Explore the vast range of titles available.

Key Points Polo-like kinases (PLKs) are a family of Ser/Thr kinases that have a pivotal role in cell cycle progression, the centrosome cycle, mitosis and cellular responses to DNA damage, which makes them attractive targets for treatments against several diseases. PLK1 is the most ancestral and best-conserved member of the family; it is found in most eukaryotic organisms, except for higher land plants. PLK4 is the most divergent member of the family. PLK2, PLK3 and PLK5 have evolved very recently, probably from a PLK1 gene duplication in vertebrates. PLK1 and PLK4 have distinct structural organizations and are phosphorylated at different residues, which correlate with different mode of actions. The amino-terminal kinase domain and carboxy-terminal polo box domains that characterize PLKs are crucial for regulation of their kinase catalytic activity in time and space, and for controlling subcellular PLK localization. Recent studies show non-canonical functions for PLKs in asymmetric cell division and cilia disassembly. PLKs function in centriole and centrosome biogenesis; PLK1 integrates various external stimuli with cell cycle inputs to coordinate mitotic progression and the centrosome cycle, whereas PLK4 drives centriole assembly. PLK2 and PLK3 have roles in DNA replication and in the DNA damage response and are also expressed in non-proliferative tissues, in which they have a role in cell differentiation and homeostasis (for example, PLK2 and PLK5 regulate neuronal activity). Members of the polo-like kinase (PLK) family are crucial regulators of cell cycle progression, centriole duplication, mitosis, cytokinesis and the DNA damage response. Recent structural and molecular studies have revealed how such processes depend on the tight regulation of PLK abundance, activity, localization and interactions with other proteins, and how dysregulation may be associated with disease. Members of the polo-like kinase (PLK) family are crucial regulators of cell cycle progression, centriole duplication, mitosis, cytokinesis and the DNA damage response. PLKs undergo major changes in abundance, activity, localization and structure at different stages of the cell cycle. They interact with other proteins in a tightly controlled spatiotemporal manner as part of a network that coordinates key cell cycle events. Their essential roles are highlighted by the fact that alterations in PLK function are associated with cancers and other diseases. Recent knowledge gained from PLK crystal structures, evolution and interacting molecules offers important insights into the mechanisms that underlie their regulation and activity, and suggests novel functions unrelated to cell cycle control for this family of kinases.

Humans and other vertebrates define body axis left-right asymmetry in the early stages of embryo development. The mechanism behind left-right establishment is not fully understood. Symmetry breaking occurs in a dedicated organ called the left-right organizer (LRO) and involves motile cilia generating fluid-flow therein. However, it has been a matter of debate whether the process of symmetry breaking relies on a chemosensory or a mechanosensory mechanism (Shinohara et al., 2012). Novel tailored manipulations for LRO fluid extraction in living zebrafish embryos allowed us to pinpoint a physiological developmental period for breaking left-right symmetry during development. The shortest critical time-window was narrowed to one hour and characterized by a mild counterclockwise flow. The experimental challenge consisted in emptying the LRO of its fluid, abrogating simultaneously flow force and chemical determinants. Our findings revealed an unprecedented recovery capacity of the embryo to re-fil and re-circulate new LRO fluid. The embryos that later developed laterality problems were found to be those that had lower anterior angular velocity and thus less anterior-posterior heterogeneity. Next, aiming to test the presence of any secreted determinant, we replaced the extracted LRO fluid by a physiological buffer. Despite some transitory flow homogenization, laterality defects were absent unless viscosity was altered, demonstrating that symmetry breaking does not depend on the nature of the fluid content but is rather sensitive to fluid mechanics. Altogether, we conclude that the zebrafish LRO is more sensitive to fluid dynamics for symmetry breaking.

Mammalian sperm delve into the female reproductive tract to fertilize the female gamete. The available information about how sperm regulate their motility during the final journey to the fertilization site is extremely limited. In this work, we investigated the structural and functional changes in the sperm flagellum after acrosomal exocytosis (AE) and during the interaction with the eggs. The evidence demonstrates that the double helix actin network surrounding the mitochondrial sheath of the midpiece undergoes structural changes prior to the motility cessation. This structural modification is accompanied by a decrease in diameter of the midpiece and is driven by intracellular calcium changes that occur concomitant with a reorganization of the actin helicoidal cortex. Midpiece contraction occurs in a subset of cells that undergo AE, and live-cell imaging during in vitro fertilization showed that the midpiece contraction is required for motility cessation after fusion is initiated. These findings provide the first evidence of the F-actin network’s role in regulating sperm motility, adapting its function to meet specific cellular requirements during fertilization, and highlighting the broader significance of understanding sperm motility.

