Endoplasmic reticulum stress in adipose tissue augments lipolysis

The endoplasmic reticulum (ER) is an organelle important for protein synthesis and folding, lipid synthesis and Ca²⁺ homoeostasis. Consequently, ER stress or dysfunction affects numerous cellular processes and has been implicated as a contributing factor in several pathophysiological conditions. Tunicamycin induces ER stress in various cell types in vitro as well as in vivo. In mice, a hallmark of tunicamycin administration is the development of fatty livers within 24–48 hrs accompanied by hepatic ER stress. We hypothesized that tunicamycin would induce ER stress in adipose tissue that would lead to increased lipolysis and subsequently to fatty infiltration of the liver and hepatomegaly. Our results show that intraperitoneal administration of tunicamycin rapidly induced an ER stress response in adipose tissue that correlated with increased circulating free fatty acids (FFAs) and glycerol along with decreased adipose tissue mass and lipid droplet size. Furthermore, we found that in addition to fatty infiltration of the liver as well as hepatomegaly, lipid accumulation was also present in the heart, skeletal muscle and kidney. To corroborate our findings to a clinical setting, we examined adipose tissue from burned patients where increases in lipolysis and the development of fatty livers have been well documented. We found that burned patients displayed significant ER stress within adipose tissue and that ER stress augments lipolysis in cultured human adipocytes. Our results indicate a possible role for ER stress induced lipolysis in adipose tissue as an underlying mechanism contributing to increases in circulating FFAs and fatty infiltration into other organs.     

Design principles of tissue organisation: How single cells coordinate across scales

Cells act as building blocks of multicellular organisms, forming higher-order structures at different biological scales. Niches, tissues and, ultimately, entire organisms consist of single cells that remain in constant communication. Emergence of developmental patterns and tissue architecture thus relies on single cells acting as a collective, coordinating growth, migration, cell fate transitions and cell type sorting. For this, information has to be transmitted forward from cells to tissues and fed back to the individual cell to allow dynamic and robust coordination. Here, we define the design principles of tissue organisation integrating chemical, genetic and mechanical cues. We also review the state-of-the-art technologies used for dissecting collective cellular behaviours at single cell– and tissue-level resolution. We finally outline future challenges that lie in a comprehensive understanding of how single cells coordinate across biological scales to insure robust development, homoeostasis and regeneration of tissues.     

Spatial, temporal and molecular hierarchies in the link between death, delamination and dorsal closure

Dead cells in most epithelia are eliminated by cell extrusion. Here, we explore whether cell delamination in the amnioserosa, a seemingly stochastic event that results in the extrusion of a small fraction of cells and known to provide a force for dorsal closure, is contingent upon the receipt of an apoptotic signal. Through the analysis of mutant combinations and the profiling of apoptotic signals in situ, we establish spatial, temporal and molecular hierarchies in the link between death and delamination. We show that although an apoptotic signal is necessary and sufficient to provide cell-autonomous instructions for delamination, its induction during natural delamination occurs downstream of mitochondrial fragmentation. We further show that apoptotic regulators can influence both delamination and dorsal closure cell non-autonomously, presumably by influencing tissue mechanics. The spatial heterogeneities in delamination frequency and mitochondrial morphology suggest that mechanical stresses may underlie the activation of the apoptotic cascade through their influence on mitochondrial dynamics. Our results document for the first time the temporal propagation of an apoptotic signal in the context of cell behaviours that accomplish morphogenesis during development. They highlight the importance of mitochondrial dynamics and tissue mechanics in its regulation. Together, they provide novel insights into how apoptotic signals can be deployed to pattern tissues.     

A finite element model of cell-matrix interactions to study the differential effect of scaffold composition on chondrogenic response to mechanical stimulation

