The induction of autophagy by mechanical stress

The ability to respond and adapt to changes in the physical environment is a universal and essential cellular property. Here we demonstrated that cells respond to mechanical compressive stress by rapidly inducing autophagosome formation. We measured this response in both Dictyostelium and mammalian cells, indicating that this is an evolutionarily conserved, general response to mechanical stress. In Dictyostelium, the number of autophagosomes increased 20-fold within 10 min of 1 kPa pressure being applied and a similar response was seen in mammalian cells after 30 min. We showed in both cell types that autophagy is highly sensitive to changes in mechanical pressure and the response is graduated, with half-maximal responses at ~0.2 kPa, similar to other mechano-sensitive responses. We further showed that the mechanical induction of autophagy is TOR-independent and transient, lasting until the cells adapt to their new environment and recover their shape. The autophagic response is therefore part of an integrated response to mechanical challenge, allowing cells to cope with a continuously changing physical environment.     

MTOR-independent induction of autophagy in trabecular meshwork cells subjected to biaxial stretch

The trabecular meshwork (TM) is part of a complex tissue that controls the exit of aqueous humor from the anterior chamber of the eye, and therefore helps maintaining intraocular pressure (IOP). Because of variations in IOP with changing pressure gradients and fluid movement, the TM and its contained cells undergo morphological deformations, resulting in distention and stretching. It is therefore essential for TM cells to continuously detect and respond to these mechanical forces and adapt their physiology to maintain proper cellular function and protect against mechanical injury. Here we demonstrate the activation of autophagy, a pro-survival pathway responsible for the degradation of long-lived proteins and organelles, in TM cells when subjected to biaxial static stretch (20% elongation), as well as in high-pressure perfused eyes (30 mm Hg). Morphological and biochemical markers for autophagy found in the stretched cells include elevated LC3-II levels, increased autophagic flux, and the presence of autophagic figures in electron micrographs. Furthermore, our results indicate that the stretch-induced autophagy in TM cells occurs in an MTOR- and BAG3-independent manner. We hypothesize that activation of autophagy is part of the physiological response that allows TM cells to cope and adapt to mechanical forces.     

Signal transduction during environmental stress: InsP8 operates within highly restricted contexts

Genetic manipulation of diphosphoinositol polyphosphate synthesis impacts many biological processes (reviewed in S.B. Shears, Biochem. J. 377, 2004, 265-280). These observations lacked a cell-signalling context, until the recent discovery that bis-diphosphoinositol tetrakisphosphate ([PP]2-InsP4 or "InsP8") accumulates rapidly in mammalian cells in response to hyperosmotic stress (X. Pesesse, K. Choi, T. Zhang, and S. B. Shears J. Biol. Chem. 279, 2004, 43378-43381). We now investigate how widely applicable is this new stress-response. [PP]2-InsP4 did not respond to mechanical strain or oxidative stress in mammalian cells. Furthermore, despite tight conservation of many molecular stress responses across the phylogenetic spectrum, we show that cellular [PP]2-InsP4 levels do not respond significantly to osmotic imbalance, heat stress and salt toxicity in Saccharomyces cerevisiae. In contrast, we show that [PP]2-InsP4 is a novel sensor of mild thermal stress in mammalian cells: [PP]2-InsP4 levels increased 3-4 fold when cells were cooled from 37 to 33 degrees C, or heated to 42 degrees C. Increases in [PP]2-InsP4 levels following heat-shock were evident <5 min, and reversible (t(1/2)=7 min) once cells were returned to 37 degrees C. These responses were blocked by pharmacological inhibition of the ERK/MEK pathway. Additional control processes may lie upstream of [PP]2-InsP4 synthesis, which was synergistically activated when heat stress and osmotic stress were combined. Our data add to the repertoire of signaling responses following thermal challenges, a topic of current interest for its possible therapeutic value.     

