Dynamics Of Cellular Interactions Research Articles

An In-Situ-Tag-Generation Proximity Labeling Technology for Recording Cellular Interactions.

Obtaining information about cellular interactions is fundamental to the elucidation of physiological and pathological processes. Proximity labeling technologies have been widely used to report cellular interactions in situ; however, the reliance on addition of tag molecules typically restricts their application to regions where tags can readily diffuse, while the application in, for example, solid tissues, is susceptible. Here, we propose an "in-situ-tag-generation mechanism" and develop the GalTag technology based on galactose oxidase (GAO) for recording cellular interactions within three-dimensional biological solid regions. GAO mounted on bait cells can in situ generate bio-orthogonal aldehyde tags as interaction reporters on prey cells. Using GalTag, we monitored the dynamics of cellular interactions and assessed the targeting ability of engineered cells. In particular, we recorded, for the first time, the footprints of Bacillus Calmette-Guérin (BCG) invasion into the bladder tissue of living mice, providing a valuable perspective to elucidate the anti-tumor mechanism of BCG.

An in‐situ‐tag‐generation proximity labeling technology for recording cellular interactions

Obtaining information about cellular interactions is fundamental to the elucidation of physiological and pathological processes. Proximity labeling technologies have been widely used to report cellular interactions in situ; however, the reliance on addition of tag molecules typically restricts their application to regions where tags can readily diffuse, while the application in, for example, solid tissues, is susceptible. Here, we propose an "in‐situ‐tag‐generation mechanism" and develop the GalTag technology based on galactose oxidase (GAO) for recording cellular interactions within three‐dimensional biological solid regions. GAO mounted on bait cells can in situ generate bio‐orthogonal aldehyde tags as interaction reporters on prey cells. Using GalTag, we monitored the dynamics of cellular interactions and assessed the targeting ability of engineered cells. In particular, we recorded, for the first time, the footprints of Bacillus Calmette‐Guérin (BCG) invasion into the bladder tissue of living mice, providing a valuable perspective to elucidate the anti‐tumor mechanism of BCG.

The Unreasonable Effectiveness of Reaction Diffusion in Vertebrate Skin Color Patterning.

In 1952, Alan Turing published the reaction-diffusion (RD) mathematical framework, laying the foundations of morphogenesis as a self-organized process emerging from physicochemical first principles. Regrettably, this approach has been widely doubted in the field of developmental biology. First, we summarize Turing's line of thoughts to alleviate the misconception that RD is an artificial mathematical construct. Second, we discuss why phenomenological RD models are particularly effective for understanding skin color patterning at the meso/macroscopic scales, without the need to parameterize the profusion of variables at lower scales. More specifically, we discuss how RD models (a) recapitulate the diversity of actual skin patterns, (b) capture the underlying dynamics of cellular interactions, (c) interact with tissue size and shape, (d) can lead to ordered sequential patterning, (e) generate cellular automaton dynamics in lizards and snakes, (f) predict actual patterns beyond their statistical features, and (g) are robust to model variations. Third, we discuss the utility of linear stability analysis and perform numerical simulations to demonstrate how deterministic RD emerges from the underlying chaotic microscopic agents.

MnO2 nanosheets and gold nanoparticles supported electrochemical detection of circulating tumor cells.

The concentration of circulating tumor cells (CTCs) in peripheral blood is strongly correlated with the progress of certain metastatic cancers. In this study, we have developed a novel and facile electrochemical biosensor for the detection of CTCs based on the use of manganese dioxide nanosheets (MnO2 NSs) and gold nanoparticles (AuNPs). Aptamer sequence of target cell is modified on the surface of AuNPs for specifical recognition. With low-speed centrifugation, numerous AuNPs@DNA can be removed from the supernatant. On the other hand, MnO2 NSs are modified on the electrode surface to capture unreacted AuNPs@DNA. The declined electrochemical signal intensity can be used to reflect the level of CTCs. This biosensor achieves a wide linear range from 10 to 104 cells mL-1 and a limit of detection as low as 3 cells mL-1. Due to the specific aptamer as the recognition element, interfering cells can be successfully distinguished and this method performs satisfactorily in clinical samples. Therefore, it has great potential to be used as a powerful tool benefiting rare cells analysis and the investigation of dynamics of cellular interactions.

Rules of Engagement: A Guide to Developing Agent-Based Models.

