Subcellular Scale Research Articles

Intermediate Filaments in Cellular Mechanoresponsiveness: Mediating Cytoskeletal Crosstalk From Membrane to Nucleus and Back.

The mammalian cytoskeleton forms a mechanical continuum that spans across the cell, connecting the cell surface to the nucleus via transmembrane protein complexes in the plasma and nuclear membranes. It transmits extracellular forces to the cell interior, providing mechanical cues that influence cellular decisions, but also actively generates intracellular forces, enabling the cell to probe and remodel its tissue microenvironment. Cells adapt their gene expression profile and morphology to external cues provided by the matrix and adjacent cells as well as to cell-intrinsic changes in cytoplasmic and nuclear volume. The cytoskeleton is a complex filamentous network of three interpenetrating structural proteins: actin, microtubules, and intermediate filaments. Traditionally the actin cytoskeleton is considered the main contributor to mechanosensitivity. This view is now shifting owing to the mounting evidence that the three cytoskeletal filaments have interdependent functions due to cytoskeletal crosstalk, with intermediate filaments taking a central role. In this Mini Review we discuss how cytoskeletal crosstalk confers mechanosensitivity to cells and tissues, with a particular focus on the role of intermediate filaments. We propose a view of the cytoskeleton as a composite structure, in which cytoskeletal crosstalk regulates the local stability and organization of all three filament families at the sub-cellular scale, cytoskeletal mechanics at the cellular scale, and cell adaptation to external cues at the tissue scale.

The impact of neuron morphology on cortical network architecture.

SummaryThe neurons in the cerebral cortex are not randomly interconnected. This specificity in wiring can result from synapse formation mechanisms that connect neurons, depending on their electrical activity and genetically defined identity. Here, we report that the morphological properties of the neurons provide an additional prominent source by which wiring specificity emerges in cortical networks. This morphologically determined wiring specificity reflects similarities between the neurons’ axo-dendritic projections patterns, the packing density, and the cellular diversity of the neuropil. The higher these three factors are, the more recurrent is the topology of the network. Conversely, the lower these factors are, the more feedforward is the network’s topology. These principles predict the empirically observed occurrences of clusters of synapses, cell type-specific connectivity patterns, and nonrandom network motifs. Thus, we demonstrate that wiring specificity emerges in the cerebral cortex at subcellular, cellular, and network scales from the specific morphological properties of its neuronal constituents.

Condensation of the Drosophila nerve cord is oscillatory and depends on coordinated mechanical interactions

During development, organs reach precise shapes and sizes. Organ morphology is not always obtained through growth; a classic counterexample is the condensation of the nervous system during Drosophila embryogenesis. The mechanics underlying such condensation remain poorly understood. Here, we characterize the condensation of the embryonic ventral nerve cord (VNC) at both subcellular and tissue scales. This analysis reveals that condensation is not a unidirectional continuous process but instead occurs through oscillatory contractions. The VNC mechanical properties spatially and temporally vary, and forces along its longitudinal axis are spatially heterogeneous. We demonstrate that the process of VNC condensation is dependent on the coordinated mechanical activities of neurons and glia. These outcomes are consistent with a viscoelastic model of condensation, which incorporates time delays and effective frictional interactions. In summary, we have defined the progressive mechanics driving VNC condensation, providing insights into how a highly viscous tissue can autonomously change shape and size.

Multi-Scale Computational Modeling of Spatial Calcium Handling From Nanodomain to Whole-Heart: Overview and Perspectives.

