Cell-cell communication through direct contact, or juxtacrine signaling, is important in development, disease, and many areas of physiology. Synthetic forms of juxtacrine signaling can be precisely controlled and operate orthogonally to native processes, making them a powerful reductionist tool with which to address fundamental questions in cell-cell communication in vivo. Here we investigate how cell-cell contact length and tissue growth dynamics affect juxtacrine signal responses through implementing a custom synthetic gene circuit in Drosophila wing imaginal discs alongside mathematical modeling to determine synthetic Notch (synNotch) activation patterns. We find that the area of contact between cells largely determines the extent of synNotch activation, leading to the prediction that the shape of the interface between signal-sending and signal-receiving cells will impact the magnitude of the synNotch response. Notably, synNotch outputs form a graded spatial profile that extends several cell diameters from the signal source, providing evidence that the response to juxtacrine signals can persist in cells as they proliferate away from source cells, or that cells remain able to communicate directly over several cell diameters. Our model suggests the former mechanism may be sufficient, since it predicts graded outputs without diffusion or long-range cell-cell communication. Overall, we identify that cell-cell contact area together with output synthesis and decay rates likely govern the pattern of synNotch outputs in both space and time during tissue growth, insights that may have broader implications for juxtacrine signaling in general.

BACKGROUND:Although quantitative single-cell analysis is frequently applied in animal systems, e.g. to identify novel drugs, similar applications on plant single cells are largely missing. We have exploited the applicability of high-throughput microscopic image analysis on plant single cells using tobacco leaf protoplasts, cell-wall free single cells isolated by lytic digestion. Protoplasts regenerate their cell wall within several days after isolation and have the potential to expand and proliferate, generating microcalli and finally whole plants after the application of suitable regeneration conditions.RESULTS:High-throughput automated microscopy coupled with the development of image processing pipelines allowed to quantify various developmental properties of thousands of protoplasts during the initial days following cultivation by immobilization in multi-well-plates. The focus on early protoplast responses allowed to study cell expansion prior to the initiation of proliferation and without the effects of shape-compromising cell walls. We compared growth parameters of wild-type tobacco cells with cells expressing the antiapoptotic protein Bcl2-associated athanogene 4 from Arabidopsis (AtBAG4).CONCLUSIONS:AtBAG4-expressing protoplasts showed a higher proportion of cells responding with positive area increases than the wild type and showed increased growth rates as well as increased proliferation rates upon continued cultivation. These features are associated with reported observations on a BAG4-mediated increased resilience to various stress responses and improved cellular survival rates following transformation approaches. Moreover, our single-cell expansion results suggest a BAG4-mediated, cell-independent increase of potassium channel abundance which was hitherto reported for guard cells only. The possibility to explain plant phenotypes with single-cell properties, extracted with the single-cell processing and analysis pipeline developed, allows to envision novel biotechnological screening strategies able to determine improved plant properties via single-cell analysis.

Under both physiological (development, regeneration) and pathological conditions (cancer metastasis), cells migrate while sensing environmental cues in the form of mechanical, chemical or electrical stimuli. In the case of bone tissue, osteoblast migration is essential in bone regeneration. Although it is known that osteoblasts respond to exogenous electric fields, the underlying mechanism of electrotactic collective movement of human osteoblasts is unclear. Here, we present a computational model that describes the osteoblast cell migration in a direct current electric field as the motion of a collection of active self-propelled particles and takes into account fluctuations in the direction of single-cell migration, finite-range cell-cell interactions, and the interaction of a cell with the external electric field. By comparing this model with in vitro experiments in which human primary osteoblasts are exposed to a direct current electric field of different field strengths, we show that cell-cell interactions and fluctuations in the migration direction promote anode-directed collective migration of osteoblasts.

Surface charges at the cell–biomaterial interface are known to determine cellular functions. Previous findings on cell signaling indicate that osteoblastic cells favor certain moderately positive surface charges, whereas highly positive charges are not tolerated. In this study, we aimed to gain deeper insights into the influence exerted by surface charges on the actin cytoskeleton and the cell shape. We analyzed surfaces with a negative, moderately positive, and highly positive zeta (ζ) potential: titanium (Ti), Ti with plasma polymerized allylamine (PPAAm), and Ti with a polydiallyldimethylammonium chloride (PDADMA) multilayer, respectively. We used the software FilaQuant for automatic actin filament quantification of osteoblastic MG-63s, analyzed the cell edge height with scanning ion conductance microscopy (SICM), and described the cellular shape via a mathematical vertex model. A significant enhancement of actin filament formation was achieved on moderately positive (+7 mV) compared with negative ζ-potentials (−87 mV). A hampered cell spreading was reflected in a diminished actin filament number and length on highly positively charged surfaces (+50 mV). Mathematical simulations suggested that in these cells, cortical tension forces dominate the cell–substrate adhesion forces. Our findings present new insights into the impact of surface charges on the overall cell shape and even intracellular structures.

