bims-ecemfi Biomed News
on ECM and fibroblasts
Issue of 2024–04–28
fifteen papers selected by
Badri Narayanan Narasimhan, University of California, San Diego
  1. Viscoelastic Extracellular Matrix Enhances Epigenetic Remodeling and Cellular Plasticity.
  2. F-actin architecture determines the conversion of chemical energy into mechanical work.
  3. Active viscoelastic models for cell and tissue mechanics.
  4. Positive and negative durotaxis - mechanisms and emerging concepts.
  5. Confinement in fibrous environments positions and orients mitotic spindles.
  6. Programming viscoelastic properties in a complexation gel composite by utilizing entropy-driven topologically frustrated dynamical state.
  7. Stimulative piezoelectric nanofibrous scaffolds for enhanced small extracellular vesicle production in 3D cultures.
  8. 3-Dimensional Hydrogel Culture System Recapitulates Key Tuberculosis Phenotypes and Demonstrates Pyrazinamide Efficacy.
  9. The actin binding sites of talin have both distinct and complementary roles in cell-ECM adhesion.
  10. A mechanobiological model of bone metastasis reveals that mechanical stimulation inhibits the pro-osteolytic effects of breast cancer cells.
  11. Open-Top Patterned Hydrogel-Laden 3D Glioma Cell Cultures for Creation of Dynamic Chemotactic Gradients to Direct Cell Migration.
  12. Mechanical stress through growth on stiffer substrates impacts animal health and longevity in C. elegans.
  13. Alteration of mechanical stresses in the murine brain by age and hemorrhagic stroke.
  14. Model-Based Design of Variable Stiffness Soft Gripper Actuated by Smart Hydrogels.
  15. Optogenetically engineered calcium oscillations promote autophagy-mediated cell death via AMPK activation.
  1. bioRxiv. 2024 Apr 17. pii: 2024.04.14.589442. [Epub ahead of print]
    Viscoelastic Extracellular Matrix Enhances Epigenetic Remodeling and Cellular Plasticity.
    Yifan Wu, Yang Song, Jennifer Soto, Tyler Hoffman, Aaron Zhang, Xiao Han, Zhiwei Fang, Joon Eoh, Luo Gu, Zhen Gu, Song Li.
      Living tissue and extracellular matrices possess viscoelastic properties, but understanding how viscoelastic matrix regulates chromatin and the epigenome is limited. Here, we find that the regulation of the epigenetic state by the viscoelastic matrix is more pronounced on softer matrices. Cells on viscoelastic matrices exhibit larger nuclei, increased nuclear lamina ruffling, loosely organized chromatin, and faster chromatin dynamics, compared to those on elastic matrices. These changes are accompanied by a global increase in euchromatic marks and a local increase in chromatin accessibility at the cis -regulatory elements associated with neuronal and pluripotent genes. Consequently, viscoelastic matrices enhanced the efficiency of reprogramming fibroblasts into neurons and induced pluripotent stem cells, respectively. Together, our findings demonstrate the key roles of matrix viscoelasticity in the regulation of epigenetic state, and uncover a new mechanism of biophysical regulation of chromatin and cell reprogramming, with implications for the design of smart materials to engineer cell fate.
    DOI:  https://doi.org/10.1101/2024.04.14.589442
  2. Nat Commun. 2024 Apr 24. 15(1): 3444
    F-actin architecture determines the conversion of chemical energy into mechanical work.
    Ryota Sakamoto, Michael P Murrell.
      Mechanical work serves as the foundation for dynamic cellular processes, ranging from cell division to migration. A fundamental driver of cellular mechanical work is the actin cytoskeleton, composed of filamentous actin (F-actin) and myosin motors, where force generation relies on adenosine triphosphate (ATP) hydrolysis. F-actin architectures, whether bundled by crosslinkers or branched via nucleators, have emerged as pivotal regulators of myosin II force generation. However, it remains unclear how distinct F-actin architectures impact the conversion of chemical energy to mechanical work. Here, we employ in vitro reconstitution of distinct F-actin architectures with purified components to investigate their influence on myosin ATP hydrolysis (consumption). We find that F-actin bundles composed of mixed polarity F-actin hinder network contraction compared to non-crosslinked network and dramatically decelerate ATP consumption rates. Conversely, linear-nucleated networks allow network contraction despite reducing ATP consumption rates. Surprisingly, branched-nucleated networks facilitate high ATP consumption without significant network contraction, suggesting that the branched network dissipates energy without performing work. This study establishes a link between F-actin architecture and myosin energy consumption, elucidating the energetic principles underlying F-actin structure formation and the performance of mechanical work.