Regulation of intracellular calcium is a critical component of cardiac electrophysiology and excitation-contraction coupling. The calcium spark, the fundamental element of the intracellular calcium transient, is initiated in specialized nanodomains which co-locate the ryanodine receptors and L-type calcium channels. However, calcium homeostasis is ultimately regulated at the cellular scale, by the interaction of spatially separated but diffusively coupled nanodomains with other sub-cellular and surface-membrane calcium transport channels with strong non-linear interactions; and cardiac electrophysiology and arrhythmia mechanisms are ultimately tissue-scale phenomena, regulated by the interaction of a heterogeneous population of coupled myocytes. Recent advances in imaging modalities and image-analysis are enabling the super-resolution reconstruction of the structures responsible for regulating calcium homeostasis, including the internal structure of nanodomains themselves. Extrapolating functional and imaging data from the nanodomain to the whole-heart is non-trivial, yet essential for translational insight into disease mechanisms. Computational modeling has important roles to play in relating structural and functional data at the sub-cellular scale and translating data across the scales. This review covers recent methodological advances that enable image-based modeling of the single nanodomain and whole cardiomyocyte, as well as the development of multi-scale simulation approaches to integrate data from nanometer to whole-heart. Firstly, methods to overcome the computational challenges of simulating spatial calcium dynamics in the nanodomain are discussed, including image-based modeling at this scale. Then, recent whole-cell models, capable of capturing a range of different structures (such as the T-system and mitochondria) and cellular heterogeneity/variability are discussed at two different levels of discretization. Novel methods to integrate the models and data across the scales and simulate stochastic dynamics in tissue-scale models are then discussed, enabling elucidation of the mechanisms by which nanodomain remodeling underlies arrhythmia and contractile dysfunction. Perspectives on model differences and future directions are provided throughout.

Combined Electron Microscopy Approaches for Arterial Glycocalyx Visualization.

Mainly constituted of glycosaminoglycans and proteoglycans, the glycocalyx is anchored in the plasma membrane, covering, in particular, the extracellular face of the arterial endothelium. Due to its complex three-dimensional (3D) architecture, the glycocalyx interacts with a wide variety of proteins, contributing to vascular permeability, the flow of mechanotransduction, and the modulation of local inflammatory processes. Alterations of glycocalyx structure mediate the endothelial dysfunction and contribute to the aggravation of peripheral vascular diseases. Therefore, the exploration of its ultrastructure becomes a priority to evaluate the degree of injury under physiopathological conditions and to assess the impact of therapeutic approaches. The objective of this study was to develop innovative approaches in electron microscopy to visualize the glycocalyx at the subcellular scale. Intravenous perfusion on rats with a fixing solution containing aldehyde fixatives enriched with lanthanum ions was performed to prepare arterial samples. The addition of lanthanum nitrate in the fixing solution allowed the enhancement of the staining of the glycocalyx for transmission electron microscopy (TEM) and to detect elastic and inelastic scattered electrons, providing complementary qualitative information. The strength of scanning electron microscopy (SEM) was used on resin-embedded serial sections, allowing rapid and efficient large field imaging and previous correlative TEM observations for ultrastructural fine details. To demonstrate the dynamic feature of the glycocalyx, 3D tomography was provided by dual-beam focus-ion-beam-SEM (FIB-SEM). These approaches allowed us to visualize and characterize the ultrastructure of the pulmonary artery glycocalyx under physiological conditions and in a rat pulmonary ischemia-reperfusion model, known to induce endothelial dysfunction. This study demonstrates the feasibility of combined SEM, TEM, and FIB-SEM tomography approaches on the same sample as the multiscale visualization and the identification of structural indicators of arterial endothelial glycocalyx integrity.

Quantitative modeling identifies critical cell mechanics driving bile duct lumen formation.

Biliary ducts collect bile from liver lobules, the smallest functional and anatomical units of liver, and carry it to the gallbladder. Disruptions in this process caused by defective embryonic development, or through ductal reaction in liver disease have a major impact on life quality and survival of patients. A deep understanding of the processes underlying bile duct lumen formation is crucial to identify intervention points to avoid or treat the appearance of defective bile ducts. Several hypotheses have been proposed to characterize the biophysical mechanisms driving initial bile duct lumen formation during embryogenesis. Here, guided by the quantification of morphological features and expression of genes in bile ducts from embryonic mouse liver, we sharpened these hypotheses and collected data to develop a high resolution individual cell-based computational model that enables to test alternative hypotheses in silico. This model permits realistic simulations of tissue and cell mechanics at sub-cellular scale. Our simulations suggest that successful bile duct lumen formation requires a simultaneous contribution of directed cell division of cholangiocytes, local osmotic effects generated by salt excretion in the lumen, and temporally-controlled differentiation of hepatoblasts to cholangiocytes, with apical constriction of cholangiocytes only moderately affecting luminal size.

