Root stem cell niche maintenance and apical meristem activity critically depend on THREONINE SYNTHASE1.

Indeterminate root growth depends on the stem cell niche (SCN) and root apical meristem (RAM) maintenance whose regulation permits plasticity in root system formation. Using a forward genetics approach, we isolated the moots koom1 ('short root' in Mayan) mutant that shows complete primary RAM exhaustion and abolished SCN activity. We identified that this phenotype is caused by a point mutation in the METHIONINE OVERACCUMULATOR2 (MTO2) gene that encodes THREONINE SYNTHASE1 and renamed the mutant as mto2-2. The amino acid profile showed drastic changes, most notorious of which was accumulation of methionine. In non-allelic mto1-1 (Arabidopsis thaliana cystathionine gamma-synthetase1) and mto3-1 (S-adenosylmethionine synthetase) mutants, both with an increased methionine level, the RAM size was similar to that of the wild type, suggesting that methionine overaccumulation itself did not cause RAM exhaustion in mto2 mutants. When mto2-2 RAM is not yet completely exhausted, exogenous threonine induced de novo SCN establishment and root growth recovery. The threonine-dependent RAM re-establishment in mto2-2 suggests that threonine is a limiting factor for RAM maintenance. In the root, MTO2 was predominantly expressed in the RAM. The essential role of threonine in mouse embryonic stem cells and in RAM maintenance suggests that common regulatory mechanisms may operate in plant and animal SCN maintenance.

Spo0M: structure and function beyond regulation of sporulation.

In this mini-review, we present a perspective on the recent findings relating Spo0M structure and function that will stimulate and guide further studies in the characterization of this interesting protein. Cell division and sporulation constitute two of the best studied processes in the model organism Bacillus subtilis; however, there are many missing pieces in the giant regulatory puzzle that governs the independent and shared networks between them. Spo0M is a little studied protein that has been related to both, cell division and sporulation, but its biochemical function and its direct interactions have not been yet defined. Structural analysis of Spo0M revealed the presence of an arrestin-like domain and an FP domain (a dimerization domain present in proteasome elements), motifs more commonly found in eukaryotic proteins. The aim of this perspective is to present open questions regarding the functional and structural features of Spo0M that make this protein a good candidate for the ancestor of arrestins in bacteria and an important element in developmental and differentiation processes of Bacillus subtilis.

On-Chip Terahertz-Frequency Measurements of Liquids.

Terahertz-frequency-range measurements can offer potential insight into the picosecond dynamics, and therefore function, of many chemical systems. There is a need to develop technologies capable of performing such measurements in aqueous and polar environments, particularly when it is necessary to maintain the full functionality of biological samples. In this study, we present a proof-of-concept technology comprising an on-chip planar Goubau line, integrated with a microfluidic channel, which is capable of low-loss, terahertz-frequency-range spectroscopic measurements of liquids. We also introduce a mathematical model that accounts for changes in the electric field distribution around the waveguide, allowing accurate, frequency-dependent liquid parameters to be extracted. We demonstrate the sensitivity of this technique by measuring a homologous alcohol series across the 0.1-0.8 THz frequency range.

Morphological, Biochemical, and Functional Study of Viral Replication Compartments Isolated from Adenovirus-Infected Cells.

