Single-cell Events Research Articles

The frequency and duration of Salmonella-macrophage adhesion events determines infection efficiency.

Salmonella enterica causes a range of important diseases in humans and a in a variety of animal species. The ability of bacteria to adhere to, invade and survive within host cells plays an important role in the pathogenesis of Salmonella infections. In systemic salmonellosis, macrophages constitute a niche for the proliferation of bacteria within the host organism. Salmonella enterica serovar Typhimurium is flagellated and the frequency with which this bacterium collides with a cell is important for infection efficiency. We investigated how bacterial motility affects infection efficiency, using a combination of population-level macrophage infection experiments and direct imaging of single-cell infection events, comparing wild-type and motility mutants. Non-motile and aflagellate bacterial strains, in contrast to wild-type bacteria, collide less frequently with macrophages, are in contact with the cell for less time and infect less frequently. Run-biased Salmonella also collide less frequently with macrophages but maintain contact with macrophages for a longer period of time than wild-type strains and infect the cells more readily. Our results suggest that uptake of S. Typhimurium by macrophages is dependent upon the duration of contact time of the bacterium with the cell, in addition to the frequency with which the bacteria collide with the cell.

Neutrophil Extracellular Trap Formation: A Single Cell Event?

In 2004, neutrophil extracellular traps (NETs) have been described as fundamental immune defense of neutrophils against various microbes. Since that time publications are increasing that characterize the stimuli and cellular mechanisms which can activate the cells to release NETs. However, it is still not entirely clear whether NET formation starts as a single cell event, that spreads by cell to cell communication or if neighboring cells undergo NET formation simultaneously caused by a common trigger. Using immunofluorescence microscopy, we here show that at lower cell density only single cells were detected that released NET structures. However, the higher the cell density, the higher was the x-fold-increase of NET formation in PMA stimulated cells compared to unstimulated cells. This might give a hint that NET formation might start as a single cell event but is able to spread due to cell communication. To fully understand the mechanisms mediating NET formation, future experiments should focus on single cell analysis to characterize the detailed cellular events that mediate formation of NETs in single cells within a population and to differentiate the signaling process that leads to NET formation in contrast to other antimicrobial strategies as phagocytosis or degranulation.

A signal integration model of thymic selection and natural regulatory T cell commitment.

The extent of TCR self-reactivity is the basis for selection of a functional and self-tolerant T cell repertoire and is quantified by repeated engagement of TCRs with a diverse pool of self-peptides complexed with self-MHC molecules. The strength of a TCR signal depends on the binding properties of a TCR to the peptide and the MHC, but it is not clear how the specificity to both components drives fate decisions. In this study, we propose a TCR signal-integration model of thymic selection that describes how thymocytes decide among distinct fates, not only based on a single TCR-ligand interaction, but taking into account the TCR stimulation history. These fates are separated based on sustained accumulated signals for positive selection and transient peak signals for negative selection. This spans up the cells into a two-dimensional space where they are either neglected, positively selected, negatively selected, or selected as natural regulatory T cells (nTregs). We show that the dynamics of the integrated signal can serve as a successful basis for extracting specificity of thymocytes to MHC and detecting the existence of cognate self-peptide-MHC. It allows to select a self-MHC-biased and self-peptide-tolerant T cell repertoire. Furthermore, nTregs in the model are enriched with MHC-specific TCRs. This allows nTregs to be more sensitive to activation and more cross-reactive than conventional T cells. This study provides a mechanistic model showing that time integration of TCR-mediated signals, as opposed to single-cell interaction events, is needed to gain a full view on the properties emerging from thymic selection.

