Quantitative videomicroscopy reveals latent control of cell-pair rotations in vivo.

Collective cell rotations are widely used during animal organogenesis. Theoretical and in vitro studies have conceptualized rotating cells as identical rigid-point objects that stochastically break symmetry to move monotonously and perpetually within an inert environment. However, it is unclear whether this notion can be extrapolated to a natural context, where rotations are ephemeral and heterogeneous cellular cohorts interact with an active epithelium. In zebrafish neuromasts, nascent sibling hair cells invert positions by rotating ≤180° around their geometric center after acquiring different identities via Notch1a-mediated asymmetric repression of Emx2. Here, we show that this multicellular rotation is a three-phasic movement that progresses via coherent homotypic coupling and heterotypic junction remodeling. We found no correlation between rotations and epithelium-wide cellular flow or anisotropic resistive forces. Moreover, the Notch/Emx2 status of the cell dyad does not determine asymmetric interactions with the surrounding epithelium. Aided by computer modeling, we suggest that initial stochastic inhomogeneities generate a metastable state that poises cells to move and spontaneous intercellular coordination of the resulting instabilities enables persistently directional rotations, whereas Notch1a-determined symmetry breaking buffers rotational noise.

Lineage tracing of Shh+ floor plate cells and dynamics of dorsal-ventral gene expression in the regenerating axolotl spinal cord.

Both development and regeneration depend on signaling centers, which are sources of locally secreted tissue-patterning molecules. As many signaling centers are decommissioned before the end of embryogenesis, a fundamental question is how signaling centers can be re-induced later in life to promote regeneration after injury. Here, we use the axolotl salamander model (Ambystoma mexicanum) to address how the floor plate is assembled for spinal cord regeneration. The floor plate is an archetypal vertebrate signaling center that secretes Shh ligand and patterns neural progenitor cells during embryogenesis. Unlike mammals, axolotls continue to express floor plate genes (including Shh) and downstream dorsal-ventral patterning genes in their spinal cord throughout life, including at steady state. The parsimonious hypothesis that Shh+ cells give rise to functional floor plate cells for regeneration had not been tested. Using HCR in situ hybridization and mathematical modeling, we first quantified the behaviors of dorsal-ventral spinal cord domains, identifying significant increases in gene expression level and floor plate size during regeneration. Next, we established a transgenic axolotl to specifically label and fate map Shh+ cells in vivo. We found that labeled Shh+ cells gave rise to regeneration floor plate, and not to other neural progenitor domains, after tail amputation. Thus, despite changes in domain size and downstream patterning gene expression, Shh+ cells retain their floor plate identity during regeneration, acting as a stable cellular source for this regeneration signaling center in the axolotl spinal cord.

Postembryonic development and aging of the appendicular skeleton in Ambystoma mexicanum.

BACKGROUND: The axolotl is a key model to study appendicular regeneration. The limb complexity resembles that of humans in structure and tissue components; however, axolotl limbs develop postembryonically. In this work, we evaluated the postembryonic development of the appendicular skeleton and its changes with aging. RESULTS: The juvenile limb skeleton is formed mostly by Sox9/Col1a2 cartilage cells. Ossification of the appendicular skeleton starts when animals reach a length of 10 cm, and cartilage cells are replaced by a primary ossification center, consisting of cortical bone and an adipocyte-filled marrow cavity. Vascularization is associated with the ossification center and the marrow cavity formation. We identified the contribution of Col1a2-descendants to bone and adipocytes. Moreover, ossification progresses with age toward the epiphyses of long bones. Axolotls are neotenic salamanders, and still ossification remains responsive to l-thyroxine, increasing the rate of bone formation. CONCLUSIONS: In axolotls, bone maturation is a continuous process that extends throughout their life. Ossification of the appendicular bones is slow and continues until the complete element is ossified. The cellular components of the appendicular skeleton change accordingly during ossification, creating a heterogenous landscape in each element. The continuous maturation of the bone is accompanied by a continuous body growth.

A dynamic cell recruitment process drives growth of the Drosophila wing by overscaling the vestigial expression pattern.