    DOI:  https://doi.org/10.1038/s41467-024-47593-x
  3. R Soc Open Sci. 2024 Apr;11(4): 231074
    Active viscoelastic models for cell and tissue mechanics.
    Bahareh Tajvidi Safa, Changjin Huang, Alexandre Kabla, Ruiguo Yang.
      Living cells are out of equilibrium active materials. Cell-generated forces are transmitted across the cytoskeleton network and to the extracellular environment. These active force interactions shape cellular mechanical behaviour, trigger mechano-sensing, regulate cell adaptation to the microenvironment and can affect disease outcomes. In recent years, the mechanobiology community has witnessed the emergence of many experimental and theoretical approaches to study cells as mechanically active materials. In this review, we highlight recent advancements in incorporating active characteristics of cellular behaviour at different length scales into classic viscoelastic models by either adding an active tension-generating element or adjusting the resting length of an elastic element in the model. Summarizing the two groups of approaches, we will review the formulation and application of these models to understand cellular adaptation mechanisms in response to various types of mechanical stimuli, such as the effect of extracellular matrix properties and external loadings or deformations.
    Keywords:  active model; cell mechanics; cell modeling; tissue mechanics; viscoelasticity
    DOI:  https://doi.org/10.1098/rsos.231074
  4. J Cell Sci. 2024 Apr 15. pii: jcs261919. [Epub ahead of print]137(8):
    Positive and negative durotaxis - mechanisms and emerging concepts.
    Mathilde Mathieu, Aleksi Isomursu, Johanna Ivaska.
      Cell migration is controlled by the coordinated action of cell adhesion, cytoskeletal dynamics, contractility and cell extrinsic cues. Integrins are the main adhesion receptors to ligands of the extracellular matrix (ECM), linking the actin cytoskeleton to the ECM and enabling cells to sense matrix rigidity and mount a directional cell migration response to stiffness gradients. Most models studied show preferred migration of single cells or cell clusters towards increasing rigidity. This is referred to as durotaxis, and since its initial discovery in 2000, technical advances and elegant computational models have provided molecular level details of stiffness sensing in cell migration. However, modeling has long predicted that, depending on cell intrinsic factors, such as the balance of cell adhesion molecules (clutches) and the motor proteins pulling on them, cells might also prefer adhesion to intermediate rigidity. Recently, experimental evidence has supported this notion and demonstrated the ability of cells to migrate towards lower rigidity, in a process called negative durotaxis. In this Review, we discuss the significant conceptual advances that have been made in our appreciation of cell plasticity and context dependency in stiffness-guided directional cell migration.
    Keywords:  Cell adhesion; Cell migration; Cytoskeleton; Durotaxis; Mechanobiology; Mechanotransduction
    DOI:  https://doi.org/10.1242/jcs.261919
  5. bioRxiv. 2024 Apr 15. pii: 2024.04.12.589246. [Epub ahead of print]
    Confinement in fibrous environments positions and orients mitotic spindles.
    Apurba Sarkar, Aniket Jana, Atharva Agashe, Ji Wang, Rakesh Kapania, Nir S Gov, Jennifer G DeLuca, Raja Paul, Amrinder S Nain.
      Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Adherent mitotic cells encounter confinement from neighboring cells or the extracellular matrix (ECM), which can cause rotation of mitotic spindles and, consequently, titling of the metaphase plate (MP). To understand the positioning and orientation of mitotic spindles under confinement by fibers (ECM-confinement), we use flexible ECM-mimicking nanofibers that allow natural rounding of the cell body while confining it to differing levels. Rounded mitotic bodies are anchored in place by actin retraction fibers (RFs) originating from adhesion clusters on the ECM-mimicking fibers. We discover the extent of ECM-confinement patterns RFs in 3D: triangular and band-like at low and high confinement, respectively. A stochastic Monte-Carlo simulation of the centrosome (CS), chromosome (CH), membrane interactions, and 3D arrangement of RFs on the mitotic body recovers MP tilting trends observed experimentally. Our mechanistic analysis reveals that the 3D shape of RFs is the primary driver of the MP rotation. Under high ECM-confinement, the fibers can mechanically pinch the cortex, causing the MP to have localized deformations at contact sites with fibers. Interestingly, high ECM-confinement leads to low and high MP tilts, which mechanistically depend upon the extent of cortical deformation, RF patterning, and MP position. We identify that cortical deformation and RFs work in tandem to limit MP tilt, while asymmetric positioning of MP leads to high tilts. Overall, we provide fundamental insights into how mitosis may proceed in fibrous ECM-confining microenvironments in vivo.
    DOI:  https://doi.org/10.1101/2024.04.12.589246
  6. Nat Commun. 2024 Apr 26. 15(1): 3569
    Programming viscoelastic properties in a complexation gel composite by utilizing entropy-driven topologically frustrated dynamical state.
    Gui Kang Wang, Yi Ming Yang, Di Jia.
      Hydrogel composites in an aqueous media with viscoelastic properties and elastic modulus that can be precisely tailored are desirable to mimic many biological tissues ranging from mucus, vitreous humor, and nucleus pulposus as well as build up biosensors. Without altering the chemistry, tuning the physical interactions and structures to govern the viscoelastic properties of the hydrogels is indispensable for their applications but quite limited. Here we design a complexation gel composite and utilize the physical principle of topologically frustrated dynamical state to tune the correlated structures between the guest polycation chains and negatively charged host gels. We precisely quantify the mesh size of the host gel and guest chain size. By designing various topologically correlated structures, a viscoelastic moduli map can be built up, ranging from tough to ultrasoft, and from elastic-like with low damping properties to viscous-like with high damping properties. We also tune the swelling ratio by using entropy effect and discover an Entropy-driven Topologically Isovolumetric Point. Our findings provide essential physics to understand the relationship between entropy-driven correlated structures and their viscoelastic properties of the complexation hydrogel composites and will have diverse applications in tissue engineering and soft biomaterials.
    DOI:  https://doi.org/10.1038/s41467-024-47969-z
  7. bioRxiv. 2024 Apr 15. pii: 2024.04.12.589114. [Epub ahead of print]
    Stimulative piezoelectric nanofibrous scaffolds for enhanced small extracellular vesicle production in 3D cultures.
    James Johnston, Hyunsu Jeon, Yun Young Choi, Gaeun Kim, Hsueh-Chia Chang, Nosang Vincent Myung, Yichun Wang.
      Small extracellular vesicles (sEVs) have great promise as effective carriers for drug delivery. However, the challenges associated with the efficient production of sEVs hinder their clinical applications. Herein, we report a stimulative 3D culture platform for enhanced sEV production. The proposed platform consists of a piezoelectric nanofibrous scaffold (PES) coupled with acoustic stimulation to enhance sEV production of cells in a 3D biomimetic microenvironment. Combining cell stimulation with a 3D culture platform in this stimulative PES enables a 49 fold increase in the production rate per cell with minimal deviations in particle size and protein composition compared with standard 2D cultures. We find that the enhanced sEV production is attributable to the activation and upregulation of crucial sEV production steps through the synergistic effect of stimulation and the 3D microenvironment. Moreover, changes in cell morphology lead to cytoskeleton redistribution through cell matrix interactions in the 3D cultures. This in turn facilitates intracellular EV trafficking, which impacts the production rate. Overall, our work provides a promising 3D cell culture platform based on piezoelectric biomaterials for enhanced sEV production. This platform is expected to accelerate the potential use of sEVs for drug delivery and broad biomedical applications.
    DOI:  https://doi.org/10.1101/2024.04.12.589114
  8. Adv Healthc Mater. 2024 Apr 24. e2304299
    3-Dimensional Hydrogel Culture System Recapitulates Key Tuberculosis Phenotypes and Demonstrates Pyrazinamide Efficacy.