Autophagy contributes to degradation of Hirano bodies

Hirano bodies are actin-rich inclusions reported most frequently in the hippocampus in association with a variety of conditions including neurodegenerative diseases, and aging. We have developed a model system for formation of Hirano bodies in Dictyostelium and cultured mammalian cells to permit detailed studies of the dynamics of these structures in living cells. Model Hirano bodies are frequently observed in membrane-enclosed vesicles in mammalian cells consistent with a role of autophagy in the degradation of these structures. Clearance of Hirano bodies by an exocytotic process is supported by images from electron microscopy showing extracellular release of Hirano bodies, and observation of Hirano bodies in the culture medium of Dictyostelium and mammalian cells. An autophagosome marker protein Atg8-GFP, was co-localized with model Hirano bodies in wild type Dictyostelium cells, but not in atg5(-) or atg1-1 autophagy mutant strains. Induction of model Hirano bodies in Dictyostelium with a high level expression of 34 kDa DeltaEF1 from the inducible discoidin promoter resulted in larger Hirano bodies and a cessation of cell doubling. The degradation of model Hirano bodies still occurred rapidly in autophagy mutant (atg5(-)) Dictyostelium, suggesting that other mechanisms such as the ubiquitin-mediated proteasome pathway could contribute to the degradation of Hirano bodies. Chemical inhibition of the proteasome pathway with lactacystin, significantly decreased the turnover of Hirano bodies in Dictyostelium providing direct evidence that autophagy and the proteasome can both contribute to degradation of Hirano bodies. Short term treatment of mammalian cells with either lactacystin or 3-methyl adenine results in higher levels of Hirano bodies and a lower level of viable cells in the cultures, supporting the conclusion that both autophagy and the proteasome contribute to degradation of Hirano bodies.     

Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement

Organs and tissues adapt to acute or chronic mechanical stress by remodeling their actin cytoskeletons. Cells that are stimulated by cyclic stretch or shear stress in vitro undergo bimodal cytoskeletal responses that include rapid reinforcement and gradual reorientation of actin stress fibers; however, the mechanism by which cells respond to mechanical cues has been obscure. We report that the application of either unidirectional cyclic stretch or shear stress to cells results in robust mobilization of zyxin from focal adhesions to actin filaments, whereas many other focal adhesion proteins and zyxin family members remain at focal adhesions. Mechanical stress also induces the rapid zyxin-dependent mobilization of vasodilator-stimulated phosphoprotein from focal adhesions to actin filaments. Thickening of actin stress fibers reflects a cellular adaptation to mechanical stress; this cytoskeletal reinforcement coincides with zyxin mobilization and is abrogated in zyxin-null cells. Our findings identify zyxin as a mechanosensitive protein and provide mechanistic insight into how cells respond to mechanical cues.     

Cellular stress responses and cancer: new mechanistic insights on anticancer effect by phytochemicals

During the tumorigenesis, cancer cells are frequently exposed to metabolic stress which is derived from altered cancer cell metabolism as well as unfavorable tumor microenvironment, such as hypoxia and glucose deprivation. Cancer cells need to respond to these stress stimuli properly through inducing cellular stress responses, such as unfolded protein response and autophagy, for cell survival. Therefore, modulation of these stress responses has been investigated as an alternative anticancer strategy, although their therapeutic clinical roles remain to be determined. In this review, we will discuss the cellular stress responses in cancer cells, the alternative anticancer strategy targeting unfolded protein response and/or autophagy, and the role of phytochemicals, which include resveratrol, genistein, curcumin, epigallocatechin-3-gallate and quercetin, in modulating the cellular stress responses.     

Analysis of hydrostatic pressure-induced changes in umbrella cell surface area

All cells experience and respond to external mechanical stimuli including shear stress, compression, and hydrostatic pressure. Cellular responses can include changes in exocytic and endocytic traffic. An excellent system to study how extracellular forces govern membrane trafficking events is the bladder umbrella cell, which lines the inner surface of the mammalian urinary bladder. It is hypothesized that umbrella cells modulate their apical plasma membrane surface area in response to hydrostatic pressure. Understanding the mechanics of this process is hampered by the lack of a suitable model system. We describe a pressure chamber that allows one to increase hydrostatic pressure in a physiological manner while using capacitance to monitor real-time changes in the apical surface area of the umbrella cell. It is demonstrated that application of hydrostatic pressure results in an increase in umbrella cell apical surface area and a change in the morphology of umbrella cells from roughly cuboidal to squamous. This process is dependent on increases in cytoplasmic Ca(2+). This system will be useful in further dissecting the mechanotransduction pathways involved in cell shape change and regulation of exocytic and endocytic traffic in umbrella cells.     

