translation elongation: recent molecular and quantitative analyses of protein synthesis.

Protein synthesis from mRNA is an energy-intensive and tightly controlled cellular process. Translation elongation is a well-coordinated, multifactorial step in translation that undergoes dynamic regulation owing to cellular state and environmental determinants. Recent studies involving genome-wide approaches have uncovered some crucial aspects of translation elongation including the mRNA itself and the nascent polypeptide chain. Additionally, these studies have fuelled quantitative and mathematical modelling of translation elongation. In this review, we provide a comprehensive overview of the key determinants of translation elongation. We discuss consequences of ribosome stalling or collision, and how the cells regulate translation in case of such events. Next, we review theoretical approaches and widely used mathematical models that have become an essential ingredient to interpret complex molecular datasets and study translation dynamics quantitatively. Finally, we review recent advances in live-cell reporter and related analysis techniques, to monitor the translation dynamics of single cells and single-mRNA molecules in real time.

Seeing around corners: Cells solve mazes and respond at a distance using attractant breakdown.

During development and metastasis, cells migrate large distances through complex environments. Migration is often guided by chemotaxis, but simple chemoattractant gradients between a source and sink cannot direct cells over such ranges. We describe how self-generated gradients, created by cells locally degrading attractant, allow single cells to navigate long, tortuous paths and make accurate choices between live channels and dead ends. This allows cells to solve complex mazes efficiently. Cells' accuracy at finding live channels was determined by attractant diffusivity, cell speed, and path complexity. Manipulating these parameters directed cells in mathematically predictable ways; specific combinations can even actively misdirect them. We propose that the length and complexity of many long-range migratory processes, including inflammation and germ cell migration, means that self-generated gradients are needed for successful navigation.

The adaptive potential of circular DNA accumulation in ageing cells.

Carefully maintained and precisely inherited chromosomal DNA provides long-term genetic stability, but eukaryotic cells facing environmental challenges can benefit from the accumulation of less stable DNA species. Circular DNA molecules lacking centromeres segregate randomly or asymmetrically during cell division, following non-Mendelian inheritance patterns that result in high copy number instability and massive heterogeneity across populations. Such circular DNA species, variously known as extrachromosomal circular DNA (eccDNA), microDNA, double minutes or extrachromosomal DNA (ecDNA), are becoming recognised as a major source of the genetic variation exploited by cancer cells and pathogenic eukaryotes to acquire drug resistance. In budding yeast, circular DNA molecules derived from the ribosomal DNA (ERCs) have been long known to accumulate with age, but it is now clear that aged yeast also accumulate other high-copy protein-coding circular DNAs acquired through both random and environmentally-stimulated recombination processes. Here, we argue that accumulation of circular DNA provides a reservoir of heterogeneous genetic material that can allow rapid adaptation of aged cells to environmental insults, but avoids the negative fitness impacts on normal growth of unsolicited gene amplification in the young population.

Motile cilia hydrodynamics: entrainment versus synchronization when coupling through flow.

Coordinated motion of cilia is a fascinating and vital aspect of very diverse forms of eukaryotic life, enabling swimming and propulsion of fluid across cellular epithelia. There are many questions still unresolved, and broadly they fall into two classes. (i) The mechanism of how cilia physically transmit forces onto each other. It is not known for many systems if the forces are mainly of hydrodynamical origin, or if elastic forces within the cytoskeleton are important. (ii) In those systems where we know that forces are purely hydrodynamical, we do not have a framework for linking our understanding of how each cilium behaves in isolation to the collective properties of two or more cilia. In this work, we take biological data of cilia dynamics from a variety of organisms as an input for an analytical and numerical study. We calculate the relative importance of external flows versus internal cilia flows on cilia coupling. This study contributes to both the open questions outlined above: firstly, we show that it is, in general, incorrect to infer cilium-cilium coupling strength on the basis of experiments with external flows, and secondly, we show a framework to recapitulate the dynamics of single cilia (the waveform) showing classes that correspond to biological systems with the same physiological activity (swimming by propulsion, versus forming collective waves). This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

CryoSTEM tomography in biology.