Mechanically induced cell deformations have been shown to influence chondrocyte response in 3D culture. However, the relationship between the mechanical stimulation and cell response is not yet fully understood. In this study a finite element model was developed to investigate cell-matrix interactions under unconfined compression conditions, using a tissue engineered encapsulating hydrogel seeded with chondrocytes. Model predictions of stress and strain distributions within the cell and on the cell boundary were shown to exhibit space-dependent responses that varied with scaffold mechanical properties, the presence of a pericellular matrix (PCM), and the cell size. The simulations predicted that when the cells were initially encapsulated into the hydrogel scaffolds, the cell size hardly affected the magnitude of the stresses and strains that were reaching the encapsulated cells. However, with the inclusion of a PCM layer, larger cells experienced enhanced stresses and strains resulting from the mechanical stimulation. It was also noted that the PCM had a stress shielding effect on the cells in that the peak stresses experienced within the cells during loading were significantly reduced. On the other hand, the PCM caused the stresses at the cell-matrix interface to increase. Based on the model predictions, the PCM modified the spatial stress distribution within and around the encapsulated cells by redirecting the maximum stresses from the periphery of the cells to the cell nucleus. In a tissue engineered cartilage exposed to mechanical loading, the formation of a neo-PCM by encapsulated chondrocytes appears to protect them from initially excessive mechanical loading. Predictive models can thus shed important insight into how chondrocytes remodel their local environment in order to redistribute mechanical signals in tissue engineered constructs.     

The Mechanochemical Basis of Cell and Tissue Regulation

This article is a summary of a lecture presented at a symposium on "Mechanics and Chemistry of Biosystems' in honor of Professor Y.C. Fung that convened at the University of California, Irvine in February 2004. The article reviews work from our laboratory that focuses on the mechanism by which mechanical and chemical signals interplay to control how individual cells decide whether to grow, differentiate, move, or die, and thereby promote pattern formation during tissue morphogenesis. Pursuit of this challenge has required development and application of new microtechnologies, theoretical formulations, computational models and bioinformatics tools. These approaches have been used to apply controlled mechanical stresses to specific cell surface molecules and to measure mechanical and biochemical responses; to control cell shape independently of chemical factors; and to handle the structural, hierarchical and informational complexity of living cells. Results of these studies have changed our view of how cells and tissues control their shape and mechanical properties, and have led to the discovery that integrins and the cytoskeleton play a central role in cellular mechanotransduction. Recognition of these critical links between mechanics and cellular biochemistry should lead to novel strategies for the development of new drugs and engineered tissues, as well as biomimetic microdevices and nanotechnologies that more effectively function within the context of living tissues.     

Morphomechanics: goals, basic experiments and models

Morphomechanics is a branch of developmental biology, studying the generation, space-time patterns and morphogenetic role of mechanical stresses (MS) which reside in embryonic tissues. All the morphogenetically active embryonic tissues studied in this respect have been shown to bear substantial mechanical stresses of tension or pressure. MS are indispensable for organized cell movements, expression of a number of developmentally important genes and the very viability of cells. Even a temporary relaxation of MS leads to an increase in the morphological variability and asymmetry of embryonic rudiments. Moreover, MS may be among the decisive links of morphogenetic feedback required for driving forth embryonic development and providing its regular space-time patterns. We hypothesize that one such feedback is based upon the tendency of cells and tissues to hyperrestore (restore with an overshoot) their MS values after any deviations, either artificial or produced by neighboring morphogenetically active tissues. This idea is supported by a number of observations and experiments performed on the tissue and individual cell levels. We describe also the models demonstrating that a number of biologically realistic stationary shapes and propagating waves can be generated by varying the parameters of the hyperrestoration feedback loop. Morphomechanics is an important and rapidly developing branch of developmental and cell biology, being complementary to other approaches.     

Modeling mechanical inhomogeneities in small populations of proliferating monolayers and spheroids

Understanding the mechanical behavior of multicellular monolayers and spheroids is fundamental to tissue culture, organism development, and the early stages of tumor growth. Proliferating cells in monolayers and spheroids experience mechanical forces as they grow and divide and local inhomogeneities in the mechanical microenvironment can cause individual cells within the multicellular system to grow and divide at different rates. This differential growth, combined with cell division and reorganization, leads to residual stress. Multiple different modeling approaches have been taken to understand and predict the residual stresses that arise in growing multicellular systems, particularly tumor spheroids. Here, we show that by using a mechanically robust agent-based model constructed with the peridynamic framework, we gain a better understanding of residual stresses in multicellular systems as they grow from a single cell. In particular, we focus on small populations of cells (1–100 s) where population behavior is highly stochastic and prior investigation has been limited. We compare the average strain energy density of cells in monolayers and spheroids using different growth and division rules and find that, on average, cells in spheroids have a higher strain energy density than cells in monolayers. We also find that cells in the interior of a growing spheroid are, on average, in compression. Finally, we demonstrate the importance of accounting for stochastic fluctuations in the mechanical environment, particularly when the cellular response to mechanical cues is nonlinear. The results presented here serve as a starting point for both further investigation with agent-based models, and for the incorporation of major findings from agent-based models into continuum scale models when explicit representation of individual cells is not computationally feasible.     