Organs mainly attain their size by cell growth and proliferation, but sometimes also grow through recruitment of undifferentiated cells. Here we investigate the participation of cell recruitment in establishing the pattern of Vestigial (Vg), the product of the wing selector gene in Drosophila. We find that the Vg pattern overscales along the dorsal-ventral (DV) axis of the wing imaginal disc, i.e., it expands faster than the DV length of the pouch. The overscaling of the Vg pattern cannot be explained by differential proliferation, apoptosis, or oriented-cell divisions, but can be recapitulated by a mathematical model that explicitly considers cell recruitment. When impairing cell recruitment genetically, we find that the Vg pattern almost perfectly scales and adult wings are approximately 20% smaller. Conversely, impairing cell proliferation results in very small wings, suggesting that cell recruitment and cell proliferation additively contribute to organ growth in this system. Furthermore, using fluorescent reporter tools, we provide direct evidence that cell recruitment is initiated between early and mid third-instar larval development. Altogether, our work quantitatively shows when, how, and by how much cell recruitment shapes the Vg pattern and drives growth of the Drosophila wing.

Familial chilblain lupus due to a gain-of-function mutation in STING.

OBJECTIVES: Familial chilblain lupus is a monogenic form of cutaneous lupus erythematosus caused by loss-of-function mutations in the nucleases TREX1 or SAMHD1. In a family without TREX1 or SAMHD1 mutation, we sought to determine the causative gene and the underlying disease pathology. METHODS: Exome sequencing was used for disease gene identification. Structural analysis was performed by homology modelling and docking simulations. Type I interferon (IFN) activation was assessed in cells transfected with STING cDNA using an IFN-ß reporter and Western blotting. IFN signatures in patient blood in response to tofacitinib treatment were measured by RT-PCR of IFN-stimulated genes. RESULTS: In a multigenerational family with five members affected with chilblain lupus, we identified a heterozygous mutation of STING, a signalling molecule in the cytosolic DNA sensing pathway. Structural and functional analyses indicate that mutant STING enhances homodimerisation in the absence of its ligand cGAMP resulting in constitutive type I IFN activation. Treatment of two affected family members with the Janus kinase (JAK) inhibitor tofacitinib led to a marked suppression of the IFN signature. CONCLUSIONS: A heterozygous gain-of-function mutation in STING can cause familial chilblain lupus. These findings expand the genetic spectrum of type I IFN-dependent disorders and suggest that JAK inhibition may be of therapeutic value.

SpoIIIE mechanism of directional translocation involves target search coupled to sequence-dependent motor stimulation.

SpoIIIE/FtsK are membrane-anchored, ATP-fuelled, directional motors responsible for chromosomal segregation in bacteria. Directionality in these motors is governed by interactions between specialized sequence-recognition modules (SpoIIIE-γ/FtsK-γ) and highly skewed chromosomal sequences (SRS/KOPS). Using a new combination of ensemble and single-molecule methods, we dissect the series of steps required for SRS localization and motor activation. First, we demonstrate that SpoIIIE/DNA association kinetics are sequence independent, with binding specificity being uniquely determined by dissociation. Next, we show by single-molecule and modelling methods that hexameric SpoIIIE binds DNA non-specifically and finds SRS by an ATP-independent target search mechanism, with ensuing oligomerization and binding of SpoIIIE-γ to SRS triggering motor stimulation. Finally, we propose a new model that provides an entirely new interpretation of previous observations for the origin of SRS/KOPS-directed translocation by SpoIIIE/FtsK.

How a reaction-diffusion signal can control spinal cord regeneration in axolotls: A modeling study.