    Vishal K Gupta, Vijaya V Vaishnavi, Mario L Arrieta-Ortiz, Abhirami P S, Jyothsna K M, Sharumathi Jeyasankar, Varun Raghunathan, Nitin S Baliga, Rachit Agarwal.
      The mortality caused by tuberculosis (TB) infections is a global concern, and there is a need to improve our understanding of the disease. Current in vitro infection models to study the disease have limitations, such as short investigation durations and divergent transcriptional signatures. This study aims to overcome these limitations by developing a 3D collagen culture system that mimics the biomechanical and extracellular matrix (ECM) of lung microenvironment (collagen fibers, stiffness comparable to in vivo conditions), as the infection primarily manifests in the lungs. The system incorporates Mycobacterium tuberculosis (Mtb) infected human THP-1 or primary monocytes/macrophages. Dual RNA sequencing revealed higher mammalian gene expression similarity with patient samples than 2D macrophage infections. Similarly, bacterial gene expression more accurately recapitulated in vivo gene expression patterns compared to bacteria in 2D infection models. Key phenotypes observed in humans, such as foamy macrophages and mycobacterial cords, were reproduced in the model. This biomaterial system overcomes challenges associated with traditional platforms by modulating immune cells and closely mimicking in vivo infection conditions, including showing efficacy with clinically relevant concentrations of anti-TB drug pyrazinamide, not seen in any other in vitro infection model, making it reliable and readily adoptable for tuberculosis studies and drug screening. This article is protected by copyright. All rights reserved.
    Keywords:  collagen hydrogels; cord formation; foamy macrophages; pyrazinamide
    DOI:  https://doi.org/10.1002/adhm.202304299
  9. PLoS Genet. 2024 Apr 25. 20(4): e1011224
    The actin binding sites of talin have both distinct and complementary roles in cell-ECM adhesion.
    Darius Camp, Bhavya Venkatesh, Veronika Solianova, Lorena Varela, Benjamin T Goult, Guy Tanentzapf.
      Cell adhesion requires linkage of transmembrane receptors to the cytoskeleton through intermediary linker proteins. Integrin-based adhesion to the extracellular matrix (ECM) involves large adhesion complexes that contain multiple cytoskeletal adapters that connect to the actin cytoskeleton. Many of these adapters, including the essential cytoskeletal linker Talin, have been shown to contain multiple actin-binding sites (ABSs) within a single protein. To investigate the possible role of having such a variety of ways of linking integrins to the cytoskeleton, we generated mutations in multiple actin binding sites in Drosophila talin. Using this approach, we have been able to show that different actin-binding sites in talin have both unique and complementary roles in integrin-mediated adhesion. Specifically, mutations in either the C-terminal ABS3 or the centrally located ABS2 result in lethality showing that they have unique and non-redundant function in some contexts. On the other hand, flies simultaneously expressing both the ABS2 and ABS3 mutants exhibit a milder phenotype than either mutant by itself, suggesting overlap in function in other contexts. Detailed phenotypic analysis of ABS mutants elucidated the unique roles of the talin ABSs during embryonic development as well as provided support for the hypothesis that talin acts as a dimer in in vivo contexts. Overall, our work highlights how the ability of adhesion complexes to link to the cytoskeleton in multiple ways provides redundancy, and consequently robustness, but also allows a capacity for functional specialization.
    DOI:  https://doi.org/10.1371/journal.pgen.1011224
  10. Cell Rep. 2024 Apr 18. pii: S2211-1247(24)00371-1. [Epub ahead of print]43(5): 114043
    A mechanobiological model of bone metastasis reveals that mechanical stimulation inhibits the pro-osteolytic effects of breast cancer cells.
    Vatsal Kumar, Syeda M Naqvi, Anneke Verbruggen, Eoin McEvoy, Laoise M McNamara.