ER stress protects from retinal degeneration

The unfolded protein response (UPR) is a specific cellular process that allows the cell to cope with the overload of unfolded/misfolded proteins in the endoplasmic reticulum (ER). ER stress is commonly associated with degenerative pathologies, but its role in disease progression is still a matter for debate. Here, we found that mutations in the ER‐resident chaperone, neither inactivation nor afterpotential A (NinaA), lead to mild ER stress, protecting photoreceptor neurons from various death stimuli in adult Drosophila. In addition, Drosophila S2 cultured cells, when pre‐exposed to mild ER stress, are protected from H₂O₂, cycloheximide‐ or ultraviolet‐induced cell death. We show that a specific ER‐mediated signal promotes antioxidant defences and inhibits caspase‐dependent cell death. We propose that an immediate consequence of the UPR not only limits the accumulation of misfolded proteins but also protects tissues from harmful exogenous stresses.     

Endophyte-mediated adjustments in host morphology and physiology and effects on host fitness traits in grasses

Endophytic fungi have been shown to increase tolerance of hosts to biotic and abiotic stresses and in some cases alter growth and development of plants. In this article we evaluate some effects that clavicipitaceous endophytes have on development and physiology of plant tissues. We postulate that oxidative stress protection is the fundamental underlying benefit conferred by many endophytes, accounting for frequently observed enhanced disease resistance, drought tolerance, heavy metal tolerance and tolerance to numerous additional oxidative stresses. We hypothesize that endophyte-mediated oxidative stress protection of the host is the result of at least two processes, including: (1) secretion of reactive oxygen species (ROS) from endophytic mycelia into plant cells; and (2) secretion of auxin from endophytic mycelia into plant cells. Both processes result in an increase in ROS in plant tissues; and stimulate plant tissues to increase activities of antioxidant systems. Auxin is suggested to function in suppression of plant cell death and may be important in maintaining the endophyte–plant symbiosis.     

Correction to: The biotechnological importance of the plant-specific NAC transcription factor family in crop improvement

Climate change, malnutrition, and food insecurity are the inevitable challenges being faced by the agriculture sector today. Plants are susceptible to extreme temperatures during the crucial phases of flowering and seed development, and elevated carbon levels also lead to yield losses. Productivity is also affected by floods and droughts. Therefore, increasing plant yield and stress tolerance are the priorities to be met through novel biotechnological interventions. The contributions of NAC genes towards enhancing plant survivability under stress is well known. Here we focus on the potential of NAC genes in the regulation of abiotic stress tolerance, secondary cell wall synthesis, lateral root development, yield potential, seed size and biomass, ROS signaling, leaf senescence, and programmed cell death. Once naturally tolerant candidate NAC genes have been identified, and the nature of their association with growth and fitness against multi-environmental stresses has been determined, they can be exploited for building inherent tolerance in future crops via transgenic technologies. An update on the latest developments is provided in this review, which summarizes the current understanding of the roles of NAC in the establishment of various stress-adaptive mechanisms in model and food crop plants.

     

Capabilities and Limitations of Tissue Size Control through Passive Mechanical Forces

Embryogenesis is an extraordinarily robust process, exhibiting the ability to control tissue size and repair patterning defects in the face of environmental and genetic perturbations. The size and shape of a developing tissue is a function of the number and size of its constituent cells as well as their geometric packing. How these cellular properties are coordinated at the tissue level to ensure developmental robustness remains a mystery; understanding this process requires studying multiple concurrent processes that make up morphogenesis, including the spatial patterning of cell fates and apoptosis, as well as cell intercalations. In this work, we develop a computational model that aims to understand aspects of the robust pattern repair mechanisms of the Drosophila embryonic epidermal tissues. Size control in this system has previously been shown to rely on the regulation of apoptosis rather than proliferation; however, to date little work has been done to understand the role of cellular mechanics in this process. We employ a vertex model of an embryonic segment to test hypotheses about the emergence of this size control. Comparing the model to previously published data across wild type and genetic perturbations, we show that passive mechanical forces suffice to explain the observed size control in the posterior (P) compartment of a segment. However, observed asymmetries in cell death frequencies across the segment are demonstrated to require patterning of cellular properties in the model. Finally, we show that distinct forms of mechanical regulation in the model may be distinguished by differences in cell shapes in the P compartment, as quantified through experimentally accessible summary statistics, as well as by the tissue recoil after laser ablation experiments.     