Abstract 13443: Function Follows Form: Shape and Substrate Stiffness Drive Maturity in Human Cardiomyocytes Differentiated From Pluripotent Stem Cells

Introduction: Heart function relies on the contractility of its muscle cells (cardiomyocytes). Human pluripotent stem cells (hPSC) can be differentiated into cardiomyocytes (hPSC-CMs). However, while their myogenic maturity increases with time, they do not resemble adult cardiomyocytes in their morphology, structural organization, mechanical output and electrophysiology. The low maturity of hPSC-CMs limits their potential to model, study and treat heart diseases. Hypothesis: Since the organization of sarcomeres regulates the mechanical output of cardiomyocytes, we hypothesized that enhanced sarcomere maturity correlates with the mechanical output of these cells and the physiology of matured cardiomyocytes. Methods: We cultured single hPSC-CMs on 2000 μm2 rectangular protein patterns on polyacrylamide substrates with a modulus of 10 kPa to resemble the morphology of ventricular cardiomyocytes and match healthy adult ventricular stiffness. We infected seeded cells with rAV CAG-LifeAct-Tag RFP to label actin and image sarcomeres in contracting live cells. We estimated forces generated during single-cell contractile events with traction force microscopy and calculated power (force x cell shortening velocity). We varied cell elongation by changing the cell aspect ratio (1:1 to 7:1) and maintaining constant area. We used substrates having stiffness of 6 kPa (embryonic myocardium), 10 kPa (adult) and 35 kPa (infarcted myocardium). Results: Optimized power output was observed in cells with a 7:1 aspect ratio. The size, organization and dynamics of sarcomeres in these elongated hPSC-CMs are more mature. Optimized power output was observed in cells with a 7:1 aspect ratio. The power generated by hPSC-CMs on 35 kPa substrates was significantly reduced, while reduced variations in the power output across different aspect ratios was observed in 6 kPa. Forces generated by patterned hPSC-CMs were related to the organization, dynamics and size of sarcomeres. Electrophysiological parameters closer to that of mature cardiomyocytes were also observed with patch-clamp measurements in these hPSC-CMs with mature sarcomere organization. Conclusions: Stiffness and shape contribute to the structural and electrophysiological maturation of hPSC-CMs.

Behaviors and strategies of bacterial navigation in chemical and nonchemical gradients.

Navigation of cells to the optimal environmental condition is critical for their survival and growth. Escherichia coli cells, for example, can detect various chemicals and move up or down those chemical gradients (i.e., chemotaxis). Using the same signaling machinery, they can also sense other external factors such as pH and temperature and navigate from both sides toward some intermediate levels of those stimuli. This mode of precision sensing is more sophisticated than the (unidirectional) chemotaxis strategy and requires distinctive molecular mechanisms to encode and track the preferred external conditions. To systematically study these different bacterial taxis behaviors, we develop a continuum model that incorporates microscopic signaling events in single cells into macroscopic population dynamics. A simple theoretical result is obtained for the steady state cell distribution in general. In particular, we find the cell distribution is controlled by the intracellular sensory dynamics as well as the dependence of the cells' speed on external factors. The model is verified by available experimental data in various taxis behaviors (including bacterial chemotaxis, pH taxis, and thermotaxis), and it also leads to predictions that can be tested by future experiments. Our analysis help reveal the key conditions/mechanisms for bacterial precision-sensing behaviors and directly connects the cellular taxis performances with the underlying molecular parameters. It provides a unified framework to study bacterial navigation in complex environments with chemical and non-chemical stimuli.

Bidirectional global spontaneous network activity precedes the canonical unidirectional circuit organization in the developing hippocampus

Spontaneous network activity is believed to sculpt developing neural circuits. Spontaneous giant depolarizing potentials (GDPs) were first identified with single-cell recordings from rat CA3 pyramidal neurons, but here we identify and characterize a large-scale spontaneous network activity we term global network activation (GNA) in the developing mouse hippocampal slices, which is measured macroscopically by fast voltage-sensitive dye imaging. The initiation and propagation of GNA in the mouse is largely GABA-independent and dominated by glutamatergic transmission via AMPA receptors. Despite the fact that signal propagation in the adult hippocampus is strongly unidirectional through the canonical trisynaptic circuit (dentate gyrus [DG] to CA3 to CA1), spontaneous GNA in the developing hippocampus originates in distal CA3 and propagates both forward to CA1 and backward to DG. Photostimulation-evoked GNA also shows prominent backward propagation in the developing hippocampus from CA3 to DG. Mouse GNA is strongly correlated to electrophysiological recordings of highly localized single-cell and local field potential events. Photostimulation mapping of neural circuitry demonstrates that the enhancement of local circuit connections to excitatory pyramidal neurons occurs over the same time course as GNA and reveals the underlying pathways accounting for GNA backward propagation from CA3 to DG. The disappearance of GNA coincides with a transition to the adult-like unidirectional circuit organization at about 2 weeks of age. Taken together, our findings strongly suggest a critical link between GNA activity and maturation of functional circuit connections in the developing hippocampus.