Agent-based models (ABM), also called individual-based models, first appeared several decades ago with the promise of nearly real-time simulations of active, autonomous individuals such as animals or objects. The goal of ABMs is to represent a population of individuals (agents) interacting with one another and their environment. Because of their flexible framework, ABMs have been widely applied to study systems in engineering, economics, ecology, and biology. This chapter is intended to guide the users in the development of an agent-based model by discussing conceptual issues, implementation, and pitfalls of ABMs from first principles. As a case study, we consider an ABM of the multi-scale dynamics of cellular interactions in a microbial community. We develop a lattice-free agent-based model of individual cells whose actions of growth, movement, and division are influenced by both their individual processes (cell cycle) and their contact with other cells (adhesion and contact inhibition).

Coupling in vitro cell culture with synchrotron SAXS to understand the bio-interaction of lipid-based liquid crystalline nanoparticles with vascular endothelial cells.

Nonlamellar lipid-based liquid crystalline (LLC) nanoparticles possessing different internal nanostructures, specifically the 3D-ordered cubosomes (V2 phase) and the 2D-ordered hexosomes (H2 phase), are of increasing interest as drug delivery systems. To facilitate their development, it is important that we understand their interactions with healthy human umbilical vein endothelial cells (HUVECs). To this end, a 3D cells-in-a-tube model that recapitulates the basic morphology (i.e. tubular lumen) and in vivo microenvironment (i.e. physiological shear stress) of blood vessels was employed as a biomimetic testing platform, and the bio-nanoparticle interactions were compared with that of the conventional 2D planar cell culture. Confocal microscopy imaging revealed internalisation of the nanoparticles into HUVECs within 2h and that the nanoparticle-cell interactions of cubosomes and hexosomes were not significantly different from one another. Low fluid shear stress conditions (i.e. venous simulation at 0.8 dynes/cm2) were shown to impose subtle effects on the degree of nanoparticle-cell interactions as compared with the static 2D culture. The unexpected similarity of cellular interactions between cubosomes and hexosomes was clarified via a real-time phase behaviour analysis using the synchrotron-based small-angle X-ray scattering (SAXS) technique. When the nanoparticles came into contact with HUVECs under circulating conditions, the cubosomes gradually evolved into hexosomes (within 16min). In contrast, the hexosomes retained their original internal structure with minimal changes to the lattice parameters. This study highlights the need to couple cellular studies with high-resolution analytics such as time-resolved SAXS analysis to ensure that particle structures are verified in situ, enabling accurate interpretation of the dynamics of cellular interactions and potential bio-induced changes of particles intended for biomedical applications. Graphical abstract.

Increasing access to microfluidics for studying fungi and other branched biological structures

BackgroundMicrofluidic systems are well-suited for studying mixed biological communities for improving industrial processes of fermentation, biofuel production, and pharmaceutical production. The results of which have the potential to resolve the underlying mechanisms of growth and transport in these complex branched living systems. Microfluidics provide controlled environments and improved optical access for real-time and high-resolution imaging studies that allow high-content and quantitative analyses. Studying growing branched structures and the dynamics of cellular interactions with both biotic and abiotic cues provides context for molecule production and genetic manipulations. To make progress in this arena, technical and logistical barriers must be overcome to more effectively deploy microfluidics in biological disciplines. A principle technical barrier is the process of assembling, sterilizing, and hydrating the microfluidic system; the lack of the necessary equipment for the preparatory process is a contributing factor to this barrier. To improve access to microfluidic systems, we present the development, characterization, and implementation of a microfluidics assembly and packaging process that builds on self-priming point-of-care principles to achieve “ready-to-use microfluidics.”ResultsWe present results from domestic and international collaborations using novel microfluidic architectures prepared with a unique packaging protocol. We implement this approach by focusing primarily on filamentous fungi; we also demonstrate the utility of this approach for collaborations on plants and neurons. In this work we (1) determine the shelf-life of ready-to-use microfluidics, (2) demonstrate biofilm-like colonization on fungi, (3) describe bacterial motility on fungal hyphae (fungal highway), (4) report material-dependent bacterial-fungal colonization, (5) demonstrate germination of vacuum-sealed Arabidopsis seeds in microfluidics stored for up to 2 weeks, and (6) observe bidirectional cytoplasmic streaming in fungi.ConclusionsThis pre-packaging approach provides a simple, one step process to initiate microfluidics in any setting for fungal studies, bacteria-fungal interactions, and other biological inquiries. This process improves access to microfluidics for controlling biological microenvironments, and further enabling visual and quantitative analysis of fungal cultures.