Axolotls are uniquely able to completely regenerate the spinal cord after amputation. The underlying governing mechanisms of this regenerative response have not yet been fully elucidated. We previously found that spinal cord regeneration is mainly driven by cell-cycle acceleration of ependymal cells, recruited by a hypothetical signal propagating from the injury. However, the nature of the signal and its propagation remain unknown. In this theoretical study, we investigated whether the regeneration-inducing signal can follow a reaction-diffusion process. We developed a computational model, validated it with experimental data, and showed that the signal dynamics can be understood in terms of reaction-diffusion mechanism. By developing a theory of the regenerating outgrowth in the limit of fast reaction-diffusion, we demonstrate that control of regenerative response solely relies on cell-to-signal sensitivity and the signal reaction-diffusion characteristic length. This study lays foundations for further identification of the signal controlling regeneration of the spinal cord.

Naturally occurring genetic variants of human caspase-1 differ considerably in structure and the ability to activate interleukin-1ß.

Caspase-1 (Interleukin-1 Converting Enzyme, ICE) is a proinflammatory enzyme that plays pivotal roles in innate immunity and many inflammatory conditions such as periodic fever syndromes and gout. Inflammation is often mediated by enzymatic activation of interleukin (IL)-1ß and IL-18. We detected seven naturally occurring human CASP1 variants with different effects on protein structure, expression, and enzymatic activity. Most mutations destabilized the caspase-1 dimer interface as revealed by crystal structure analysis and homology modeling followed by molecular dynamics simulations. All variants demonstrated decreased or absent enzymatic and IL-1ß releasing activity in vitro, in a cell transfection model, and as low as 25% of normal ex vivo in a whole blood assay of samples taken from subjects with variant CASP1, a subset of whom suffered from unclassified autoinflammation. We conclude that decreased enzymatic activity of caspase-1 is compatible with normal life and does not prevent moderate and severe autoinflammation.

Molecular dynamics of water in the neighborhood of aquaporins.

Present knowledge obtained by molecular dynamics (MD) simulation studies regarding the dynamics of water, both in the vicinity of biological membranes and within the proteinaceous water channels, also known as aquaporins (AQPs), is reviewed. A brief general summary of the water models most extensively employed in MD simulations (SPC, SPC/E, TIP3P, TIP4P), indicating their most relevant pros and cons, is likewise provided. Structural considerations of water are also discussed, based on different order parameters, which can be extracted from MD simulations as well as from experiments. Secondly, the behaviour of water in the neighbourhood of membranes by means of molecular dynamics simulations is addressed. Consequently, the comparison with previous experimental evidence is pointed out. In living cells, water is transported across the plasma membrane through the lipid bilayer and the aforementioned AQPs, which motivates this review to focus mostly on MD simulation studies of water within AQPs. Relevant contributions explaining peculiar properties of these channels are discussed, such as selectivity and gating. Water models used in these studies are also summarised. Finally, based on the information presented here, further MD studies are encouraged.

Mechanisms of interaction between Candida albicans and Streptococcus mutans: an experimental and mathematical modelling study.

OBJECTIVE: To evaluate the mechanisms of microbial interaction between the oral pathogens Candida albicans and Streptococcus mutans. MATERIALS AND METHODS: Growth kinetics for the two micro-organisms, cultured individually or together, were followed experimentally for 36 h. The different growth curves were analysed by means of mathematical modelling. RESULTS: Under the experimental conditions, S. mutans final concentration, when grown individually, was 5-times that of C. albicans. Contrarily, when both micro-organisms grew together, this ratio was inversed and C. albicans final concentration was even higher than that of S. mutans. When both micro-organisms share the niche, a model including linear competition among one another was best suited to reproduce the experimental observations. The results of this model show that the initial growth rates of both species are positively influenced by their mutual interaction. However, at longer incubation times, C. albicans prevents bacterial growth and achieves concentrations 4-times higher than when grown individually. CONCLUSIONS: The results suggest that C. albicans biofilm formation could be potentiated by the presence of S. mutans by two mechanisms: synergically at short times and by competition at longer periods.

Fluctuations in tissue growth portray homeostasis as a critical state and long-time non-Markovian cell proliferation as Markovian.