UNLABELLED: Adenovirus (Ad) replication compartments (RC) are nuclear microenvironments where the viral genome is replicated and a coordinated program of late gene expression is established. These virus-induced nuclear sites seem to behave as central hubs for the regulation of virus-host cell interactions, since proteins that promote efficient viral replication as well as factors that participate in the antiviral response are coopted and concentrated there. To gain further insight into the activities of viral RC, here we report, for the first time, the morphology, composition, and activities of RC isolated from Ad-infected cells. Morphological analyses of isolated RC particles by superresolution microscopy showed that they were indistinguishable from RC within infected cells and that they displayed a dynamic compartmentalization. Furthermore, the RC-containing fractions (RCf) proved to be functional, as they directed de novo synthesis of viral DNA and RNA as well as RNA splicing, activities that are associated with RC in vivo. A detailed analysis of the production of viral late mRNA from RCf at different times postinfection revealed that viral mRNA splicing occurs in RC and that the synthesis, posttranscriptional processing, and release from RC to the nucleoplasm of individual viral late transcripts are spatiotemporally separate events. The results presented here demonstrate that RCf are a powerful system for detailed study into RC structure, composition, and activities and, as a result, the determination of the molecular mechanisms that induce the formation of these viral sites of adenoviruses and other nuclear-replicating viruses. IMPORTANCE: RC may represent molecular hubs where many aspects of virus-host cell interaction are controlled. Here, we show by superresolution microscopy that RCf have morphologies similar to those of RC within Ad-infected cells and that they appear to be compartmentalized, as nucleolin and DBP display different localization in the periphery of these viral sites. RCf proved to be functional, as they direct de novo synthesis of viral DNA and mRNA, allowing the detailed study of the regulation of viral genome replication and expression. Furthermore, we show that the synthesis and splicing of individual viral late mRNA occurs in RC and that they are subject to different temporal patterns of regulation, from their synthesis to their splicing and release from RC to the nucleoplasm. Hence, RCf represent a novel system to study molecular mechanisms that are orchestrated in viral RC to take control of the infected cell and promote an efficient viral replication cycle.

Free-space terahertz radiation from a LT-GaAs-on-quartz large-area photoconductive emitter.

We report on large-area photoconductive terahertz (THz) emitters with a low-temperature-grown GaAs (LT-GaAs) active layer fabricated on quartz substrates using a lift-off transfer process. These devices are compared to the same LT-GaAs emitters when fabricated on the growth substrate. We find that the transferred devices show higher optical-to-THz conversion efficiencies and significantly larger breakdown fields, which we attribute to reduced parasitic current in the substrate. Through these improvements, we demonstrate a factor of ~8 increase in emitted THz field strength at the maximum operating voltage. In addition we find improved performance when these devices are used for photoconductive detection, which we explain through a combination of reduced parasitic substrate currents and reduced space-charge build-up in the device.

Measurement of single-cell dynamics.

Populations of cells are almost always heterogeneous in function and fate. To understand the plasticity of cells, it is vital to measure quantitatively and dynamically the molecular processes that underlie cell-fate decisions in single cells. Early events in cell signalling often occur within seconds of the stimulus, whereas intracellular signalling processes and transcriptional changes can take minutes or hours. By contrast, cell-fate decisions, such as whether a cell divides, differentiates or dies, can take many hours or days. Multiparameter experimental and computational methods that integrate quantitative measurement and mathematical simulation of these noisy and complex processes are required to understand the highly dynamic mechanisms that control cell plasticity and fate.

On-chip picosecond pulse detection and generation using graphene photoconductive switches.

We report on the use of graphene for room temperature on-chip detection and generation of pulsed terahertz (THz) frequency radiation, exploiting the fast carrier dynamics of light-generated hot carriers, and compare our results with conventional low-temperature-grown gallium arsenide (LT-GaAs) photoconductive (PC) switches. Coupling of picosecond-duration pulses from a biased graphene PC switch into Goubau line waveguides is also demonstrated. A Drude transport model based on the transient photoconductance of graphene is used to describe the mechanism for both detection and generation of THz radiation.

Niflumic acid disrupts marine spermatozoan chemotaxis without impairing the spatiotemporal detection of chemoattractant gradients.