Adaptation of cellular mechanical behavior to mechanical loading for osteoblastic cells

Numerous cellular biochemical responses to mechanical loading are transient, indicating a cell's ability to adapt its behavior to a new mechanical environment. Since load-induced cellular deformation can initiate these biochemical responses, the overall goal of this study was to investigate the adaptation of global, or whole-cell, mechanical behavior, i.e., cellular deformability, in response to mechanical loading for osteoblastic cells. Confluent cell cultures were subjected to 1 or 2 Pa flow-induced shear stress for 2 h. Whole-cell mechanical behavior was then measured for individual cells using an atomic force microscope. Compared to cells maintained under static conditions, whole-cell stiffness was 1.36-fold (p=0.006) and 1.70-fold (p<0.001) greater for cells exposed to 1 and 2 Pa shear loading, respectively. The increase in shear stress magnitude from 1 to 2 Pa also caused a statistically significant, 1.25-fold increase in cell stiffness (p=0.02). Increases in cell stiffness were not altered in either flow group for 70 min after flow was terminated (p=0.15). Flow-induced rearrangement of the actin cytoskeleton was also maintained for at least 90 min after flow was terminated. Taken together, these findings support the hypothesis that cells become mechanically adapted to their mechanical environment via cytoskeletal modifications. Accordingly, cellular mechanical adaptation may play a key role in regulation of cellular mechanosensitivity and the related effects on tissue structure and function.     

Differential effects of endoplasmic reticulum stress-induced autophagy on cell survival

Autophagy is a cellular response to adverse environment and stress, but its significance in cell survival is not always clear. Here we show that autophagy could be induced in the mammalian cells by chemicals, such as A23187, tunicamycin, thapsigargin, and brefeldin A, that cause endoplasmic reticulum stress. Endoplasmic reticulum stress-induced autophagy is important for clearing polyubiquitinated protein aggregates and for reducing cellular vacuolization in HCT116 colon cancer cells and DU145 prostate cancer cells, thus mitigating endoplasmic reticulum stress and protecting against cell death. In contrast, autophagy induced by the same chemicals does not confer protection in a normal human colon cell line and in the non-transformed murine embryonic fibroblasts but rather contributes to cell death. Thus the impact of autophagy on cell survival during endoplasmic reticulum stress is likely contingent on the status of cells, which could be explored for tumor-specific therapy.     

Evidence for a Mechanically Induced Oxidative Burst

Rapid release of H2O2 may constitute an initial defense response mounted by a plant. Inauguration of this oxidative burst is known to occur upon stimulation with chemical elicitors, but the possibility of mechanical elicitation arising from pathogen penetration/weakening of the cell wall has never been examined. To introduce an adjustable mechanical stress on the plasma membrane, cultured soybean (Glycine max Merr. cv Kent) cells were subjected to defined changes in medium osmolarity. Dilution of the medium with water or resuspension of cells in sucrose solutions of reduced osmolarity yielded an oxidative burst similar to those stimulated by chemical elicitors. Furthermore, the magnitude of oxidant biosynthesis and osmotic stress correlated directly. Upon return of the cells to normal tonicity, the oxidative burst abruptly halted, indicating that its expression depended on maintenance of the osmotic stress and not on any external chemical signal. To confirm the ability of soybean cells to respond to a mechanical stimulus with induction of an oxidative burst, cells were subjected to direct physical pressure. Application of pressure yielded a characteristic oxidative burst. Because neither these cells nor those subjected to osmotic pressure were damaged by their treatments, we conclude that plant cells can detect mechanical disturbances and initiate a classical defense reaction in response.     

Autophagy, nutrition and immunology

Turnover of cellular components in lysosomes or autophagy is an essential mechanism for cellular quality control. Added to this cleaning role, autophagy has recently been shown to participate in the dynamic interaction of cells with the surrounding environment by acting as a point of integration of extracellular cues. In this review, we focus on the relationship between autophagy and two types of environmental factors: nutrients and pathogens. We describe their direct effect on autophagy and discuss how the autophagic reaction to these stimuli allows cells to accommodate the requirements of the cellular response to stress, including those specific to the immune responses.     