Surface Coverage-Regulated Cellular Interaction of Ultrasmall Luminescent Gold Nanoparticles.

Investigations for accurately controlling the interaction between functional nanoparticles (NPs) and living cells set a long-thought benefit in nanomedicine and disease diagnostics. Here, we reveal a surface coverage-dependent cellular interaction by comparing the membrane binding and uptake of three ultrasmall luminescent gold NPs (AuNPs) with different surface coverages. Lower surface coverage leads to fast cellular interaction and strong membrane binding but low cellular uptake, whereas high surface coverage induces slow cellular interaction and low membrane binding but major cellular uptake. The slight number increase of cell-penetrating peptide on the surface of AuNPs shows improved cellular interaction dynamics and internalization through direct cellular membrane penetration. Furthermore, the different intrinsic emissions resulted from the surface coverage variation, especially the pH-responsive dual emissions, make the AuNPs powerful optical probes for subcellular imaging and tracking. The findings advance the fundamental understanding of the cellular interaction mechanisms of ultrasmall AuNPs and provide a feasible strategy for the design of functional NPs with tunable cellular interaction by surface regulation.

Enumeration of circulating tumor cells and investigation of cellular responses using aptamer-immobilized AlGaN/GaN high electron mobility transistor sensor array

This study reports the fabrication and characterization of electrical double layer gated AlGaN/GaN High electron mobility (HEMT) biosensor array to capture, detect and count circulating tumor cells (CTCs) of colorectal cancer (CRC). GaN HEMT chips were assembled into a sensor array on a thermo-curable polymer substrate in a simple and robust packaging process. The array had multiple aptamer immobilized sensing sites and was highly sensitive and selective with up to single cell resolution. The present method can detect cells in their native electrolyte composition, in a very short time with extremely low cost compared to the conventional circulating tumor cell assays. The effect of cellular binding in different test environments was further studied and reported. The electrical response of the sensor was discovered to be relevant to the transmembrane potential of the cell. This whole cell sensor array has the potential to be used as a point-of-care diagnostic tool in varied applications such as detection of rare cells, pathogens and studying the dynamics of cellular interactions.

Simultaneous MR imaging for tissue engineering in a rat model of stroke.

In situ tissue engineering within a stroke cavity is gradually emerging as a novel therapeutic paradigm. Considering the varied lesion topology within each subject, the placement and distribution of cells within the lesion cavity is challenging. The use of multiple cell types to reconstruct damaged tissue illustrates the complexity of the process, but also highlights the challenges to provide a non-invasive assessment. The distribution of implanted cells within the lesion cavity and crucially the contribution of neural stem cells and endothelial cells to morphogenesis could be visualized simultaneously using two paramagnetic chemical exchange saturation transfer (paraCEST) agents. The development of sophisticated imaging methods is essential to guide delivery of the building blocks for in situ tissue engineering, but will also be essential to understand the dynamics of cellular interactions leading to the formation of de novo tissue.

MALDI-MS and NanoSIMS imaging techniques to study cnidarian–dinoflagellate symbioses

Cnidarian–dinoflagellate photosynthetic symbioses are fundamental to biologically diverse and productive coral reef ecosystems. The hallmark of this symbiotic relationship is the ability of dinoflagellate symbionts to supply their cnidarian host with a wide range of nutrients. Many aspects of this association nevertheless remain poorly characterized, including the exact identity of the transferred metabolic compounds, the mechanisms that control their exchange across the host–symbiont interface, and the precise subcellular fate of the translocated materials in cnidarian tissues. This lack of knowledge is mainly attributed to difficulties in investigating such metabolic interactions both in situ, i.e. on intact symbiotic associations, and at high spatial resolution. To address these issues, we illustrate the application of two in situ and high spatial resolution molecular and ion imaging techniques–matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and the nano-scale secondary-ion mass spectrometry (NanoSIMS) ion microprobe. These imaging techniques provide important new opportunities for the detailed investigation of many aspects of cnidarian–dinoflagellate associations, including the dynamics of cellular interactions.