Observing Real-Time Molecular Event Dynamics of Apoptosis in Living Cancer Cells using Nuclear-Targeted Plasmonically Enhanced Raman Nanoprobes

Apoptosis is a biological process that plays important roles in embryogenesis, aging, and various diseases. During the process of apoptosis, cells undergo a series of morphological and molecular events such as blebbing, cell shrinkage, proteolysis, and nuclear DNA fragmentation. Investigating these events on a molecular level is crucial for gaining a more complete understanding of the intricate mechanism of apoptosis; however, the simultaneous direct observation of morphological and molecular events in real-time on a single living cell scale still remains a challenge. Herein, we directly monitored morphological and molecular events during cellular apoptosis in real-time after the treatment of an apoptosis-inducing agent, by utilizing our previously described plasmonically enhanced Rayleigh/Raman spectroscopic technique. Spectroscopic analysis of the DNA/protein composition around the cell nucleus revealed the occurrence and dynamics of three apoptotic molecular events: protein denaturation, proteolysis, and DNA fragmentation. The molecular event dynamics were used to create a temporal profile of apoptotic events in single cells. It is found that the sequence of events occurring in the apoptotic process induced by hydrogen peroxide addition is protein denaturation through disulfide bond breakage as well as DNA fragmentation, followed in time by protein unraveling with hydrophobic amino acid exposure, and finally protein degradation. These results demonstrate the potential of using this time-dependent plasmonically enhanced vibrational imaging technique to study the detailed mechanism of other apoptosis molecular pathways induced by different agents (e.g., anticancer drugs). A note is given in the conclusion discussing the expected large difference between the SERS spectrum of biological molecules in solution and that observed in live cells which are enhanced by the plasmonic field of the aggregated nanoparticles.

Single-Cell States in the Estrogen Response of Breast Cancer Cell Lines

Estrogen responsive breast cancer cell lines have been extensively studied to characterize transcriptional patterns in hormone-responsive tumors. Nevertheless, due to current technological limitations, genome-wide studies have typically been limited to population averaged data. Here we obtain, for the first time, a characterization at the single-cell level of the states and expression signatures of a hormone-starved MCF-7 cell system responding to estrogen. To do so, we employ a recently proposed model that allows for dissecting single-cell states from time-course microarray data. We show that within 32 hours following stimulation, MCF-7 cells traverse, most likely, six states, with a faster early response followed by a progressive deceleration. We also derive the genome-wide transcriptional profiles of such single-cell states and their functional characterization. Our results support a scenario where estrogen promotes cell cycle progression by controlling multiple, sequential regulatory steps, whose single-cell events are here identified.