Combining dynamic stretch and tunable stiffness to probe cell mechanobiology in vitro

Cells have the ability to actively sense their mechanical environment and respond to both substrate stiffness and stretch by altering their adhesion, proliferation, locomotion, morphology, and synthetic profile. In order to elucidate the interrelated effects of different mechanical stimuli on cell phenotype in vitro, we have developed a method for culturing mammalian cells in a two-dimensional environment at a wide range of combined levels of substrate stiffness and dynamic stretch. Polyacrylamide gels were covalently bonded to flexible silicone culture plates and coated with monomeric collagen for cell adhesion. Substrate stiffness was adjusted from relatively soft (G′ = 0.3 kPa) to stiff (G′ = 50 kPa) by altering the ratio of acrylamide to bis-acrylamide, and the silicone membranes were stretched over circular loading posts by applying vacuum pressure to impart near-uniform stretch, as confirmed by strain field analysis. As a demonstration of the system, porcine aortic valve interstitial cells (VIC) and human mesenchymal stem cells (hMSC) were plated on soft and stiff substrates either statically cultured or exposed to 10% equibiaxial or pure uniaxial stretch at 1Hz for 6 hours. In all cases, cell attachment and cell viability were high. On soft substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates (p<0.05). Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. hMSCs exhibited a less pronounced response than VICs, likely due to a lower stiffness threshold for spreading on static gels. These preliminary data demonstrate that inhibition of spreading due to a lack of matrix stiffness surrounding a cell may be overcome by externally applied stretch suggesting similar mechanotransduction mechanisms for sensing stiffness and stretch.     

Physically-Induced Cytoskeleton Remodeling of Cells in Three-Dimensional Culture

Characterizing how cells in three-dimensional (3D) environments or natural tissues respond to biophysical stimuli is a longstanding challenge in biology and tissue engineering. We demonstrate a strategy to monitor morphological and mechanical responses of contractile fibroblasts in a 3D environment. Cells responded to stretch through specific, cell-wide mechanisms involving staged retraction and reinforcement. Retraction responses occurred for all orientations of stress fibers and cellular protrusions relative to the stretch direction, while reinforcement responses, including extension of cellular processes and stress fiber formation, occurred predominantly in the stretch direction. A previously unreported role of F-actin clumps was observed, with clumps possibly acting as F-actin reservoirs for retraction and reinforcement responses during stretch. Responses were consistent with a model of cellular sensitivity to local physical cues. These findings suggest mechanisms for global actin cytoskeleton remodeling in non-muscle cells and provide insight into cellular responses important in pathologies such as fibrosis and hypertension.     

Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium

Chemotaxis-competent cells respond to a variety of ligands by activating second messenger pathways leading to changes in the actin/myosin cytoskeleton and directed cell movement. We demonstrate that Dictyostelium Akt/PKB, a homologue of mammalian Akt/PKB, is very rapidly and transiently activated by the chemoattractant cAMP. This activation takes place through G protein-coupled chemoattractant receptors via a pathway that requires homologues of mammalian p110 phosphoinositide-3 kinase. pkbA null cells exhibit aggregation-stage defects that include aberrant chemotaxis, a failure to polarize properly in a chemoattractant gradient and aggregation at low densities. Mechanistically, we demonstrate that the PH domain of Akt/PKB fused to GFP transiently translocates to the plasma membrane in response to cAMP with kinetics similar to those of Akt/PKB kinase activation and is localized to the leading edge of chemotaxing cells in vivo. Our results indicate Akt/PKB is part of the regulatory network required for sensing and responding to the chemoattractant gradient that mediates chemotaxis and aggregation.     

Dissecting the function of Atg1 complex in Dictyostelium autophagy reveals a connection with the pentose phosphate pathway enzyme transketolase

The network of protein–protein interactions of the Dictyostelium discoideum autophagy pathway was investigated by yeast two-hybrid screening of the conserved autophagic proteins Atg1 and Atg8. These analyses confirmed expected interactions described in other organisms and also identified novel interactors that highlight the complexity of autophagy regulation. The Atg1 kinase complex, an essential regulator of autophagy, was investigated in detail here. The composition of the Atg1 complex in D. discoideum is more similar to mammalian cells than to Saccharomyces cerevisiae as, besides Atg13, it contains Atg101, a protein not conserved in this yeast. We found that Atg101 interacts with Atg13 and genetic disruption of these proteins in Dictyostelium leads to an early block in autophagy, although the severity of the developmental phenotype and the degree of autophagic block is higher in Atg13-deficient cells. We have also identified a protein containing zinc-finger B-box and FNIP motifs that interacts with Atg101. Disruption of this protein increases autophagic flux, suggesting that it functions as a negative regulator of Atg101. We also describe the interaction of Atg1 kinase with the pentose phosphate pathway enzyme transketolase (TKT). We found changes in the activity of endogenous TKT activity in strains lacking or overexpressing Atg1, suggesting the presence of an unsuspected regulatory pathway between autophagy and the pentose phosphate pathway in Dictyostelium that seems to be conserved in mammalian cells.     