Sophisticated Adaptations of Gregarina cuneata (Apicomplexa) Feeding Stages for Epicellular Parasitism

BackgroundGregarines represent a very diverse group of early emerging apicomplexans, parasitising numerous invertebrates and urochordates, and are considered of little practical significance. Recently, they have gained more attention since some analyses showed that cryptosporidia are more closely related to the gregarines than to coccidia.Methodology/Principal FindingsUsing a combined microscopic approach, this study points out the spectacular strategy of Gregarina cuneata for attachment to host tissue and nutrient acquisition while parasitising the intestine of yellow mealworm larvae, and reveals the unusual dynamics of cellular interactions between the host epithelium and parasite feeding stages. Trophozoites of G. cuneata develop epicellularly, attached to the luminal side of the host epithelial cell by an epimerite exhibiting a high degree of morphological variability. The presence of contractile elements in the apical region of feeding stages indicates that trophozoite detachment from host tissue is an active process self-regulated by the parasite. A detailed discussion is provided on the possibility of reversible retraction and protraction of the eugregarine apical end, facilitating eventual reattachment to another host cell in better physiological conditions. The gamonts, found in contact with host tissue via a modified protomerite top, indicate further adaptation of parasite for nutrient acquisition via epicellular parasitism while keeping their host healthy. The presence of eugregarines in mealworm larvae even seems to increase the host growth rate and to reduce the death rate despite often heavy parasitisation.Conclusions/SignificanceImproved knowledge about the formation of host-parasite interactions in deep-branching apicomplexans, including gregarines, would offer significant insights into the fascinating biology and evolutionary strategy of Apicomplexa. Gregarines exhibit an enormous diversity in cell architecture and dimensions, depending on their parasitic strategy and the surrounding environment. They seem to be a perfect example of a coevolution between a group of parasites and their hosts.

Quantifying cellular interaction dynamics in 3D fluorescence microscopy data

The wealth of information available from advanced fluorescence imaging techniques used to analyze biological processes with high spatial and temporal resolution calls for high-throughput image analysis methods. Here, we describe a fully automated approach to analyzing cellular interaction behavior in 3D fluorescence microscopy images. As example application, we present the analysis of drug-induced and S1P(1)-knockout-related changes in bone-osteoclast interactions. Moreover, we apply our approach to images showing the spatial association of dendritic cells with the fibroblastic reticular cell network within lymph nodes and to microscopy data regarding T-B lymphocyte synapse formation. Such analyses that yield important information about the molecular mechanisms determining cellular interaction behavior would be very difficult to perform with approaches that rely on manual/semi-automated analyses. This protocol integrates adaptive threshold segmentation, object detection, adaptive color channel merging, and neighborhood analysis and permits rapid, standardized, quantitative analysis and comparison of the relevant features in large data sets.

IL-12 Produced by Dendritic Cells Augments CD8+ T Cell Activation through the Production of the Chemokines CCL1 and CCL17

IL-12 family members are an important link between innate and adaptive immunity. IL-12 drives Th1 responses by augmenting IFN-gamma production, which is key for clearance of intracellular pathogens. IL-23 promotes the development of IL-17-producing CD4(+) T cells that participate in the control of extracellular pathogens and the induction of autoimmunity. However, recent studies have shown that these cytokines can modulate lymphocyte migration and cellular interactions. Therefore, we sought to determine the individual roles of IL-12 and IL-23 in naive CD8(+) T cell activation by addressing their ability to influence IFN-gamma production and cellular interaction dynamics during priming by Listeria monocytogenes-infected dendritic cells (DC). We found that IL-12 was the major cytokine influencing the level of IFN-gamma production by CD8(+) T cells while IL-23 had little effect on this response. In addition, we observed that IL-12 promoted longer duration conjugation events between CD8(+) T cells and DC. This enhanced cognate interaction time correlated with increased production of the chemokines CCL1 and CCL17 by WT but not IL-12-deficient DC. Neutralization of both chemokines resulted in reduced interaction time and IFN-gamma production, demonstrating their importance in priming naive CD8(+) T cells. Our study demonstrates a novel mechanism through which IL-12 augments naive CD8(+) T cell activation by facilitating chemokine production, thus promoting more stable cognate interactions during priming.

Two‐photon imaging of effector T‐cell behavior: lessons from a tumor model

Recent advances in two-photon microscopy have provided a new way of visualizing the behavior of fluorescently tagged cells within their natural microenvironment. This technology has allowed for generating a detailed picture of the cellular interaction dynamics operant in the activation of T cells and B cells during primary immune responses within secondary lymphoid organs. In contrast, relatively little is known about the migratory and interactive behavior of effector T cells within peripheral organs. We have recently developed a two-photon microscopy model that enables tracking of cytotoxic T cells within tumors. We have demonstrated that tumor-infiltrating T lymphocytes (TILs) follow random migratory paths and that their migratory properties depend on signals from the T-cell receptor. We further showed that TILs underwent short- and long-term interactions with tumor cells as well as macrophages. Recently, we succeeded in dynamic imaging of the distribution of fluorescently tagged molecules within TILs at subcellular resolution, which will be instrumental for defining the composition of the lytic synapse as well as the targeted release of cytotoxic granules by these cells. The purpose of this review is to put our findings into the context of the current literature and to point out the molecular cues mediating effector T-cell function as candidates for future investigation.