Our current understanding of vein development in leaves is based on canalization of the plant hormone auxin into self-reinforcing streams which determine the sites of vascular cell differentiation. By comparison, how auxin biosynthesis affects leaf vein patterning is less well understood. Here, after observing that inhibiting polar auxin transport rescues the sparse leaf vein phenotype in auxin biosynthesis mutants, we propose that the processes of auxin biosynthesis and cellular auxin efflux work in concert during vein development. By using computational modeling, we show that localized auxin maxima are able to interact with mechanical forces generated by the morphological constraints which are imposed during early primordium development. This interaction is able to explain four fundamental characteristics of midvein morphology in a growing leaf: (i) distal cell division; (ii) coordinated cell elongation; (iii) a midvein positioned in the center of the primordium; and (iv) a midvein which is distally branched. Domains of auxin biosynthetic enzyme expression are not positioned by auxin canalization, as they are observed before auxin efflux proteins polarize. This suggests that the site-specific accumulation of auxin, as regulated by the balanced action of cellular auxin efflux and local auxin biosynthesis, is crucial for leaf vein formation.

Under both physiological (development, regeneration) and pathological conditions (cancer metastasis), cells migrate while sensing environmental cues in the form of mechanical, chemical or electrical stimuli. Although it is known that osteoblasts respond to exogenous electric fields, the underlying mechanism of electrotactic collective movement of human osteoblasts is unclear. Theoretical approaches to study electrotactic cell migration until now mainly used reaction-diffusion models, and did not consider the effect of electric field on single-cell motility, or incorporate spatially dependent cell-to-cell interactions. Here, we present a computational model that takes into account cell interactions and describes cell migration in direct current electric field. We compare this model with in vitro experiments in which human primary osteoblasts are exposed to direct current electric field of varying field strength. Our results show that cell-cell interactions and fluctuations in the migration direction together lead to anode-directed collective migration of osteoblasts. Author summary Electrotactic migration of cells involves directed movement of a large number of single cells under the influence of external electric field. Influencing the migration behaviour of osteoblasts by external direct current electric field offers a promising approach towards building highly effective implants for bone regeneration. We present a computational model for electrotactic migration of osteoblasts subject to external direct current electric field. Our model considers individual cells that interact with each other and the external electric field, and, replicates the experimental observations, based on single-cell analysis, of the response of osteoblasts to electrical stimulation of varying strengths for 7 hours. Our results suggest that tracking trajectories of individual cells provide a way of determining the role of various interactions of a cell in collective migration. Our model provides a framework that links single cell response to the large scale collective dynamics.

Mesenchymal stem cell dynamics involve cell proliferation and cell differentiation into cells of distinct functional type, such as osteoblasts, adipocytes, or chondrocytes. Electrically active implants influence these dynamics for the regeneration of the cells in damaged tissues. How applied electric field influences processes of individual stem cells is a problem mostly unaddressed. The mathematical approaches to study stem cell dynamics have focused on the stem cell population as a whole, without resolving individual cells and intracellular processes. In this paper, we present a theoretical framework to describe the dynamics of a population of stem cells, taking into account the processes of the individual cells. We study the influence of the applied electric field on the cellular processes. We test our mean-field theory with the experiments from the literature, involving in vitro electrical stimulation of stem cells. We show that a simple model can quantitatively describe the experimentally observed time-course behavior of the total number of cells and the total alkaline phosphate activity in a population of mesenchymal stem cells. Our results show that the stem cell differentiation rate is dependent on the applied electrical field, confirming published experimental findings. Moreover, our analysis supports the cell density-dependent proliferation rate. Since the experimental results are averaged over many cells, our theoretical framework presents a robust and sensitive method for determining the effect of applied electric fields at the scale of the individual cell. These results indicate that the electric field stimulation may be effective in promoting bone regeneration by accelerating osteogenic differentiation.

Under both physiological (development, regeneration) and pathological conditions 15 (cancer metastasis), cells migrate while sensing environmental cues in the form of physical, 16 chemical or electrical gradients. Although it is known that osteoblasts respond to exogenous 17 electric fields, the underlying mechanism of electrotactic collective movement of human 18 osteoblasts is unclear. Theoretical approaches to study electrotactic cell migration until now 19 mainly used reaction-diffusion models, and did not consider the affect of electric field on 20 single-cell motility, or incorporate spatially dependent cell-to-cell interactions. Here, we present a 21 computational model that takes into account cell interactions and describes cell migration in 22 direct current electric field. We compare this model with in vitro experiments, in which human 23 primary osteoblasts are exposed to direct current electric field of varying field strength. Our 24 results show that cell-cell interactions and fluctuations in the migration direction together leads 25 to anode-directed collective migration of osteoblasts. 26

Systems biology orientates signaling pathways in their biological context. This aim invariably requires models that ignore extraneous factors and focus on the most crucial pathways of any given process. The developing embryo encapsulates many important processes in plant development; understanding their interaction will be key to designing crops able to maximize yield in an ever-more challenging world. Here, we briefly summarize the role of auxin during embryo development. We highlight recent advances in our understanding of auxin signaling and discuss implications for a systems understanding of development.