In many broadcast-spawning marine organisms, oocytes release chemicals that guide conspecific spermatozoa towards them through chemotaxis. In the sea urchin Lytechinus pictus, the chemoattractant peptide speract triggers a train of fluctuations of intracellular Ca(2+) concentration in the sperm flagella. Each transient Ca(2+) elevation leads to a momentary increase in flagellar bending asymmetry, known as a chemotactic turn. Furthermore, chemotaxis requires a precise spatiotemporal coordination between the Ca(2+)-dependent turns and the form of chemoattractant gradient. Spermatozoa that perform Ca(2+)-dependent turns while swimming down the chemoattractant gradient, and conversely suppress turning events while swimming up the gradient, successfully approach the center of the gradient. Previous experiments in Strongylocentrotus purpuratus sea urchin spermatozoa showed that niflumic acid (NFA), an inhibitor of several ion channels, drastically altered the speract-induced Ca(2+) fluctuations and swimming patterns. In this study, mathematical modeling of the speract-dependent Ca(2+) signaling pathway suggests that NFA, by potentially affecting hyperpolarization-activated and cyclic nucleotide-gated channels, Ca(2+)-regulated Cl(-) channels and/or Ca(2+)-regulated K(+) channels, may alter the temporal organization of Ca(2+) fluctuations, and therefore disrupt chemotaxis. We used a novel automated method for analyzing sperm behavior and we identified that NFA does indeed disrupt chemotactic responses of L. pictus spermatozoa, although the temporal coordination between the Ca(2+)-dependent turns and the form of chemoattractant gradient is unaltered. Instead, NFA disrupts sperm chemotaxis by altering the arc length traveled during each chemotactic turning event. This alteration in the chemotactic turn trajectory disorientates spermatozoa at the termination of the turning event. We conclude that NFA disrupts chemotaxis without affecting how the spermatozoa decode environmental cues.

On-chip Raman spectroscopy of live single cells for the staging of oesophageal adenocarcinoma progression.

The absence of early diagnosis contributes to oesophageal cancer being the sixth most common cause of global cancer-associated deaths, with a 5-year survival rate of < 20%. Barrett's oesophagus is the main pre-cancerous condition to adenocarcinoma development, characterised by the morphological transition of oesophageal squamous epithelium to metaplastic columnar epithelium. Early tracking and treatment of oesophageal adenocarcinoma could dramatically improve with diagnosis and monitoring of patients with Barrett's Oesophagus. Current diagnostic methods involve invasive techniques such as endoscopies and, with only a few identified biomarkers of disease progression, the detection of oesophageal adenocarcinoma is costly and challenging. In this work, single-cell Raman spectroscopy was combined with microfluidic techniques to characterise the development of oesophageal adenocarcinoma through the progression of healthy epithelial, Barrett's oesophagus and oesophageal adenocarcinoma cell lines. Principal component analysis and linear discriminant analysis were used to classify the different stages of cancer progression. with the ability to differentiate between healthy and cancerous cells with an accuracy of 97%. Whilst the approach could also separate the dysplastic stages from healthy or cancer with high accuracy-the intra-class separation was approximately 68%. Overall, these results highlight the potential for rapid and reliable diagnostic/prognostic screening of Barrett's Oesophagus patients.

Extending resolution within a single imaging frame.

The resolution of fluorescence microscopy images is limited by the physical properties of light. In the last decade, numerous super-resolution microscopy (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on the Mean Shift theory, which extends spatial resolution of single fluorescence images beyond the diffraction limit of light. MSSR works on low and high fluorophore densities, is not limited by the architecture of the optical setup and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image stacks, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other SRM approaches. Along with its wide accessibility, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.

Tuning sperm chemotaxis by calcium burst timing.

Marine invertebrate oocytes establish chemoattractant gradients that guide spermatozoa towards their source. In sea urchin spermatozoa, this relocation requires coordinated motility changes initiated by Ca(2+)-driven alterations in sperm flagellar curvature. We discovered that Lytechinus pictus spermatozoa undergo chemotaxis in response to speract, an egg-derived decapeptide previously noted to stimulate non-chemotactic motility alterations in Strongylocentrotus purpuratus spermatozoa. Sperm of both species responded to speract gradients with a sequence of turning episodes that correlate with transient flagellar Ca(2+) increases, yet only L. pictus spermatozoa accumulated at the gradient source. Detailed analysis of sperm behavior revealed that L. pictus spermatozoa selectively undergo Ca(2+) fluctuations while swimming along negative speract gradients while S. purpuratus sperm generate Ca(2+) fluctuations in a spatially non-selective manner. This difference is attributed to the selective suppression of Ca(2+) fluctuations of L. pictus spermatozoa as they swim towards the source of the chemoattractant gradient. This is the first study to compare and characterize the motility components that differ in chemotactic and non-chemotactic spermatozoa. Tuning of Ca(2+) fluctuations and associated turning episodes to the chemoattractant gradient polarity is a central feature of sea urchin sperm chemotaxis and may be a feature of sperm chemotaxis in general.