Coupling mechanical deformations and planar cell polarity to create regular patterns in the zebrafish retina

The orderly packing and precise arrangement of epithelial cells is essential to the functioning of many tissues, and refinement of this packing during development is a central theme in animal morphogenesis. The mechanisms that determine epithelial cell shape and position, however, remain incompletely understood. Here, we investigate these mechanisms in a striking example of planar order in a vertebrate epithelium: The periodic, almost crystalline distribution of cone photoreceptors in the adult teleost fish retina. Based on observations of the emergence of photoreceptor packing near the retinal margin, we propose a mathematical model in which ordered columns of cells form as a result of coupling between planar cell polarity (PCP) and anisotropic tissue-scale mechanical stresses. This model recapitulates many observed features of cone photoreceptor organization during retinal growth and regeneration. Consistent with the model's predictions, we report a planar-polarized distribution of Crumbs2a protein in cone photoreceptors in both unperturbed and regenerated tissue. We further show that the pattern perturbations predicted by the model to occur if the imposed stresses become isotropic closely resemble defects in the cone pattern in zebrafish lrp2 mutants, in which intraocular pressure is increased, resulting in altered mechanical stress and ocular enlargement. Evidence of interactions linking PCP, cell shape, and mechanical stresses has recently emerged in a number of systems, several of which show signs of columnar cell packing akin to that described here. Our results may hence have broader relevance for the organization of cells in epithelia. Whereas earlier models have allowed only for unidirectional influences between PCP and cell mechanics, the simple, phenomenological framework that we introduce here can encompass a broad range of bidirectional feedback interactions among planar polarity, shape, and stresses; our model thus represents a conceptual framework that can address many questions of importance to morphogenesis.     

Cell flow and tissue polarity patterns

Planar tissue polarity is a fundamental feature of many epithelia. Large-scale cell polarity patterns govern the orientation of external structures such as hairs and cilia. Tissue polarity patterns arise from the collective organization of cells, which are polarized individually. Such cell and tissue polarities are reflected in anisotropic distributions of proteins of the planar cell polarity (PCP) pathway. Here we give an overview on recent progress in understanding how large-scale patterns of tissue polarity are controlled. We highlight the role of active mechanical events in the organization of polarity patterns during the development of the pupal fly wing. Patterns of cell flow are generated by mechanical stresses exerted on the tissue as well as by oriented cell divisions and neighbor exchanges. We discuss how the resulting tissue shear controls polarity orientation. We argue that the often-observed alignment of PCP either parallel or perpendicular to the long axis of developing tissues is a characteristic consequence of shear-induced polarity alignment. This principle allows for the versatile and robust generation of polarity patterns in tissues.     

A “sweet-spot” for fluid-induced oscillations in the conditioning of stem cell-based engineered heart valve tissues

Fluid-induced shear stresses are involved in the development of cardiovascular tissues. In a tissue engineering framework, this stimulus has also been considered as a mechanical regulator of stem cell differentiation. We recently demonstrated that the fluid-oscillating effect in combination with a physiologically-relevant shear stress magnitude contributes to the formation of stem cell-derived de novo heart valve tissues. However, the range of oscillations necessary to induce favorable gene expression and engineered tissue formation is unknown. In this study, we took a computational approach to establish a range of oscillatory shear stresses that may optimize in vitro valvular tissue growth. Taking a biomimetic approach, three physiologically-relevant flow waveforms from the human: (i) aorta, (ii) pulmonary artery and (iii) superior vena cava were utilized to simulate pulsatile flow conditions within a bioreactor that housed 3 tissue specimens. Results were compared to non-physiological pulsatile flow (NPPF) and cyclic flexure-steady flow (Flex-Flow) conditions. The oscillatory shear index (OSI) was used to quantify the fluid-induced oscillations occurring on the specimen surfaces. The range of mean OSI under the physiological conditions investigated was found to be 0.18 ≤ OSI ≤ 0.23. On the other hand, NPPF and Flex-Flow environments yielded a mean OSI of 0.37 and 0.11 respectively, which were 46% higher and 45% lower than physiological conditions. Moreover, we subsequently conducted OSI-based human bone marrow stem cell (HBMSC) culture experiments which resulted in preferential valvular gene expression and phenotype (significant upregulation of BMP, KLF2A, CD31 and α-SMA using an OSI of 0.23 in comparison to a lower OSI of 0.10 or a higher OSI of 0.38; p < .05). These findings suggest that a distinct range or a “sweet-spot” for physiological OSI exists in the mechanical conditioning of tissue engineered heart valves grown from stem cell sources. We conclude that in vitro heart valve matrix development could be further enhanced by simultaneous exposure of the engineered tissues to physiologically-relevant magnitudes of both fluid-induced oscillations and shear stresses.     