Stimulated Raman scattering microscopy with spectral phasor analysis: applications in assessing drug-cell interactions.

Statins have displayed significant, although heterogeneous, anti-tumour activity in breast cancer disease progression and recurrence. They offer promise as a class of drugs, normally used for cardiovascular disease control, that could have a significant impact on the treatment of cancer. Understanding their mode of action and accurately assessing their efficacy on live cancer cells is an important and significant challenge. Stimulated Raman scattering (SRS) microscopy is a powerful, label-free imaging technique that can rapidly characterise the biochemical responses of live cell populations following drug treatment. Here, we demonstrate multi-wavelength SRS imaging together with spectral phasor analysis to characterise a panel of breast cancer cell lines (MCF-7, SK-BR-3 and MDA-MB-231 cells) treated with two clinically relevant statins, atorvastatin and rosuvastatin. Label-free SRS imaging within the high wavenumber region of the Raman spectrum (2800–3050 cm−1) revealed the lipid droplet distribution throughout populations of live breast cancer cells using biocompatible imaging conditions. A spectral phasor analysis of the hyperspectral dataset enables rapid differentiation of discrete cellular compartments based on their intrinsic SRS characteristics. Applying the spectral phasor method to studying statin treated cells identified a lipid accumulating phenotype in cell populations which displayed the lowest sensitivity to statin treatment, whilst a weaker lipid accumulating phenotype was associated with a potent reduction in cell viability. This study provides an insight into potential resistance mechanisms of specific cancer cells towards treatment with statins. Label-free SRS imaging provides a novel and innovative technique for phenotypic assessment of drug-induced effects across different cellular populations and enables effective analysis of drug–cell interactions at the subcellular scale.

Bilaterally Asymmetric Helical Myofibrils in Ascidian Tadpole Larvae.

The locomotor system is highly bilateral at the macroscopic level. Homochirality of biological molecules is fully compatible with the bilateral body. However, whether and how single-handed cells contribute to the bilateral locomotor system is obscure. Here, exploiting the small number of cells in the swimming tadpole larva of the ascidian Ciona, we analyzed morphology of the tail at cellular and subcellular scales. Quantitative phase-contrast X-ray tomographic microscopy revealed a high-density midline structure ventral to the notochord in the tail. Muscle cell nuclei on each side of the notochord were roughly bilaterally aligned. However, fluorescence microscopy detected left-right asymmetry of myofibril inclination relative to the longitudinal axis of the tail. Zernike phase-contrast X-ray tomographic microscopy revealed the presence of left-handed helices of myofibrils in muscle cells on both sides. Therefore, the locomotor system of ascidian larvae harbors symmetry-breaking left-handed helical cells, while maintaining bilaterally symmetrical cell alignment. These results suggest that bilateral animals can override cellular homochirality to generate the bilateral locomotor systems at the supracellular scale.

Different characteristics in the hygroscopicity of the graded hierarchical bamboo structure

Bamboo is mainly composed of fiber and parenchyma cells, and the gradient fiber distribution structure endows bamboo with excellent mechanical properties and improves its heterogeneity. Little is known about the real-time distribution and distribution mechanism of water on the gradient structure. In this study, the real-time moisture content of primeval bamboo with different density and proportions of fiber cells was tested. The mathematical models were used to calculate the content and distribution of adsorbed water in monolayered and multilayered bamboo and the differences in hygroscopicity of gradient structures were analyzed at cell level, sub-cellular level and molecular scale The results showed that microfibrillar aggregates of parenchyma cells differ at the subcellular scale, and parenchyma cells were more sensitive to changes in ambient humidity than fibers. When the humidity was higher than 40%, the moisture content of the different parts of bamboo differed considerably. When the humidity was 90%, the moisture content of the Inner, Middle, and Outer were 28.81%, 25.57%, and 18.55% respectively. And the adsorption isotherm were analyzed using the Hailwood–Horrobin (H–H) models. The monolayer moisture content in Inner was between 3.00% -3.50%. Under a high humidity, the multilayer adsorbed water content of Inner, Middle, and Outer reached 20.40–23.30%, 17.50–20.40%, and 14.60–17.00%.