Strategies for locating the female gamete: the importance of measuring sperm trajectories in three spatial dimensions.

The spermatozoon must find its female gamete partner and deliver its genetic material to generate a new individual. This requires that the spermatozoon be motile and endowed with sophisticated swimming strategies to locate the oocyte. A common strategy is chemotaxis, in which spermatozoa detect and follow a gradient of chemical signals released by the egg and its associated structures. Decoding the female gamete's positional information is a process that spermatozoa undergo in a three-dimensional (3D) space; however, due to their speed and small size, this process has been studied almost exclusively in spermatozoa restricted to swimming in two dimensions (2D). This review examines the relationship between the mechanics of sperm propulsion and the physiological function of these cells in 3D. It also considers whether it is possible to derive all the 3D sperm swimming characteristics by extrapolating from 2D measurements. It is concluded that full insight into flagellar beat dynamics, swimming paths and chemotaxis under physiological conditions will eventually require quantitative imaging of flagellar form, ion flux changes, cell trajectories and modelling of free-swimming spermatozoa in 3D.

Tuning sperm chemotaxis.

Sperm chemotaxis is a long-term puzzle and most of our knowledge comes from studying marine animals that are external fertilizers. Sperm are attracted by diffusible chemical factors (chemoattractants) released from the egg which redirect their swimming paths towards their source. This redirection is driven by increases in flagellar curvature that correlate with transient flagellar Ca(2+) increases. Recent experimental and modelling results provide insights into the signal flow underlying the translation of an external chemical gradient into an intracellular molecular and motor response. A fundamental element of sea-urchin sperm chemotaxis lies in the ability of these cells to suppress Ca(2+)-mediated increases in flagellar curvature while experiencing an increasing chemoattractant gradient. The article considers this new evidence and summarizes the known underlying cellular mechanisms and behavioural strategies that sperm use to locate and fertilize the oocyte.

Real-time analysis of the role of Ca(2+) in flagellar movement and motility in single sea urchin sperm.

Eggs of many marine and mammalian species attract sperm by releasing chemoattractants that modify the bending properties of flagella to redirect sperm paths toward the egg. This process, called chemotaxis, is dependent on extracellular Ca(2+). We used stroboscopic fluorescence imaging to measure intracellular Ca(2+) concentration ([Ca(2+)]i) in the flagella of swimming sea urchin sperm. Uncaging of cyclic GMP induced Ca(2+) entry via at least two distinct pathways, and we identified a nimodipine-sensitive pathway, compartmentalized in the flagella, as a key regulator of flagellar bending and directed motility changes. We found that, contrary to current models, the degree of flagellar bending does not vary in proportion to the overall [Ca(2+)]i. Instead we propose a new model whereby flagella bending is increased by Ca(2+) flux through the nimodipine-sensitive pathway, and is unaffected by [Ca(2+)]i increases through alternative pathways.

Sperm chemotaxis is driven by the slope of the chemoattractant concentration field.

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.

Single live-cell imaging for systems biology.

Understanding how mammalian cells function requires a dynamic perspective. However, owing to the complexity of signalling networks, these non-linear systems can easily elude human intuition. The central aim of systems biology is to improve our understanding of the temporal complexity of cell signalling pathways, using a combination of experimental and computational approaches. Live-cell imaging and computational modelling are compatible techniques which allow quantitative analysis of cell signalling pathway dynamics. Non-invasive imaging techniques, based on the use of various luciferases and fluorescent proteins, trace cellular events such as gene expression, protein-protein interactions and protein localization in cells. By employing a number of markers in a single assay, multiple parameters can be measured simultaneously in the same cell. Following acquisition using specialized microscopy, analysis of multi-parameter time-lapse images facilitates the identification of important qualitative and quantitative relationships-linking intracellular signalling, gene expression and cell fate.

Sperm-activating peptides in the regulation of ion fluxes, signal transduction and motility.