Apoptotic force: active mechanical function of cell death during morphogenesis

Apoptosis, or programmed cell death, is an essential process for the elimination of unnecessary cells during embryonic development, tissue homeostasis, and certain pathological conditions. Recently, an active mechanical function of apoptosis called apoptotic force has been demonstrated during a tissue fusion process of Drosophila embryogenesis. The mechanical force produced during apoptosis is used not only to force dying cells out from tissues in order to keep tissue integrity, but also to change the morphology of neighboring cells to fill the space originally occupied by the dying cell. Furthermore, the occurrence of apoptosis correlates with tissue movement and tension of the tissue. This finding suggests that apoptotic forces might be harnessed throughout cell death-related morphogenesis; however, this concept remains to be fully investigated. While the investigation of this active mechanical function of apoptosis has just begun, here we summarize the current understandings of this novel function of apoptosis, and discuss some possible developmental processes in which apoptosis may play a mechanical role. The concept of apoptotic force prompts a necessity to rethink the role of programmed cell death during morphogenesis.     

Techniques for assessing 3-D cell–matrix mechanical interactions in vitro and in vivo

Cellular interactions with extracellular matrices (ECM) through the application of mechanical forces mediate numerous biological processes including developmental morphogenesis, wound healing and cancer metastasis. They also play a key role in the cellular repopulation and/or remodeling of engineered tissues and organs. While 2-D studies can provide important insights into many aspects of cellular mechanobiology, cells reside within 3-D ECMs in vivo, and matrix structure and dimensionality have been shown to impact cell morphology, protein organization and mechanical behavior. Global measurements of cell-induced compaction of 3-D collagen matrices can provide important insights into the regulation of overall cell contractility by various cytokines and signaling pathways. However, to understand how the mechanics of cell spreading, migration, contraction and matrix remodeling are regulated at the molecular level, these processes must also be studied in individual cells. Here we review the evolution and application of techniques for imaging and assessing local cell–matrix mechanical interactions in 3-D culture models, tissue explants and living animals.     

Tensile and compressive stresses in tracheids are induced by swelling based on geometrical constraints of the wood cell

Plants are able to pre-stress their tissues in order to actuate their organs. Here, we demonstrate with two tissue types of the secondary xylem of conifers (normal wood and compression wood of spruce (Picea abies)) that either tensile or compressive stresses can develop in the longitudinal direction during the swelling of the cell wall. This dramatic difference appears to be due mostly to differences in cell geometry and cellulose fibril orientation. A mechanical model was developed to demonstrate swelling experiments with the help of sodium iodide experiments. The reversal of longitudinal extension can be predicted, based on the orientation of the (nearly inextensible) cellulose fibrils and the shape of the cell.     

Knockdown of cathepsin D in zebrafish fertilized eggs determines congenital myopathy

CD (cathepsin D) is a ubiquitous lysosomal hydrolase involved in a variety of pathophysiological functions, including protein turnover, activation of pro-hormones, cell death and embryo development. CD-mediated proteolysis plays a pivotal role in tissue and organ homoeostasis. Altered expression and compartmentalization of CD have been observed in diseased muscle fibres. Whether CD is actively involved in muscle development, homoeostasis and dystrophy remains to be demonstrated. Zebrafish (Danio rerio) is emerging as a valuable ‘in vivo’ vertebrate model for muscular degeneration and congenital myopathies. In this work, we report on the perturbance of the somitic musculature development in zebrafish larvae caused by MPO (morpholino)-mediated silencing of CD in oocytes at the time of fertilization. Restoring CD expression, using an MPO-non-matching mutated mRNA, partially rescued the normal phenotype, confirming the indispensable role of CD in the correct development and integrity of the somitic musculature. This is the first report showing a congenital myopathy caused by CD deficiency in a vertebrate experimental animal model.