Electron cryo-tomography using the scanning transmission modality (STEM) enables 3D reconstruction of unstained, vitrified specimens as thick as 1µm or more. Contrast is related to mass/thickness and atomic number, providing quantifiable chemical characterization and mass mapping of intact prokaryotic and eukaryotic cells. Energy dispersive X-ray spectroscopy by STEM provides a simple, on-the-spot chemical identification of the elemental composition in sub-cellular organic bodies or mineral deposits. This chapter provides basic background and practical information for performing cryo-STEM tomography on vitrified biological cells.

Cell Form and Function: Interpreting and Controlling the Shape of Adherent Cells.

Beautiful images of animal cells cultured on surfaces are ubiquitous in biological research, but these shapes also carry valuable information about the cells and the organism that they came from. Cell morphology is an emergent property of the cellular phenotype as well as of the physiological and signaling state of the cell. Many functional changes in cells cause stereotypical changes in cellular morphology, and some changes in shape can also cause characteristic changes in cellular phenotype. Thus, controlling cell shape through substrate engineering may emerge as another mechanism to modulate cell function for human health. This review summarizes current understanding of the morphology-phenotype connection, and surveys progress in the effort to interpret and control cell morphology.

Was the Mitochondrion Necessary to Start Eukaryogenesis?

Arguments based on cell energetics favour the view that a mitochondrion capable of oxidative phosphorylation was a prerequisite for the evolution of other features of the eukaryotic cell, including increased volume, genome size and, eventually, phagotrophy. Contrary to this we argue that: (i) extant amitochondriate eukaryotes possess voluminous phagotrophic cells with large genomes; (ii) picoeukaryotes demonstrate that phagotrophy is feasible at prokaryotic cell sizes; and (iii) the assumption that evolution of complex features requires extra ATP, often mentioned in this context, is unfounded and should not be used in such considerations. We claim that the diversity of cell organisations and functions observed today in eukaryotes gives no reason to postulate that a mitochondrion must have preceded phagocytosis in eukaryogenesis.

Eukaryotic cell survival mechanisms: Disease relevance and therapeutic intervention.

Cell responds to stress by activating various modes of stress responses which aim for minimal damage to cells and speedy recovery from the insults. However, unresolved stresses exceeding the tolerance limit lead to cell death (apoptosis, autophagy etc.) that helps to get rid of damaged cells and protect cell integrity. Furthermore, aberrant stress responses are the hallmarks of several pathophysiologies (neurodegeneration, metabolic diseases, cancer etc.). The catastrophic remodulation of stress responses is observed in cancer cells in favor of their uncontrolled growth. Whereas pro-survival stress responses redirected to death signaling provokes excessive cell death in neurodegeneration. Clear understanding of such mechanistic link to disease progression is required in order to modulate these processes for new therapeutic targets. The current review explains this with respect to novel drug discoveries and other breakthroughs in therapeutics.

Spontaneous oscillation and fluid-structure interaction of cilia.

The exact mechanism to orchestrate the action of hundreds of dynein motor proteins to generate wave-like ciliary beating remains puzzling and has fascinated many scientists. We present a 3D model of a cilium and the simulation of its beating in a fluid environment. The model cilium obeys a simple geometric constraint that arises naturally from the microscopic structure of a real cilium. This constraint allows us to determine the whole 3D structure at any instant in terms of the configuration of a single space curve. The tensions of active links, which model the dynein motor proteins, follow a postulated dynamical law, and together with the passive elasticity of microtubules, this dynamical law is responsible for the ciliary motions. In particular, our postulated tension dynamics lead to the instability of a symmetrical steady state, in which the cilium is straight and its active links are under equal tensions. The result of this instability is a stable, wave-like, limit cycle oscillation. We have also investigated the fluid-structure interaction of cilia using the immersed boundary (IB) method. In this setting, we see not only coordination within a single cilium but also, coordinated motion, in which multiple cilia in an array organize their beating to pump fluid, in particular by breaking phase synchronization.

Dynamic Architecture of Eukaryotic DNA Replication Forks In Vivo, Visualized by Electron Microscopy.