Cells Go Solo

[ Read the Introduction to this Special Section ][1] ![Figure][2] CREDIT: DINA FADDAH AND RUDOLF JAENISCH/THE WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH The scientific literature contains an enormous body of work in which large numbers of cells have been broken open and homogenized to prepare samples for biochemical characterization and, certainly, much has been learned from such studies. But more recently, it has become possible to monitor events in single cells, thus allowing investigators to test whether existing “averaged” readings of the state of many cells from traditional large-scale assays accurately represent the behavior of the individual cells being studied. Such single-cell measurements are providing a wealth of information—sometimes unanticipated and often previously obscured—about how cells respond to perturbations or signals. In this special issue, three Reviews provide examples of fundamental insights into cellular regulation that are revealed when it is possible to measure enzymatic activity, transcriptional responses, or the metabolic state in individual cells. An obvious advantage of single-cell measurements is the ability to measure variations or “noise” in the responses of the individual cells to similar or identical conditions. In many instances, it is possible to monitor the time course of cellular responses. Gene transcription can be particularly noisy, with bursts of RNA synthesis occurring in some cells but not others of the same population. Thus, fundamental questions arise about the nature of these systems. Perhaps variation in response is advantageous in conserving resources or in assuring that some cells survive in a changing environment. Or it may be that biophysical constraints of small numbers of molecules and the characteristics of the enzymes at work dictate such variability as unavoidable. Sanchez and Golding review recent work in model systems, from bacteria to animal cells, that attempts to resolve whether the kinetics of transcription are encoded in the architecture of promoter sequences in DNA—and might therefore vary throughout the genome—or are determined by physical or biophysical properties that would impose more global constraints throughout the cell. Levine et al. explore another previously hidden phenomenon. Continuous measurements of protein activation show that many undergo asynchronous pulsatile responses, which are obscured in average measurements from a population of cells. They discuss how cellular circuits are wired to produce such responses and what the advantages of such control systems might be. Zenobi highlights methodological advances, particularly in mass spectrometry, that are enabling quantitation of the abundance of molecular components of single cells. Challenges abound for the goal of making simultaneous measurements to characterize the rapid ly changing metabolic state of individual cells. But the promise of new insights across a broad range of disciplines is sustaining a steady effort to tap into the large store of new knowledge lying hidden within the confines of single cells. [1]: http://www.sciencemag.org/content/342/6163/1187 [2]: pending:yes

Single-cell analysis of K562 cells: An imatinib-resistant subpopulation is adherent and has upregulated expression of BCR-ABL mRNA and protein

In chronic myeloid leukemia (CML) cells from different stages of maturation may have differential expression of BCR-ABL at both messenger RNA (mRNA) and protein level. However, the significance of such differential expression to clinical disease behavior is unknown. Using the CML-derived, BCR-ABL expressing cell line, K562, distinct plastic-adherent (K562/Adh) and nonadherent (K562/NonAdh) subpopulations were established and then analyzed both as single cells and as bulk cell populations. BCR-ABL mRNA was upregulated in K562/Adh compared with K562/NonAdh cells in both single cell and bulk population analyses (p < 0.0001). Similarly, phosphorylation of BCR protein was upregulated in K562/Adh, compared with K562/NonAdh cells (63.42% vs. 23.1%; p = 0.007), and these two K562 subpopulations were found to express significantly different microRNA species. Furthermore, treatment with the BCR-ABL tyrosine kinase inhibitor, imatinib, reduced cell viability more rapidly in K562/NonAdh compared with K562/Adh cells (p < 0.005) both at single and bulk cell levels. This discovery of an adherent subpopulation of K562 cells with increased BCR-ABL mRNA, increased phosphorylated BCR protein expression, differential microRNA expression, and increased imatinib resistance suggests that a similar subpopulation of cells can also mediate clinical resistance to imatinib during treatment of patients with CML.

67 : Assessment of β2-adrenergic receptors on leukocyte subpopulations using imaging cytometry