Tissue growth is an emerging phenomenon that results from the cell-level interplay between proliferation and apoptosis, which is crucial during embryonic development, tissue regeneration, as well as in pathological conditions such as cancer. In this theoretical article, we address the problem of stochasticity in tissue growth by first considering a minimal Markovian model of tissue size, quantified as the number of cells in a simulated tissue, subjected to both proliferation and apoptosis. We find two dynamic phases, growth and decay, separated by a critical state representing a homeostatic tissue. Since the main limitation of the Markovian model is its neglect of the cell cycle, we incorporated a refractory period that temporarily prevents proliferation immediately following cell division, as a minimal proxy for the cell cycle, and studied the model in the growth phase. Importantly, we obtained from this last model an effective Markovian rate, which accurately describes general trends of tissue size. This study shows that the dynamics of tissue growth can be theoretically conceptualized as a Markovian process where homeostasis is a critical state flanked by decay and growth phases. Notably, in the growing non-Markovian model, a Markovian-like growth process emerges at large time scales.

Aggregation of non-polar solutes in water at different pressures and temperatures: the role of hydrophobic interaction.

Due to the importance of the hydrophobic interaction in protein folding, we decided to study the effect of pressure and temperature on the phase transitions of non-polar solutes in water, and thereby their solubility, using molecular dynamics simulations. The main results are: (1) within a certain range, temperature induces the aggregation of Lennard-Jones particles in water; and (2) pressure induces disaggregation of the formed clusters. From the simulated data, a non-monotonic coexistence curve for the binary system was obtained, from which a critical point of T(c) = 383 ± 9 K and p(c) = 937 ± 11 bar was determined. The results are in accordance with previous experimental evidence involving transitions of hydrocarbons in water mixtures, and protein unfolding.

Size matters: tissue size as a marker for a transition between reaction-diffusion regimes in spatio-temporal distribution of morphogens.

The reaction-diffusion model constitutes one of the most influential mathematical models to study distribution of morphogens in tissues. Despite its widespread use, the effect of finite tissue size on model-predicted spatio-temporal morphogen distributions has not been completely elucidated. In this study, we analytically investigated the spatio-temporal distributions of morphogens predicted by a reaction-diffusion model in a finite one-dimensional domain, as a proxy for a biological tissue, and compared it with the solution of the infinite-domain model. We explored the reduced parameter, the tissue length in units of a characteristic reaction-diffusion length, and identified two reaction-diffusion regimes separated by a crossover tissue size estimated in approximately three characteristic reaction-diffusion lengths. While above this crossover the infinite-domain model constitutes a good approximation, it breaks below this crossover, whereas the finite-domain model faithfully describes the entire parameter space. We evaluated whether the infinite-domain model renders accurate estimations of diffusion coefficients when fitted to finite spatial profiles, a procedure typically followed in fluorescence recovery after photobleaching (FRAP) experiments. We found that the infinite-domain model overestimates diffusion coefficients when the domain is smaller than the crossover tissue size. Thus, the crossover tissue size may be instrumental in selecting the suitable reaction-diffusion model to study tissue morphogenesis.

Photosensitivity and cGAS-Dependent IFN-1 Activation in Patients with Lupus and TREX1 Deficiency.

The exonuclease TREX1 safeguards the cells against DNA accumulation in the cytosol and thereby prevents innate immune activation and autoimmunity. TREX1 mutations lead to chronic DNA damage and cell-intrinsic IFN-1 response. Associated disease phenotypes include Aicardi‒Goutières syndrome, familial chilblain lupus, and systemic lupus erythematosus. Given the role of UV light in lupus pathogenesis, we assessed sensitivity to UV light in patients with lupus and TREX1 mutation by phototesting, which revealed enhanced photosensitivity. TREX1-deficient fibroblasts and keratinocytes generated increased levels of ROS in response to UV irradiation as well as increased levels of 8-oxo-guanine lesions after oxidative stress. Likewise, the primary UV-induced DNA lesions cyclobutane pyrimidine dimers were induced more strongly in TREX1-deficient cells. Further analysis revealed that single-stranded DNA regions, frequently formed during DNA replication and repair, promote cyclobutane pyrimidine dimer formation. Together, this resulted in a strong UV-induced DNA damage response that was associated with a cGAS-dependent IFN-1 activation. In conclusion, these findings link chronic DNA damage to photosensitivity and IFN-1 production in TREX1 deficiency and explain the induction of disease flares on UV exposure in patients with lupus and TREX1 mutation.