The DNA replication process can be heavily perturbed by several different conditions of genotoxic stress, particularly relevant for cancer onset and therapy. The combination of psoralen crosslinking and electron microscopy has proven instrumental to reveal the fine architecture of in vivo DNA replication intermediates and to uncover their remodeling upon specific conditions of genotoxic stress. The replication structures are stabilized in vivo (by psoralen crosslinking) prior to extraction and enrichment procedures, allowing their visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize and interpret in vivo replication intermediates of eukaryotic genomic DNA, and includes an improved method for enrichment of replication intermediates, compared to previously used BND-cellulose columns.

The Physiology of Phagocytosis in the Context of Mitochondrial Origin.

How mitochondria came to reside within the cytosol of their host has been debated for 50 years. Though current data indicate that the last eukaryote common ancestor possessed mitochondria and was a complex cell, whether mitochondria or complexity came first in eukaryotic evolution is still discussed. In autogenous models (complexity first), the origin of phagocytosis poses the limiting step at eukaryote origin, with mitochondria coming late as an undigested growth substrate. In symbiosis-based models (mitochondria first), the host was an archaeon, and the origin of mitochondria was the limiting step at eukaryote origin, with mitochondria providing bacterial genes, ATP synthesis on internalized bioenergetic membranes, and mitochondrion-derived vesicles as the seed of the eukaryote endomembrane system. Metagenomic studies are uncovering new host-related archaeal lineages that are reported as complex or phagocytosing, although images of such cells are lacking. Here we review the physiology and components of phagocytosis in eukaryotes, critically inspecting the concept of a phagotrophic host. From ATP supply and demand, a mitochondrion-lacking phagotrophic archaeal fermenter would have to ingest about 34 times its body weight in prokaryotic prey to obtain enough ATP to support one cell division. It would lack chemiosmotic ATP synthesis at the plasma membrane, because phagocytosis and chemiosmosis in the same membrane are incompatible. It would have lived from amino acid fermentations, because prokaryotes are mainly protein. Its ATP yield would have been impaired relative to typical archaeal amino acid fermentations, which involve chemiosmosis. In contrast, phagocytosis would have had great physiological benefit for a mitochondrion-bearing cell.

Determining whether observed eukaryotic cell migration indicates chemotactic responsiveness or random chemokinetic motion.

Chemotaxis, the motion of cells directed by a gradient of chemoattractant molecules, guides cells in immune response, development, wound healing, and cancer. Unfortunately, this process is difficult to distinguish from chemokinesis, i.e., stimulated random cell motion. Chemotaxis is frequently inferred by determining how many cells cross a boundary in a chemotaxis assay, for example how many cells crawl into a chemoattractant-infused filter, or how many cells enter a defined region in an under-agarose assay or agarose spot assay. To mitigate possible ambiguity in whether motion observed in these assays is directed by the chemoattractant gradient or by chemokinesis, we developed a mathematical model to determine when such methods indeed indicate directed motion of cells. In contrast to previous analyses of chemotaxis assays, we report not just the gradients that arise in the assays but also resulting cell motion. We applied the model to data obtained from rigorous measurements and show, as examples, that MDA-MB-231 breast-cancer cells are at least 20 times less sensitive to gradients of EGF or CXCL12 than neutrophils are to formyl peptides; we then used this information to determine the extent to which gradient sensing increases the rate of boundary crossing relative to a random-motility control. Results show, for example, that in the filter assay, 2-4 times as many neutrophils pass through the filter when exposed to a gradient as when the gradient is absent. However, in the other combinations of cells and assays we considered, only 10-20% more cells are counted as having migrated in a directed, rather than random, motility condition. We also discuss the design of appropriate controls for these assays, which is difficult for the under-agarose and agarose spot assays. Moreover, although straightforward to perform with the filter assay, reliable controls are often not done. Consequently, we infer that chemotaxis is frequently over-reported, especially for cells like MDA-MB-231 cells, which move slowly and are relatively insensitive to gradients. Such results provide insights into the use of chemotaxis assays, particularly if one wants to acquire and analyze quantitative data.

The Evolutionary Landscape of Dbl-Like RhoGEF Families: Adapting Eukaryotic Cells to Environmental Signals.