Spermatozoa of marine invertebrates are attracted to their conspecific female gamete by diffusive molecules, called chemoattractants, released from the egg investments in a process known as chemotaxis. The information from the egg chemoattractant concentration field is decoded into intracellular Ca2+ concentration ([Ca2+]i) changes that regulate the internal motors that shape the flagellum as it beats. By studying sea urchin species-specific differences in sperm chemoattractant-receptor characteristics we show that receptor density constrains the steepness of the chemoattractant concentration gradient detectable by spermatozoa. Through analyzing different chemoattractant gradient forms, we demonstrate for the first time that Strongylocentrotus purpuratus sperm are chemotactic and this response is consistent with frequency entrainment of two coupled physiological oscillators: i) the stimulus function and ii) the [Ca2+]i changes. We demonstrate that the slope of the chemoattractant gradients provides the coupling force between both oscillators, arising as a fundamental requirement for sperm chemotaxis.

The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins for in vivo localization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered “net like” structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.

Intracellular calcium ([Ca2+]i) is a basic and ubiquitous cellular signal controlling a wide variety of biological processes. A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of [Ca2+]i spikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelope and temporal organisation of the [Ca2+]i-spike trains. The signalling network comprises an initial membrane hyperpolarisation produced by an Upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules leading to intraflagellar pH (pHi), voltage and [Ca2+]i fluctuations. The Upstream module outputs were fitted to kinetic data on cGMP activity and early membrane potential changes measured in bulk cell populations. Two candidate modules featuring voltage-dependent Ca2+-channels link these outputs to the downstream dynamics and can independently explain the typical decaying envelope and the progressive spacing of the spikes. In the first module, [Ca2+]i-spike trains require the concerted action of a classical CaV-like channel and a potassium channel, BK (Slo1), whereas the second module relies on pHi-dependent CatSper dynamics articulated with voltage-dependent neutral sodium-proton exchanger (NHE). We analysed the dynamics of these two modules alone and in mixed scenarios. We show that the [Ca2+]i dynamics observed experimentally after sustained alkalinisation can be reproduced by a model featuring the CatSper and NHE module but not by those including the pH-independent CaV and BK module or proportionate mixed scenarios. We conclude in favour of the module containing CatSper and NHE and highlight experimentally testable predictions that would corroborate this conclusion.

Rotavirus genome replication and assembly take place in cytoplasmic electron dense inclusions termed viroplasms (VPs). Previous conventional optical microscopy studies observing the intracellular distribution of rotavirus proteins and their organization in VPs have lacked molecular-scale spatial resolution, due to inherent spatial resolution constraints. In this work we employed super-resolution microscopy to reveal the nanometric-scale organization of VPs formed during rotavirus infection, and quantitatively describe the structural organization of seven viral proteins within and around the VPs. The observed viral components are spatially organized as five concentric layers, in which NSP5 localizes at the center of the VPs, surrounded by a layer of NSP2 and NSP4 proteins, followed by an intermediate zone comprised of the VP1, VP2, VP6. In the outermost zone, we observed a ring of VP4 and finally a layer of VP7. These findings show that rotavirus VPs are highly organized organelles. Rotaviruses are small viruses that can infect cells in the intestine. They are responsible for most cases of severe infectious diarrhea, the most common cause of death among young children in developing countries. Controlling the spread of rotavirus infections is difficult, even with high levels of hygiene, so effective treatments are essential to curtail the virus’ infections. Understanding how new rotaviral particles are made in infected cells is one of the first steps toward developing new therapies. Once rotaviruses enter the cells, proteins from the virus and the cell aggregate into compact spheres called viroplasms to make new viral particles. Studying these viroplasms used to be difficult because they are too small to see with the resolution of standard microscopes. In recent years, advances in microscopy and mathematical methods have focused on breaking the existing resolution limits, leading to the development of super-resolution microscopy. This new technique has made it possible to study objects with sizes in the order of a billionth of a meter, known as nanoscopic structures, including viroplasms. Garcés et al. use super-resolution microscopy to determine how viral proteins are arranged in the viroplasm and gain a better understanding of how the viruses are assembled. The images revealed that, in infected monkey kidney cells, rotavirus proteins inside the viroplasm form highly organized concentric layers. This arrangement is reliably repeated in viroplasms of different sizes, indicating that the organization of the proteins is likely set up when the viroplasm starts to form. These findings make use of new microscopy, image analysis and statistical tools to study rotaviruses, providing a new framework to understand many aspects of rotaviral biology. Additionally, the result showing that proteins organize consistently in viroplasms is a first step towards understanding how the machinery that makes new rotaviruses works, which could lead to future treatments for severe infectious diarrhea.