Micro- and Nano-Scales Three-Dimensional Characterisation of Softwood.

Understanding the mechanical response of cellular biological materials to environmental stimuli is of fundamental importance from an engineering perspective in composites. To provide a deep understanding of their behaviour, an exhaustive analytical and experimental protocol is required. Attention is focused on softwood but the approach can be applied to a range of cellular materials. This work presents a new non-invasive multi-scale approach for the investigation of the hygro-mechanical behaviour of softwood. At the TOMCAT beamline of the Paul Scherrer Institute, in Switzerland, the swelling behaviour of softwood was probed at the cellular and sub-cellular scales by means of 3D high-resolution phase-contrast X-ray imaging. At the cellular scale, new findings in the anisotropic and reversible swelling behaviour of softwood and in the origin of swelling hysteresis of porous materials are explained from a mechanical perspective. However, the mechanical and moisture properties of wood highly depend on sub-cellular features of the wood cell wall, such as bordered pits, yielding local deformations during a full hygroscopic loading protocol.

Controlled Glioma Cell Migration and Confinement Using Biomimetic‐Patterned Hydrogels

Cancer cell migration is significantly influenced by the physical properties (e.g., mechanics, topography) of the local extracellular matrix (ECM). However, how cells guide their movement in a complex, heterogeneous landscape while interpreting various physical factors, especially those with sizes on the subcellular length scale, is not completely understood. Herein, a photodegradable hydrogel is fabricated with control over the spatial distribution of features with different physical characteristics. On the surface of this hydrogel, patterns of features with sizes and spacings on the subcellular to cellular length scale are generated using a digital micromirror device, and the influence of the organization of these patterns on cellular migratory behavior is investigated. The size and spatial arrangement of hard (or stiff) and soft domains, more so than their topography, is found to have a significant impact on the migration speed. Using this approach, the maximum migration speed is observed on hydrogels with 10 μm pattern sizes spaced 10 μm apart. In this regime, the cells are confined and interact with both hard and soft regions at their periphery. Together, this study of cell–ECM interactions at the interface of hard and soft domains provides insights into how cancer cells interact with their environment.

Report of the 2021 meeting of the International Society of Protistologists.

THE International Society of Protistologists (ISOP) met online from 26-30 July, 2021. The Program and Abstract book for this is available here (File S1). An in-person international meeting was not possible due to the continued COVID-19 pandemic. The meeting therefore took place using the society's Zoom license, which is available for use by ISOP members for other online protist meetings.

Meta-analytic electric field modeling and developing multi-scale models

Abstract In this talk, I will present two recent developments of my lab using electric field modeling. First, I will talk about our efforts in combining electric field modeling with traditional meta-analysis on the effects of tDCS on working memory. Due to differences in electrode montages and stimulation intensities across different studies, results are difficult to aggregate for meta-analytic inferences. To overcome these limitations, we have developed a novel meta-analytic method relating behavioral effect sizes to electric field strength, to identify brain regions underlying the largest tDCS-induced WM improvement. Simulations on 69 studies targeting left prefrontal cortex showed that tDCS electric field strength in lower dorsolateral prefrontal cortex (Brodmann area 45/47) relates most strongly to improved WM performance. This brain region could be a target area for future tDCS studies. Our metanalytic framework can be applied to other stimulation modalities and behavioral measures. In the second part, I will talk about our efforts developing multi-scale models for TMS. While the induced electric field is the key factor to determine dosing and target engagement, it is only a first step to model the physiological response. To advance these efforts we have developed a new multi-scale pipeline (NeMo-TMS) for modeling TMS effects across spatial scales. On the macro-scale, we simulate TMS electric fields using SimNIBS. Afterwards, simulated electric fields from the previous step are coupled with morphologically realistic neuronal models in the NEURON environment. These neuron-scale simulations allow the investigation of membrane voltage (depolarization/hyperpolarization), action potential initiation and propagation, field intensity, and orientation necessary for modulating neuron response, etc. Finally, we incorporate the membrane voltage data to simulate the calcium concentration induced by voltage-gated calcium-channels at the subcellular scale by solving the calcium dynamics equations. This allows us to model effects of rTMS protocols on somatic calcium accumulation, important for neural plasticity.