The dynamics of cell morphology in eukaryotes is largely controlled by small GTPases of the Rho family. Rho GTPases are activated by guanine nucleotide exchange factors (RhoGEFs), of which diffuse B-cell lymphoma (Dbl)-like members form the largest family. Here, we surveyed Dbl-like sequences from 175 eukaryotic genomes and illuminate how the Dbl family evolved in all eukaryotic supergroups. By combining probabilistic phylogenetic approaches and functional domain analysis, we show that the human Dbl-like family is made of 71 members, structured into 20 subfamilies. The 71 members were already present in ancestral jawed vertebrates, but several members were subsequently lost in specific clades, up to 12% in birds. The jawed vertebrate repertoire was established from two rounds of duplications that occurred between tunicates, cyclostomes, and jawed vertebrates. Duplicated members showed distinct tissue distributions, conserved at least in Amniotes. All 20 subfamilies have members in Deuterostomes and Protostomes. Nineteen subfamilies are present in Porifera, the first phylum that diverged in Metazoa, 14 in Choanoflagellida and Filasterea, single-celled organisms closely related to Metazoa and three in Fungi, the sister clade to Metazoa. Other eukaryotic supergroups show an extraordinary variability of Dbl-like repertoires as a result of repeated and independent gain and loss events. Last, we observed that in Metazoa, the number of Dbl-like RhoGEFs varies in proportion of cell signaling complexity. Overall, our analysis supports the conclusion that Dbl-like RhoGEFs were present at the origin of eukaryotes and evolved as highly adaptive cell signaling mediators.

Membranes, energetics, and evolution across the prokaryote-eukaryote divide.

The evolution of the eukaryotic cell marked a profound moment in Earth's history, with most of the visible biota coming to rely on intracellular membrane-bound organelles. It has been suggested that this evolutionary transition was critically dependent on the movement of ATP synthesis from the cell surface to mitochondrial membranes and the resultant boost to the energetic capacity of eukaryotic cells. However, contrary to this hypothesis, numerous lines of evidence suggest that eukaryotes are no more bioenergetically efficient than prokaryotes. Thus, although the origin of the mitochondrion was a key event in evolutionary history, there is no reason to think membrane bioenergetics played a direct, causal role in the transition from prokaryotes to eukaryotes and the subsequent explosive diversification of cellular and organismal complexity.

Historical Perspective of Eukaryotic DNA Replication.

The replication of the genome of a eukaryotic cell is a complex process requiring the ordered assembly of multiprotein replisomes at many chromosomal sites. The process is strictly controlled during the cell cycle to ensure the complete and faithful transmission of genetic information to progeny cells. Our current understanding of the mechanisms of eukaryotic DNA replication has evolved over a period of more than 30 years through the efforts of many investigators. The aim of this perspective is to provide a brief history of the major advances during this period.

Phosphorescence based O2 sensors - Essential tools for monitoring cell and tissue oxygenation and its impact on metabolism.

Oxygenation condition at the cellular level is a critical factor in tissue physiology and common pathophysiological states including cancer, metabolic disorders, ischemia-reperfusion injury and inflammation. O2 and ROS signalling and hypoxia research are rapidly growing areas spanning life and biomedical sciences, but still many current cell and tissue models and experimental set ups lack physiological relevance, particularly precise control of cellular O2. Quenched-phosphorescence O2 sensing enables implementation of such in situ control of cellular O2 and the creation of physiological conditions in respiring samples analysed in vitro. The advantages of optical O2 sensing are the non-invasive, contactless, real-time, quantitative monitoring of O2 concentration, which can be performed in the gas or liquid phase, macroscopically or microscopically, by point measurement or in imaging mode, with sub-cellular spatial resolution, in a flexible manner and with various cell and tissue models. Significantly, this same technology can also be used to probe the metabolism of cells and tissue under specific oxygenation conditions and their responses to changing conditions. Here we describe the range of available O2 sensing systems and tools, their analytical capabilities, uses in cell/tissue physiology and hypoxia research, and strategies for integration in routine experimental procedures.