Echinoderm sperm use cyclic nucleotides (CNs) as essential second messengers to locate and swim towards the egg. Sea urchin sperm constitute a rich source of membrane-bound guanylyl cyclase (mGC), which was first cloned from sea urchin testis by the group of David Garbers. His group also identified speract, the first sperm-activating peptide (SAP) to be isolated from the egg investment (or egg jelly). This decapeptide stimulates sperm mGC causing a fast transient increase in cGMP that triggers an orchestrated set of physiological responses including: changes in: membrane potential, intracellular pH (pHi), intracellular Ca2+ concentration ([Ca2+]i) and cAMP levels. Evidence from several groups indicated that cGMP activation of a K+ selective channel was the first ion permeability change in the signaling cascade induced by SAPs, and recently the candidate gene was finally identified. Each of the 4 repeated, 6 trans-membrane segments of this channel contains a cyclic nucleotide binding domain. Together they comprise a single polypeptide chain like voltage-gated Na+ or Ca2+ channels. This new type of channel, named tetraKCNG, appears to belong to the exclusive club of novel protein families expressed only in sperm and its progenitors. SAPs also induce fluctuations in flagellar [Ca2+]i that correlate with changes in flagellar form and regulate sperm trajectory. The motility changes depend on [Ca2+]i influx through specific Ca2+ channels and not on the overall [Ca2+]i in the sperm flagellum. All cilia and flagella have a conserved axonemal structure and thus understanding how Ca2+ regulates cilia and flagella beating is a fundamental question.

Nanoscale organization of rotavirus replication machineries.

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.

Analysis of the Binding of Expansin Exl1, from Pectobacterium carotovorum, to Plant Xylem and Comparison to EXLX1 from Bacillus subtilis.

The plant xylem is a preferred niche for some important bacterial phytopathogens, some of them encoding expansin proteins, which bind plant cell walls. Yet, the identity of the substrate for bacterial expansins within the plant cell wall and the nature of its interaction with it are poorly known. Here, we determined the localization of two bacterial expansins with differing isoelectric points (and with differing binding patterns to cell wall extracts) on plant tissue through in vitro fluorophore labeling and confocal imaging. Differential localization was observed, in which Exl1 from Pectobacterium carotovorum located into the intercellular spaces between xylem vessels and adjacent cells of the plant xylem, whereas EXLX1 from Bacillus subtilis bound cell walls of most cell types. In isolated vascular tissue, however, both PcExl1 and BsEXLX1 preferentially bound to tracheary elements over the xylem fibers, even though both are composed of secondary cell walls. Fluorescence correlation spectroscopy, employed to analyze the interaction of expansins with isolated xylem, indicates that binding is governed by more than one factor, which could include interaction with more than one type of polymer in the fibers, such as cellulose and hemicellulose or pectin. Binding to different polysaccharides could explain the observed reduction of cellulolytic and xylanolytic activities in the presence of expansin, possibly because of competition for the substrate. Our findings are relevant for the comprehensive understanding of the pathogenesis by P. carotovorum during xylem invasion, a process in which Exl1 might be involved.

Rapid cell separation with minimal manipulation for autologous cell therapies.

The ability to isolate specific, viable cell populations from mixed ensembles with minimal manipulation and within intra-operative time would provide significant advantages for autologous, cell-based therapies in regenerative medicine. Current cell-enrichment technologies are either slow, lack specificity and/or require labelling. Thus a rapid, label-free separation technology that does not affect cell functionality, viability or phenotype is highly desirable. Here, we demonstrate separation of viable from non-viable human stromal cells using remote dielectrophoresis, in which an electric field is coupled into a microfluidic channel using shear-horizontal surface acoustic waves, producing an array of virtual electrodes within the channel. This allows high-throughput dielectrophoretic cell separation in high conductivity, physiological-like fluids, overcoming the limitations of conventional dielectrophoresis. We demonstrate viable/non-viable separation efficacy of >98% in pre-purified mesenchymal stromal cells, extracted from human dental pulp, with no adverse effects on cell viability, or on their subsequent osteogenic capabilities.