Introduction: β2-Adrenergic receptors (AR) are the primary subtype of adrenergic receptors found on blood leukocytes, and are recognized as key mediators of interaction between the sympathetic nervous system and immune system. This biological pathway is central in understanding neuroimmune signaling in a number of disease states, spanning mental disorders to traumatic brain injury. Traditionally, β2-ARs have been primarily assessed using radio-ligand binding assays, where results are limited in their ability to provide in depth cell-signaling information on specific leukocyte subtypes. As a result, current knowledge of β2-AR distribution, reactivity and potential implications in disease pathology or drug therapy are limited. This study evaluated β2-AR expression on circulating leukocyte subsets with various commercially available antibodies (Abs) using imaging cytometry. Methods: Whole blood was labeled with CD14 and CD16 surface markers for leukocyte subtype phenotyping. Four different β2-AR antibodies, sc-81578 – FITC (Santa Cruz), sc-81577 – PE (Santa Cruz), PA5-19649 (Pierce Biotechnology, USA) and MCA2784 (Serotec, USA) were assessed. DAPI stain was added to all samples to determine nuclear morphology. Based on cell area and aspect ratio, 100,000 single-cell events were acquired on an ImageStreamX Mark II® Cytometer (Amnis, Seattle, WA). Monocytes and lymphocytes were identified using side scatter (SSC) and CD14 expression. Neutrophils and eosinophils were identified using SSC and CD16. Results: β2−AR expression levels on leukocyte subpopulations in whole blood were detectable using image cytometery. Leukocyte subsets identified according to surface markers, size, granularity, cell and nuclear morphology displayed varying amounts of β2-AR expression. Furthermore, the PA5-19649 Ab performed best in terms of binding specificity and signal strength. Conclusion: Imaging cytometry is a valuable technique for evaluating β2-ARs on leukocyte subsets. The capability of this technology to provide in-depth, high throughput cell signaling information offers an attractive alternative to current methods used in the assessment of adrenergic receptors.

High-Throughput Microfluidic Single-Cell Digital Polymerase Chain Reaction

Here we present an integrated microfluidic device for the high-throughput digital polymerase chain reaction (dPCR) analysis of single cells. This device allows for the parallel processing of single cells and executes all steps of analysis, including cell capture, washing, lysis, reverse transcription, and dPCR analysis. The cDNA from each single cell is distributed into a dedicated dPCR array consisting of 1020 chambers, each having a volume of 25 pL, using surface-tension-based sample partitioning. The high density of this dPCR format (118,900 chambers/cm(2)) allows the analysis of 200 single cells per run, for a total of 204,000 PCR reactions using a device footprint of 10 cm(2). Experiments using RNA dilutions show this device achieves shot-noise-limited performance in quantifying single molecules, with a dynamic range of 10(4). We performed over 1200 single-cell measurements, demonstrating the use of this platform in the absolute quantification of both high- and low-abundance mRNA transcripts, as well as micro-RNAs that are not easily measured using alternative hybridization methods. We further apply the specificity and sensitivity of single-cell dPCR to performing measurements of RNA editing events in single cells. High-throughput dPCR provides a new tool in the arsenal of single-cell analysis methods, with a unique combination of speed, precision, sensitivity, and specificity. We anticipate this approach will enable new studies where high-performance single-cell measurements are essential, including the analysis of transcriptional noise, allelic imbalance, and RNA processing.

Prevalent coordination of mitochondrial DNA transcription and initiation of replication with the cell cycle

Nuclear (nDNA) and mitochondrial DNA (mtDNA) communication is essential for cell function, but it remains unclear whether the replication of these genomes is linked. We inspected human cells with a novel fluorescence in situ hybridization protocol (mitochondrial Transcription and Replication Imaging Protocol) that identifies mitochondrial structures engaged in initiation of mtDNA replication and unique transcript profiles, and reconstruct the temporal series of mitochondrial and nuclear events in single cells during the cell cycle. We show that mtDNA transcription and initiation of replication are prevalently coordinated with the cell cycle, preceding nuclear DNA synthesis, and being reactivated towards the end of S-phase. This coordination is achieved by modulating the fraction of mitochondrial structures that intiate mtDNA synthesis and/or contain transcript at a given time. Thus, although replication of the mitochondrial genome is active through the entire cell cycle, but in a limited fraction of mitochondrial structures, peaks of these activities are synchronized with nDNA synthesis. After release from blockage of mtDNA replication with either nocodazole or double thymidine treatment, prevalent mtDNA and nDNA synthesis occurred simultaneously, indicating that mitochondrial coordination with the nuclear phase can be adjusted in response to physiological alterations. These findings will help redefine other nuclear–mitochondrial links in cell function.