A new stabilizing craniotomy–duratomy technique for single-cell anatomo-electrophysiological exploration of living intact brain networks

Standard large craniotomies induce undesirable brain motions during intracellular recordings in whole animal preparations. Practically all of the papers available in the literature outline a number of specific methodological approaches designed to avoid this inconvenience. Our study describes a new craniotomy–duratomy, which consists of the maintenance of a thin bone membrane and dura mater surrounding the small hole opened for lowering the recording micropipette. This new surgical preparation avoids brain movements by keeping the brain’s volume constant within the cranial cavity and does not require additional technical procedures. It is an all-purpose surgical technique, although it was developed in anaesthetized rats while studying spatio-temporal dynamics of cellular interactions associated with thalamocortical oscillations. It significantly improves both the precision of stereotaxic approaches and the success rate of single-cell recordings (e.g., current-clamp intracellular and paired recordings) compared to standard craniotomy/electrophysiology techniques.

Cellular interactions in the rat somatosensory thalamocortical system during normal and epileptic 5–9 Hz oscillations

In Genetic Absence Epilepsy Rats from Strasbourg (GAERS), generalized spike-and-wave (SW) discharges (5-9 SW s(-1)) develop during quiet immobile wakefulness from a natural, medium-voltage, 5-9 Hz rhythm. This study examines the spatio-temporal dynamics of cellular interactions in the somatosensory thalamocortical system underlying the generation of normal and epileptic 5-9 Hz oscillations. Paired single-unit and multi-unit recordings between the principal elements of this circuit and intracellular recordings of thalamic, relay and reticular, neurones were conducted in neuroleptanalgesied GAERS and control, non-epileptic, rats. The identity of the recorded neurones was established following juxtacellular or intracellular marking. At least six major findings have emerged from this study. (1) In GAERS, generalized spike-and-wave discharges were correlated with synchronous rhythmic firings in related thalamic relay and reticular neurones. (2) Usually, corticothalamic discharges phase-led related relay and reticular firings. (3) A depolarizing wave emerging from a barrage of EPSPs was the cause of both relay and reticular discharges. (4) In some relay cells, which had a relatively high membrane input resistance, the depolarizing wave had the shape of a ramp, which could trigger a low-threshold Ca2+ spike. (5) In reticular cells, the EPSP barrage could further trigger voltage-dependent depolarizations. (6) The epilepsy-related thalamic, relay and reticular, intracellular activities were similar to the normal-related thalamic activities. Overall, these findings strongly suggest that, during absence seizures, corticothalamic neurones play a primary role in the synchronized excitation of thalamic relay and reticular neurones. The present study further suggests that absence-related spike-and-wave discharges correspond to hypersynchronous wake-related physiological oscillations.

Selected contribution: a three-dimensional model for assessment of in vitro toxicity in balaena mysticetus renal tissue.

This study established two- and three-dimensional renal proximal tubular cell cultures of the endangered species bowhead whale (Balaena mysticetus), developed SV40-transfected cultures, and cloned the 61-amino acid open reading frame for the metallothionein protein, the primary binding site for heavy metal contamination in mammals. Microgravity research, modulations in mechanical culture conditions (modeled microgravity), and shear stress have spawned innovative approaches to understanding the dynamics of cellular interactions, gene expression, and differentiation in several cellular systems. These investigations have led to the creation of ex vivo tissue models capable of serving as physiological research analogs for three-dimensional cellular interactions. These models are enabling studies in immune function, tissue modeling for basic research, and neoplasia. Three-dimensional cellular models emulate aspects of in vivo cellular architecture and physiology and may facilitate environmental toxicological studies aimed at elucidating biological functions and responses at the cellular level. Marine mammals occupy a significant ecological niche (72% of the Earth's surface is water) in terms of the potential for information on bioaccumulation and transport of terrestrial and marine environmental toxins in high-order vertebrates. Few ex vivo models of marine mammal physiology exist in vitro to accomplish the aforementioned studies. Techniques developed in this investigation, based on previous tissue modeling successes, may serve to facilitate similar research in other marine mammals.