SummaryThe plant hormone auxin (indole-3-acetic acid, IAA) has a profound influence over plant cell growth and differentiation. Current understanding of vein development in leaves is based on the canalization of auxin into self-reinforcing streams which determine the sites of vascular cell differentiation. However, the role of auxin biosynthesis during leaf development in the context of leaf vein patterning has not been much studied so far. Here we characterize the context specific importance of auxin biosynthesis, auxin transport and mechanical regulations in a growing leaf. We show that domains of auxin biosynthesis predict the positioning of vascular cells. In mutants that have reduced capacity in auxin biosynthesis, leaf vein formation is decreased. While exogenous application of auxin does not compensate the loss of vein formation in auxin biosynthesis mutants, inhibition of polar auxin transport does compensate the vein-less phenotype, suggesting that the site-specific accumulation of auxin, which is likely to be mainly caused by the local auxin biosynthesis, is important for leaf vein formation. Our computational model of midvein development brings forth the interplay of cell stiffness and auxin dependent cell division. We propose that local auxin biosynthesis has the integral role in leaf vascular development.HighlightsBuilt spatially and temporally resolved auxin biosynthesis map in growing leaf primordium of Arabidopsis.Expression domains of auxin biosynthetic enzymes within primordia strongly correlated with leaf vein initiation.Results show that domains of auxin biosynthesis within primordia drive leaf vein initiation and patterning.Highlights and eTOC BlurbUsing modelling and a spatiotemporal analysis of auxin biosynthesis and transport, Kneuper et al. show that tissue specific auxin biosynthesis defines places of vein initiation hence underlining the importance of auxin concentration in vein initiation.

How epithelial cell behaviors are coordinately regulated to sculpt tissue architecture is a fundamental question in biology. Kupffer’s vesicle (KV), a transient organ with a fluid-filled lumen, provides a simple system to investigate the interplay between intrinsic cellular mechanisms and external forces during epithelial morphogenesis. Using 3-dimensional (3D) analyses of single cells we identify asymmetric cell volume changes along the anteroposterior axis of KV that coincide with asymmetric cell shape changes. Blocking ion flux prevents these cell volume changes and cell shape changes. Vertex simulations suggest cell shape changes do not depend on lumen expansion. Consistent with this prediction, asymmetric changes in KV cell volume and shape occur normally when KV lumen growth fails due to leaky cell adhesions. These results indicate ion flux mediates cell volume changes that contribute to asymmetric cell shape changes in KV, and that these changes in epithelial morphology are separable from lumen-generated forces.

Receptor-mediated endocytosis is a mechanism for import and distribution of nutrient and signaling cargo into a series of intracellular organelles, endosomes, with distinct biochemical characteristics. Endosomes form a dynamic network by undergoing fusion and fission, exchanging and redistributing cargo. Direct learning dynamic characteristic of individual endosomes in live cells is challenging problem. We have developed model to derive endocitic cargo traffic properties from microscopic dynamic of individual endosomes [1]. By reverse engineering this model allowed learn microscopic kinetic characteristic of endocytic network from set of snap-shot images. We developed software FitModelPDE2 (based on Pluk, C++, OpenCL) for fitting integral-PDE model to experimental data. Fitting of model to the experimental data revealed contribution of different processes governing endocytic system and suggested that some model parameters are functions of cargo progression. Applying “free-shape-function” approach we learned the changes of kinetic characteristic of endocytic network that accompany cargo progression..

BACKGROUND:Endocytosis allows the import and distribution of cargo into a series of endosomes with distinct morphological and biochemical characteristics. Our current understanding of endocytic cargo trafficking is based on the kinetics of net cargo transport between endosomal compartments without considering individual endosomes. However, endosomes form a dynamic network of membranes undergoing fusion and fission, thereby continuously exchanging and redistributing cargo. The macroscopic kinetic properties, i.e., the properties of the endosomal network as a whole, result from the collective behaviors of many individual endosomes, a problem so far largely unaddressed. RESULTS:Here, we developed a general theoretical framework to describe the dynamics of cargo distributions in the endosomal network. We combined the theory with quantitative experiments to study how the macroscopic kinetic properties of the endosomal network emerge from microscopic processes at the level of individual endosomes. We compared our theory predictions to experimental data in which dynamic distributions of endocytosed low-density lipoprotein (LDL) were quantified. CONCLUSIONS:Our theory can quantitatively describe the observed cargo distributions as a function of time. Remarkably, the theory allows determining microscopic kinetic parameters such as the fusion rate between endosomes from still images of cargo distributions at different times of internalization. We show that this method is robust and sensitive because cargo distributions result from an average over many stochastic events in many cells. Our results provide theoretical and experimental support to the "funnel model" of endosome progression and suggest that the conversion of early to late endosomes is the major mode of LDL trafficking.