The motility of spermatozoa of both Lytechinus pictus and Strongylocentrotus purpuratus sea urchin species is modulated by the egg-derived decapeptide speract via an oscillatory [Ca2+]-dependent signaling pathway. Comprehension of this pathway is hence directly related to the understanding of regulated sperm swimming. Niflumic acid (NFA), a nonsteroidal anti-inflammatory drug alters several ion channels. Though unspecific, NFA profoundly affects how sea urchin sperm respond to speract, increasing the [Ca2+]i oscillation period, amplitude, peak and average level values of the responses in immobilized and swimming cells. A previous logical network model we developed for the [Ca2+] dynamics of speract signaling cascade in sea urchin sperm allows integrated dissection of individual and multiple actions of NFA. Among the channels affected by NFA are: hyperpolarization-activated and cyclic nucleotide gated Na+ channels (HCN), [Ca2+]-dependent Cl- channels (CaCC) and [Ca2+]-dependent K+ channels (CaKC), all present in the sea urchin genome. Here, using our model we investigated the effect of blocking in silico HCN and CaCC channels suggested by experiments. Regarding CaKC channels, arguments can be provided for either their blockage or activation by NFA. Our study yielded two scenarios compliant with experimental observations: i) under CaKC inhibition, this [Ca2+]-dependent K+ channel should be different from the Slo1 channel and ii) under activation of the CaKC channel, another [Ca2+] channel not considered previously in the network is required, such as the pH-dependent CatSper channel. Additionally, our findings predict cause-effect relations resulting from a selective inhibition of those channels. Knowledge of these relations may be of consequence for a variety of electrophysiological studies and have an impact on drug related investigations. Our study contributes to a better grasp of the network dynamics and suggests further experimental work.

Spo0M has been previously reported as a regulator of sporulation in Bacillus subtilis; however, little is known about the mechanisms through which it participates in sporulation, and there is no information to date that relates this protein to other processes in the bacterium. In this work we present evidence from proteomic, protein-protein interaction, morphological, subcellular localization microscopy and bioinformatics studies which indicate that Spo0M function is not necessarily restricted to sporulation, and point towards its involvement in other stages of the vegetative life cycle. In the current study, we provide evidence that Spo0M interacts with cytoskeletal proteins involved in cell division, which suggest a function additional to that previously described in sporulation. Spo0M expression is not restricted to the transition phase or sporulation; rather, its expression begins during the early stages of growth and Spo0M localization in B. subtilis depends on the bacterial life cycle and could be related to an additional proposed function. This is supported by our discovery of homologs in a broad distribution of bacterial genera, even in non-sporulating species. Our work paves the way for re-evaluation of the role of Spo0M in bacterial cell.

The emergence of left–right (LR) asymmetry in vertebrates is a prime example of a highly conserved fundamental process in developmental biology. Details of how symmetry breaking is established in different organisms are, however, still not fully understood. In the zebrafish (Danio rerio), it is known that a cilia-mediated vortical flow exists within its LR organizer, the so-called Kupffer’s vesicle (KV), and that it is directly involved in early LR determination. However, the flow exhibits spatio-temporal complexity; moreover, its conversion to asymmetric development has proved difficult to resolve despite a number of recent experimental advances and numerical efforts. In this paper, we provide further theoretical insight into the essence of flow generation by putting together a minimal biophysical model which reduces to a set of singular solutions satisfying the imposed boundary conditions; one that is informed by our current understanding of the fluid flow in the KV, that satisfies the requirements for left–right symmetry breaking, but which is also amenable to extensive parametric analysis. Our work is a step forward in this direction. By finding the general conditions for the solution to the fluid mechanics of a singular rotlet within a rigid sphere, we have enlarged the set of available solutions in a way that can be easily extended to more complex configurations. These general conditions define a suitable set for which to apply the superposition principle to the linear Stokes problem and, hence, by which to construct a continuous set of solutions that correspond to spherically constrained vortical flows generated by arbitrarily displaced infinitesimal rotations around any three-dimensional axis.