Inseparable tandem: evolution chooses ATP and Ca2+ to control life, death and cellular signalling.

From the very dawn of biological evolution, ATP was selected as a multipurpose energy-storing molecule. Metabolism of ATP required intracellular free Ca(2+) to be set at exceedingly low concentrations, which in turn provided the background for the role of Ca(2+) as a universal signalling molecule. The early-eukaryote life forms also evolved functional compartmentalization and vesicle trafficking, which used Ca(2+) as a universal signalling ion; similarly, Ca(2+) is needed for regulation of ciliary and flagellar beat, amoeboid movement, intracellular transport, as well as of numerous metabolic processes. Thus, during evolution, exploitation of atmospheric oxygen and increasingly efficient ATP production via oxidative phosphorylation by bacterial endosymbionts were a first step for the emergence of complex eukaryotic cells. Simultaneously, Ca(2+) started to be exploited for short-range signalling, despite restrictions by the preset phosphate-based energy metabolism, when both phosphates and Ca(2+) interfere with each other because of the low solubility of calcium phosphates. The need to keep cytosolic Ca(2+) low forced cells to restrict Ca(2+) signals in space and time and to develop energetically favourable Ca(2+) signalling and Ca(2+) microdomains. These steps in tandem dominated further evolution. The ATP molecule (often released by Ca(2+)-regulated exocytosis) rapidly grew to be the universal chemical messenger for intercellular communication; ATP effects are mediated by an extended family of purinoceptors often linked to Ca(2+) signalling. Similar to atmospheric oxygen, Ca(2+) must have been reverted from a deleterious agent to a most useful (intra- and extracellular) signalling molecule. Invention of intracellular trafficking further increased the role for Ca(2+) homeostasis that became critical for regulation of cell survival and cell death. Several mutually interdependent effects of Ca(2+) and ATP have been exploited in evolution, thus turning an originally unholy alliance into a fascinating success story.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'.

Mitochondria, the Cell Cycle, and the Origin of Sex via a Syncytial Eukaryote Common Ancestor.

Theories for the origin of sex traditionally start with an asexual mitosing cell and add recombination, thereby deriving meiosis from mitosis. Though sex was clearly present in the eukaryote common ancestor, the order of events linking the origin of sex and the origin of mitosis is unknown. Here, we present an evolutionary inference for the origin of sex starting with a bacterial ancestor of mitochondria in the cytosol of its archaeal host. We posit that symbiotic association led to the origin of mitochondria and gene transfer to host's genome, generating a nucleus and a dedicated translational compartment, the eukaryotic cytosol, in which-by virtue of mitochondria-metabolic energy was not limiting. Spontaneous protein aggregation (monomer polymerization) and Adenosine Tri-phosphate (ATP)-dependent macromolecular movement in the cytosol thereby became selectable, giving rise to continuous microtubule-dependent chromosome separation (reduction division). We propose that eukaryotic chromosome division arose in a filamentous, syncytial, multinucleated ancestor, in which nuclei with insufficient chromosome numbers could complement each other through mRNA in the cytosol and generate new chromosome combinations through karyogamy. A syncytial (or coenocytic, a synonym) eukaryote ancestor, or Coeca, would account for the observation that the process of eukaryotic chromosome separation is more conserved than the process of eukaryotic cell division. The first progeny of such a syncytial ancestor were likely equivalent to meiospores, released into the environment by the host's vesicle secretion machinery. The natural ability of archaea (the host) to fuse and recombine brought forth reciprocal recombination among fusing (syngamy and karyogamy) progeny-sex-in an ancestrally meiotic cell cycle, from which the simpler haploid and diploid mitotic cell cycles arose. The origin of eukaryotes was the origin of vertical lineage inheritance, and sex was required to keep vertically evolving lineages viable by rescuing the incipient eukaryotic lineage from Muller's ratchet. The origin of mitochondria was, in this view, the decisive incident that precipitated symbiosis-specific cell biological problems, the solutions to which were the salient features that distinguish eukaryotes from prokaryotes: A nuclear membrane, energetically affordable ATP-dependent protein-protein interactions in the cytosol, and a cell cycle involving reduction division and reciprocal recombination (sex).