Forecasting Avalanches in Branched Actomyosin Networks with Network Science and Machine Learning.

We explored the dynamic and structural effects of actin-related proteins 2/3 (Arp2/3) on actomyosin networks using mechanochemical simulations of active matter networks. On the nanoscale, the Arp2/3 complex alters the topology of actomyosin by nucleating a daughter filament at an angle with respect to a mother filament. At a subcellular scale, they orchestrate the formation of a branched actomyosin network. Using a coarse-grained approach, we sought to understand how an actomyosin network temporally and spatially reorganizes itself by varying the concentration of the Arp2/3 complexes. Driven by motor dynamics, the network stalls at a high concentration of Arp2/3 and contracts at a low Arp2/3 concentration. At an intermediate Arp2/3 concentration, however, the actomyosin network is formed by loosely connected clusters that may collapse suddenly when driven by motors. This physical phenomenon is called an "avalanche" largely due to the marginal instability inherent to the morphology of a branched actomyosin network when the Arp2/3 complex is present. While embracing the data science approaches, we unveiled the higher-order patterns in the branched actomyosin networks and discovered a sudden change in the "social" network topology of actomyosin, which is a new type of avalanche in addition to the two types of avalanches associated with a sudden change in the size or shape of the whole actomyosin network, as shown in a previous investigation. Our new finding promotes the importance of using network theory and machine learning models to forecast avalanches in actomyosin networks. The mechanisms of the Arp2/3 complexes in shaping the architecture of branched actomyosin networks obtained in this paper will help us better understand the emergent reorganization of the topology in dense actomyosin networks that are difficult to detect in experiments.

Sodium channels and the intercalated disk - it is all about location, location, location.

Sodium channels and the intercalated disk - it is all about location, location, location.

An Energetic Approach to Modeling Cytoskeletal Architecture in Maturing Cardiomyocytes.

Through a variety of mechanisms, a healthy heart is able to regulate its structure and dynamics across multiple length scales. Disruption of these mechanisms can have a cascading effect, resulting in severe structural and/or functional changes that permeate across different length scales. Due to this hierarchical structure, there is interest in understanding how the components at the various scales coordinate and influence each other. However, much is unknown regarding how myofibril bundles are organized within a densely packed cell and the influence of the subcellular components on the architecture that is formed. To elucidate potential factors influencing cytoskeletal development, we proposed a computational model that integrated interactions at both the cellular and subcellular scale to predict the location of individual myofibril bundles that contributed to the formation of an energetically favorable cytoskeletal network. Our model was tested and validated using experimental metrics derived from analyzing single-cell cardiomyocytes. We demonstrated that our model-generated networks were capable of reproducing the variation observed in experimental cells at different length scales as a result of the stochasticity inherent in the different interactions between the various cellular components. Additionally, we showed that incorporating length-scale parameters resulted in physical constraints that directed cytoskeletal architecture toward a structurally consistent motif. Understanding the mechanisms guiding the formation and organization of the cytoskeleton in individual cardiomyocytes can aid tissue engineers toward developing functional cardiac tissue.

Multiview tiling light sheet microscopy for 3D high-resolution live imaging.