Sample introduction of single selenized yeast cells (Saccharomyces cerevisiae) by micro droplet generation into an ICP-sector field mass spectrometer for label-free detection of trace elements

We have applied a micro droplet generator (μDG) for sample introduction of single selenized yeast cells into a sector field ICP-MS, which was operated in a fast scanning mode with sampling rates of up to 10 kHz, to measure single cells time resolved with 100 μs integration time. Selenized yeast cells have been used as a model system for preliminary investigation. The single cells to be measured have been embedded into droplets and it will be shown that the time duration of a single cell event always is about 400 to 500 μs, and thus comparable to the time duration of a droplet without a cell. A fixed droplet generation rate of 50 Hz produced equidistant signals in time of each droplet event and was advantageous to separate contribution from background and blank from the analytical signal. Open vessel digestion and a multielement analysis were performed with washed yeast cells and absolute amounts per single cell were determined for Na (0.91 fg), Mg (9.4 fg), Fe (5.9 fg), Cu (0.54 fg), Zn (1.2 fg) and Se (72 fg). Signal intensities from single cells have been measured for the elements Cu, Zn and Se, and histograms were calculated for about 1000 cell events. The mean elemental sensitivities measured here range from 0.7 counts per ag (Se) to 10 counts per ag (Zn) with RSD's from 49% (Zn) to 69% (Se) for about 1000 cell events.

GPU-based real-time detection and analysis of biological targets using solid-state nanopores

The emergence of nanoscale devices has provided robust interfaces to biomolecules that faithfully transduce and define fundamental interactions of living systems. Measuring single-event behavior of important targets like DNA, and diseased cells has been achieved with a number of devices and systems. An important dimension to these systems, often discounted, is real-time computational decision-making from measured data. This paper describes an adaptive approach that can record single-molecule or single-cell events in real-time and automatically analyze patterns from the measured data. The automated analysis of measured data is done using a static threshold technique and two variations of a dynamic threshold technique: baseline-tracker and moving average filtering. Dynamic techniques for threshold detection enable noise suppression in the measured data and precise detection of patterns, but at the cost of more complex software as compared to static technique. To mitigate the computational overhead, a real-time system is implemented that uses advanced I/O techniques to minimize the execution stalls, thus enabling the system to process data significantly faster than the electrical measurement setup. Furthermore, the algorithms are implemented on programmable graphics processing units for parallel pattern detection. Our implementation provides five times faster data acquisition and pattern detection than the maximum sampling rate of the electrical measurement setup.

Automation Highlights from the Literature

Automation Highlights from the Literature

Intracellular protein trafficking kinetics in chronic myeloid leukemia stem cells using a microfluidic platform

The oncogenic fusion protein BCR-ABL is produced by chronic myeloid leukemia (CML) cells and functions as an abnormal, constitutively active tyrosine kinase that interferes with normal migratory and apoptotic behaviour of cells. Small molecule tyrosine kinase inhibitors (TKIs), such as dasatinib, eliminate CML progenitor cells, but fail to target the stem cell fraction resulting in persistent disease. In order to achieve a cure for CML in the majority of patients, we need an improved understanding of intracellular signalling dynamics, including the shuttling of BCR-ABL between cytosolic and nuclear compartments. In the past, the instability of BCR-ABL in assays using conventional immunohistochemical techniques has made this difficult and has not allowed for reliable analysis at the single cell level. Here we show how the utilization of rapid on-chip cell fixation within a microfluidic platform provides a means to immunofluorescently analyze the spatiotemporal localization of both BCR-ABL and c-ABL, as well as the linked apoptosis mediator, BCL-XL, in arrays of single CD34+ CML stem/progenitor cells, without cell loss. We demonstrate this proceeds up to 4 times faster than benchtop methods. Our results indicate that whilst both BCR-ABL and c-ABL shuttle from the cytoplasm to the nucleus following dasatinib treatment, the temporal dynamics are not synchronized. The microfluidic platform has the potential to provide insights into the intracellular signalling events in single cells. The ability to examine signalling events and assess BCR-ABL expression/activity in isolated cells in "real-time" may help elucidate the characteristics of rare CML stem cell events, which lead to the resistance of CML stem cells to TKIs.