Vectorcardiography-derived index allows a robust quantification of ventricular electrical synchrony.

Alteration of muscle activation sequence is a key mechanism in heart failure with reduced ejection fraction. Successful cardiac resynchronization therapy (CRT), which has become standard therapy in these patients, is limited by the lack of precise dyssynchrony quantification. We implemented a computational pipeline that allows assessment of ventricular dyssynchrony by vectorcardiogram reconstruction from the patient's electrocardiogram. We defined a ventricular dyssynchrony index as the distance between the voltage and speed time integrals of an individual observation and the linear fit of these variables obtained from a healthy population. The pipeline was tested in a 1914-patient population. The dyssynchrony index showed minimum values in heathy controls and maximum values in patients with left bundle branch block (LBBB) or with a pacemaker (PM). We established a critical dyssynchrony index value that discriminates electrical dyssynchronous patterns (LBBB and PM) from ventricular synchrony. In 10 patients with PM or CRT devices, dyssynchrony indexes above the critical value were associated with high time to peak strain standard deviation, an echocardiographic measure of mechanical dyssynchrony. Our index proves to be a promising tool to evaluate ventricular activation dyssynchrony, potentially enhancing the selection of candidates for CRT, device configuration during implantation, and post-implant optimization.

Tig1 regulates proximo-distal identity during salamander limb regeneration.

Salamander limb regeneration is an accurate process which gives rise exclusively to the missing structures, irrespective of the amputation level. This suggests that cells in the stump have an awareness of their spatial location, a property termed positional identity. Little is known about how positional identity is encoded, in salamanders or other biological systems. Through single-cell RNAseq analysis, we identified Tig1/Rarres1 as a potential determinant of proximal identity. Tig1 encodes a conserved cell surface molecule, is regulated by retinoic acid and exhibits a graded expression along the proximo-distal axis of the limb. Its overexpression leads to regeneration defects in the distal elements and elicits proximal displacement of blastema cells, while its neutralisation blocks proximo-distal cell surface interactions. Critically, Tig1 reprogrammes distal cells to a proximal identity, upregulating Prod1 and inhibiting Hoxa13 and distal transcriptional networks. Thus, Tig1 is a central cell surface determinant of proximal identity in the salamander limb.

Spatiotemporal control of cell cycle acceleration during axolotl spinal cord regeneration.

Axolotls are uniquely able to resolve spinal cord injuries, but little is known about the mechanisms underlying spinal cord regeneration. We previously found that tail amputation leads to reactivation of a developmental-like program in spinal cord ependymal cells (Rodrigo Albors et al., 2015), characterized by a high-proliferation zone emerging 4 days post-amputation (Rost et al., 2016). What underlies this spatiotemporal pattern of cell proliferation, however, remained unknown. Here, we use modeling, tightly linked to experimental data, to demonstrate that this regenerative response is consistent with a signal that recruits ependymal cells during ~85 hours after amputation within ~830 µm of the injury. We adapted Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) technology to axolotls (AxFUCCI) to visualize cell cycles in vivo. AxFUCCI axolotls confirmed the predicted appearance time and size of the injury-induced recruitment zone and revealed cell cycle synchrony between ependymal cells. Our modeling and imaging move us closer to understanding bona fide spinal cord regeneration.

Negative feedback of extracellular ADP on ATP release in goldfish hepatocytes: a theoretical study.