Light-sheet or selective plane illumination microscopy (SPIM) is ideally suited for in toto imaging of living specimens at high temporal-spatial resolution. In SPIM, the light scattering that occurs during imaging of opaque specimens brings about limitations in terms of resolution and the imaging field of view. To ameliorate this shortcoming, the illumination beam can be engineered into a highly confined light sheet over a large field of view and multi-view imaging can be performed by applying multiple lenses combined with mechanical rotation of the sample. Here, we present a Multiview tiling SPIM (MT-SPIM) that combines the Multi-view SPIM (M-SPIM) with a confined, multi-tiled light sheet. The MT-SPIM provides high-resolution, robust and rotation-free imaging of living specimens. We applied the MT-SPIM to image nuclei and Myosin II from the cellular to subcellular spatial scale in early Drosophila embryogenesis. We show that the MT-SPIM improves the axial-resolution relative to the conventional M-SPIM by a factor of two. We further demonstrate that this axial resolution enhancement improves the automated segmentation of Myosin II distribution and of nuclear volumes and shapes.

Misic, a general deep learning-based method for the high-throughput cell segmentation of complex bacterial communities.

Studies of bacterial communities, biofilms and microbiomes, are multiplying due to their impact on health and ecology. Live imaging of microbial communities requires new tools for the robust identification of bacterial cells in dense and often inter-species populations, sometimes over very large scales. Here, we developed MiSiC, a general deep-learning-based 2D segmentation method that automatically segments single bacteria in complex images of interacting bacterial communities with very little parameter adjustment, independent of the microscopy settings and imaging modality. Using a bacterial predator-prey interaction model, we demonstrate that MiSiC enables the analysis of interspecies interactions, resolving processes at subcellular scales and discriminating between species in millimeter size datasets. The simple implementation of MiSiC and the relatively low need in computing power make its use broadly accessible to fields interested in bacterial interactions and cell biology.

Engineering modeling frameworks for microbial food safety at various scales.

The landscape of mathematical model-based understanding of microbial food safety is wide and deep, covering interdisciplinary fields of food science, microbiology, physics, and engineering. With rapidly growing interest in such model-based approaches that increasingly include more fundamental mechanisms of microbial processes, there is a need to build a general framework that steers this evolutionary process by synthesizing literature spread over many disciplines. The framework proposed here shows four interconnected, complementary levels of microbial food processes covering sub-cellular scale, microbial population scale, food scale, and human population scale (risk). A continuum of completely mechanistic to completely empirical models, widely-used and emerging, are integrated into the framework; well-known predictive microbiology modeling being a part of this spectrum. The framework emphasizes fundamentals-based approaches that should get enriched over time, such as the basic building blocks of microbial population scale processes (attachment, migration, growth, death/inactivation and communication) and of food processes (e.g., heat and moisture transfer). A spectrum of models are included, for example, microbial population modeling covers traditional predictive microbiology models to individual-based models and cellular automata. The models are shown in sufficient quantitative detail to make obvious their coupling, or their integration over various levels. Guidelines to combine sub-processes over various spatial and time scales into a complete interdisciplinary and multiphysics model (i.e., a system) are provided, covering microbial growth/inactivation/transport and physical processes such as fluid flow and heat transfer. As food safety becomes increasingly predictive at various scales, this synthesis should provide its roadmap. This big picture and framework should be futuristic in driving novel research and educationalapproaches.

Cell Migration Regulated by Spatially Controlled Stiffness inside Composition‐Tunable Three‐Dimensional Dextran Hydrogels

AbstractStiffness of the extracellular matrix plays an important role in regulating cell migration. In this paper, two types of 3D dextran hydrogel, with the stiffness distributed homogeneously and heterogeneously in subcellular scales, have been designed and fabricated to serve as macromolecule scaffolds in which C2C12 cells, an immortalized mouse myoblast cell line, can migrate in a rationally controllable manner. The experimental results indicate that heterogeneous gels can support higher migration velocities, and effective supporting time‐stage for enhancing migration depended on the bulk stiffness that can be rationally and elaborately tuned. Moreover, migration velocity for both types of gels, in most cases, tends to decrease first and then increase from day 0 to day 6. These results show advantages for regulation of cell migration using this easy‐to‐implement heterogeneous cross‐linking approach. Further, associated principles regarding the relationship among cell migration, gel stiffness distribution, and expression of F‐actin and myosin II are also discussed. These studies support rational control of cell migration velocity in heterogeneously cross‐linked hydrogels that are widely applicable in biomaterial field.