Impedance measurement technique for high-sensitivity cell detection in microstructures with non-uniform conductivity distribution

Particle detection in microstructures is a key procedure required by modern lab-on-a-chip devices. Unfortunately, state of the art approaches to impedance measuring as applied to cell detection do not perform well in regions characterized by non-homogeneous physical parameters due, for example, to the presence of air-liquid interfaces or when the particle-electrode distance is relatively high. This paper presents a robust impedance measurement technique and a circuit for detecting cells flowing in microstructures such as microchannels and microwells. Our solution makes use of an innovative three-electrode measurement scheme with asymmetric polarization in order to increase cell detection ability in microstructures featuring large electrode distances of up to 100 μm as well as to limit signal loss due to cell position relative to the electrodes. Compared to standard techniques, numerical simulations show that, with the proposed approach, the cell detection sensitivity is increased by more than 40%. In addition, we propose a custom circuit based on division instead of difference between signals, as in standard differential circuits, so as to reduce the baseline signal drift induced by non-homogeneous conductivity. A simplified analytical model shows an increase in the signal-to-noise-ratio comprised in the range 3.9-5.9. Experimental results, carried out using an open-microwell device made with flexible printed circuit board technology, are in agreement with simulations, suggesting a six-fold increase of the signal-to-noise ratio compared to the differential measurement technique. We were thus able to successfully monitor the process of isolating K562 leukemia cells inside open-microwells determining all single-cell events with no false positive detection.

Zebrafish Neural Tube Morphogenesis Requires Scribble-Dependent Oriented Cell Divisions

How control of subcellular events in single cells determines morphogenesis on the scale of the tissue is largely unresolved. The stereotyped cross-midline mitoses of progenitors in the zebrafish neural keel provide a unique experimental paradigm for defining the role and control of single-cell orientation for tissue-level morphogenesis in vivo. We show here that the coordinated orientation of individual progenitor cell division in the neural keel is the cellular determinant required for morphogenesis into a neural tube epithelium with a single straight lumen. We find that Scribble is required for oriented cell division and that its function in this process is independent of canonical apicobasal and planar polarity pathways. We identify a role for Scribble in controlling clustering of α-catenin foci in dividing progenitors. Loss of either Scrib or N-cadherin results in abnormally oriented mitoses, reduced cross-midline cell divisions, and similar neural tube defects. We propose that Scribble-dependent nascent cell-cell adhesion clusters between neuroepithelial progenitors contribute to define orientation of their cell division. Finally, our data demonstrate that while oriented mitoses of individual cells determine neural tube architecture, the tissue can in turn feed back on its constituent cells to define their polarization and cell division orientation to ensure robust tissue morphogenesis.

PARP1 suppresses homologous recombination events in mice in vivo

Recent studies suggest that PARP1 inhibitors, several of which are currently in clinical trial, may selectively kill BRCA1/2 mutant cancers cells. It is thought that the success of this therapy is based on immitigable lethal DNA damage in the cancer cells resultant from the concurrent loss or inhibition of two DNA damage repair pathways: single-strand break (SSB) repair and homologous recombination repair (HRR). Presumably, inhibition of PARP1 activity obstructs the repair of SSBs and during DNA replication, these lesions cause replication fork collapse and are transformed into substrates for HRR. In fact, several previous studies have indicated a hyper-recombinogenic phenotype in the absence of active PARP1 in vitro or in response to DNA damaging agents. In this study, we demonstrate an increased frequency of spontaneous HRR in vivo in the absence of PARP1 using the pun assay. Furthermore, we found that the HRR events that occur in Parp1 nullizygous mice are associated with a significant increase in large, clonal events, as opposed to the usually more frequent single cell events, suggesting an effect in replicating cells. In conclusion, our data demonstrates that PARP1 inhibits spontaneous HRR events, and supports the model of DNA replication transformation of SSBs into HRR substrates.