A mathematical model was built to account for the kinetic of extracellular ATP (ATPe) and extracellular ADP (ADPe) concentrations from goldfish hepatocytes exposed to hypotonicity. The model was based on previous experimental results on the time course of ATPe accumulation, ectoATPase activity, and cell viability [Pafundo et al., 2008]. The kinetic of ATPe is controlled by a lytic ATP flux, a non-lytic ATP flux, and ecto-ATPase activity, whereas ADPe kinetic is governed by a lytic ADP flux and both ecto-ATPase and ecto-ADPase activities. Non-lytic ATPe efflux was included as a diffusion equation modulated by ATPe activation (positive feedback) and ADPe inhibition (negative feedback). The model yielded physically meaningful and stable steady-state solutions, was able to fit the experimental time evolution of ATPe and simulated the concomitant kinetic of ADPe. According to the model during the first minute of hypotonicity the concentration of ATPe is mainly governed by both lytic and non-lytic ATP efflux, with almost no contribution from ecto-ATPase activity. Later on, ecto-ATPase activity becomes important in defining the time dependent decay of ATPe levels. ADPe inhibition of the non-lytic ATP efflux was strong, whereas ATPe activation was minimal. Finally, the model was able to predict the consequences of partial inhibition of ecto-ATPase activity on the ATPe kinetic, thus emulating the exposure of goldfish cells to hypotonic medium in the presence of the ATP analog AMP-PCP. The model predicts this analog to both inhibit ectoATPase activity and increase non-lytic ATP release.

Analysis of the source of heterogeneity in the osmotic response of plant membrane vesicles.

Plasma membrane vesicles have been widely employed to understand the biophysics of water movements, especially when active aquaporins are present. In general, water permeability coefficients in these preparations outcome from the analysis of the osmotic response of the vesicles by means of light scattering. As from now, this is possible by following a theoretical approach that assumes that scattered light follows a single exponential function and that this behavior is the consequence of vesicle volume changes due to an osmotic challenge. However, some experimental data do not necessarily fit to single exponentials but to double ones. It is argued that the observed double exponential behavior has two possible causes: different vesicle population in terms of permeability or in terms of size distribution. As classical models cannot identify this source of heterogeneity, a mathematical modeling approach was developed based on phenomenological equations of water transport. In the three comparative models presented here, it was assumed that water moves according to an osmotic mechanism across the vesicles, and there is no solute movement across them. Interestingly, when tested in a well described plasma membrane vesicle preparation, the application of these models indicates that the source of heterogeneity in the osmotic response is vesicles having different permeability, clearly discarding the variable size effect. In conclusion, the mathematical approach presented here allows to identify the source of heterogeneity; this information being of particular interest, especially when studying gating mechanisms triggered in water channel activity.

Kinetics of extracellular ATP from goldfish hepatocytes: a lesson from mathematical modeling.

In goldfish hepatocytes, hypotonic exposure leads to cell swelling, followed by a compensatory shrinkage termed RVD. It has been previously shown that ATP is accumulated in the extracellular medium of swollen cells in a non-linear fashion, and that extracellular ATP (ATPe) is an essential intermediate to trigger RVD. Thus, to understand how RVD proceeds in goldfish hepatocytes, we developed two mathematical models accounting for the experimental ATPe kinetics reported recently by Pafundo et al. in Am. J. Physiol. 294, R220-R233, 2008. Four different equations for ATPe fluxes were built to account for the release of ATP by lytic (J(L)) and nonlytic mechanisms (J(NL)), ATPe diffusion (J(D)), and ATPe consumption by ectonucleotidases (J(V)). Particular focus was given to J(NL), defined as the product of a time function (J(R)) and a positive feedback mechanism whereby ATPe amplifies J(NL). Several J (R) functions (Constant, Step, Impulse, Gaussian, and Lognormal) were studied. Models were tested without (model 1) or with (model 2) diffusion of ATPe. Mathematical analysis allowed us to get a general expression for each of the models. Subsequently, by using model dependent fit (simulations) as well as model analysis at infinite time, we observed that: - use of J(D) does not lead to improvements of the models. - Constant and Step time functions are only applicable when J(R)=0 (and thus, J(NL)=0), so that the only source of ATPe would be J(L), a result incompatible with experimental data. - use of impulse, Gaussian, and lognormal J(R)s in the models led to reasonable good fits to experimental data, with the lognormal function in model 1 providing the best option. Finally, the predictive nature of model 1 loaded with a lognormal J(R) was tested by simulating different putative in vivo scenarios where J(V) and J(NL) were varied over ample ranges.