On the evolutionary conservation of the cell death pathway: mitochondrial release of an apoptosis-inducing factor during Dictyostelium discoideum cell death

Mitochondria play a pivotal role in apoptosis in multicellular organisms by releasing apoptogenic factors such as cytochrome c that activate the caspases effector pathway, and apoptosis-inducing factor (AIF) that is involved in a caspase-independent cell death pathway. Here we report that cell death in the single-celled organism Dictyostelium discoideum involves early disruption of mitochondrial transmembrane potential (DeltaPsim) that precedes the induction of several apoptosis-like features, including exposure of the phosphatidyl residues at the external surface of the plasma membrane, an intense vacuolization, a fragmentation of DNA into large fragments, an autophagy, and the release of apoptotic corpses that are engulfed by neighboring cells. We have cloned a Dictyostelium homolog of mammalian AIF that is localized into mitochondria and is translocated from the mitochondria to the cytoplasm and the nucleus after the onset of cell death. Cytoplasmic extracts from dying Dictyostelium cells trigger the breakdown of isolated mammalian and Dictyostelium nuclei in a cell-free system, and this process is inhibited by a polyclonal antibody specific for Dictyostelium discoideum apoptosis-inducing factor (DdAIF), suggesting that DdAIF is involved in DNA degradation during Dictyostelium cell death. Our findings indicate that the cell death pathway in Dictyostelium involves mitochondria and an AIF homolog, suggesting the evolutionary conservation of at least part of the cell death pathway in unicellular and multicellular organisms.     

Lipopolysaccharide induction of autophagy is associated with enhanced bactericidal activity in Dictyostelium discoideum

Innate immune cells respond to microbial invaders using pattern recognition receptors that detect conserved microbial patterns. Among the cellular processes stimulated downstream of pattern recognition machinery is the initiation of autophagy, which plays protective roles against intracellular microbes. We have shown recently that Dictyostelium discoideum, which takes up bacteria for nutritive purposes, may employ pattern recognition machinery to respond to bacterial prey, as D. discoideum cells upregulate bactericidal activity upon stimulation by lipopolysaccharide (LPS). Here we extend these findings, showing that LPS treatment leads to induction of autophagosomal maturation in cells responding to the bacteria Staphylococcus aureus. Cells treated with the autophagy-inducing drug rapamycin clear internalized bacteria at an accelerated rate, while LPS-enhanced clearance of bacteria is reduced in cells deficient for the autophagy-related genes atg1 and atg9. These findings link microbial pattern recognition with autophagy in the social amoeba D. discoideum.     

Role for YakA,cAMP, and protein kinase A in regulation of stress responses of Dictyostelium discoideum cells

The Dictyostelium protein kinase YakA is required for the growth-to-development transition. During growth YakA controls the cell cycle, regulating the intervals between cell divisions. When starved for nutrients Dictyostelium cells arrest growth and undergo changes in gene expression, decreasing vegetative mRNAs and inducing the expression of pkaC. YakA is an effector of these changes, being necessary for the decrease of vegetative mRNA expression and the increase of protein kinase A (PKA) activity that will ultimately regulate expression of adenylyl cyclase, cAMP synthesis, and the induction of development. We report a role for this kinase in the response to nitrosoative or oxidative stress of Dictyostelium cells. Hydrogen peroxide and sodium nitroprusside arrest the growth of cells and trigger cAMP synthesis and activation of PKA in a manner similar to the well-established response to nutrient starvation. We have found that yakA null cells are hypersensitive to nitrosoative/oxidative stress and that a second-site mutation in pkaC suppresses this sensitivity. The response to different stresses has been investigated and YakA, cAMP, and PKA have been identified as components of the pathway that regulate the growth arrest that follows treatment with compounds that generate reactive oxygen species. The effect of different types of stress was evaluated in Dictyostelium and the YakA/PKA pathway was also implicated in the response to heat stress.