      Bone is highly susceptible to cancer metastasis, and both tumor and bone cells enable tumor invasion through a "vicious cycle" of biochemical signaling. Tumor metastasis into bone also alters biophysical cues to both tumor and bone cells, which are highly sensitive to their mechanical environment. However, the mechanobiological feedback between these cells that perpetuate this cycle has not been studied. Here, we develop highly advanced in vitro and computational models to provide an advanced understanding of how tumor growth is regulated by the synergistic influence of tumor-bone cell signaling and mechanobiological cues. In particular, we develop a multicellular healthy and metastatic bone model that can account for physiological mechanical signals within a custom bioreactor. These models successfully recapitulated mineralization, mechanobiological responses, osteolysis, and metastatic activity. Ultimately, we demonstrate that mechanical stimulus provided protective effects against tumor-induced osteolysis, confirming the importance of mechanobiological factors in bone metastasis development.
    Keywords:  CP: Cancer; bone metastasis; computational models; in vitro models; mechanobiology; multicellular models; osteoblasts; osteoclastogenesis; osteolysis; tumor growth; vicious cycle
    DOI:  https://doi.org/10.1016/j.celrep.2024.114043
  11. ACS Biomater Sci Eng. 2024 Apr 23.
    Open-Top Patterned Hydrogel-Laden 3D Glioma Cell Cultures for Creation of Dynamic Chemotactic Gradients to Direct Cell Migration.
    Aditya Rane, Steven Tate, Jenna L Sumey, Qing Zhong, Hui Zong, Benjamin Purow, Steven R Caliari, Nathan S Swami.
      The laminar flow profiles in microfluidic systems coupled to rapid diffusion at flow streamlines have been widely utilized to create well-controlled chemical gradients in cell cultures for spatially directing cell migration. However, within hydrogel-based closed microfluidic systems of limited depth (≤0.1 mm), the biomechanical cues for the cell culture are dominated by cell interactions with channel surfaces rather than with the hydrogel microenvironment. Also, leaching of poly(dimethylsiloxane) (PDMS) constituents in closed systems and the adsorption of small molecules to PDMS alter chemotactic profiles. To address these limitations, we present the patterning and integration of a PDMS-free open fluidic system, wherein the cell-laden hydrogel directly adjoins longitudinal channels that are designed to create chemotactic gradients across the 3D culture width, while maintaining uniformity across its ∼1 mm depth to enhance cell-biomaterial interactions. This hydrogel-based open fluidic system is assessed for its ability to direct migration of U87 glioma cells using a hybrid hydrogel that includes hyaluronic acid (HA) to mimic the brain tumor microenvironment and gelatin methacrylate (GelMA) to offer the adhesion motifs for promoting cell migration. Chemotactic gradients to induce cell migration across the hydrogel width are assessed using the chemokine CXCL12, and its inhibition by AMD3100 is validated. This open-top hydrogel-based fluidic system to deliver chemoattractant cues over square-centimeter-scale areas and millimeter-scale depths can potentially serve as a robust screening platform to assess emerging glioma models and chemotherapeutic agents to eradicate them.
    Keywords:  cell migration; glioma; hydrogel; microfluidics; tumor microenvironment
    DOI:  https://doi.org/10.1021/acsbiomaterials.4c00041
  12. bioRxiv. 2024 Apr 12. pii: 2024.04.11.589121. [Epub ahead of print]
    Mechanical stress through growth on stiffer substrates impacts animal health and longevity in C. elegans.
    Maria Oorloff, Adam Hruby, Maxim Averbukh, Athena Alcala, Naibedya Dutta, Toni Castro Torres, Darius Moaddeli, Matthew Vega, Juri Kim, Andrew Bong, Aeowynn J Coakley, Daniel Hicks, Jing Wang, Tiffany Wang, Sally Hoang, Kevin M Tharp, Gilberto Garcia, Ryo Higuchi-Sanabria.
      Mechanical stress is a measure of internal resistance exhibited by a body or material when external forces, such as compression, tension, bending, etc. are applied. The study of mechanical stress on health and aging is a continuously growing field, as major changes to the extracellular matrix and cell-to-cell adhesions can result in dramatic changes to tissue stiffness during aging and diseased conditions. For example, during normal aging, many tissues including the ovaries, skin, blood vessels, and heart exhibit increased stiffness, which can result in a significant reduction in function of that organ. As such, numerous model systems have recently emerged to study the impact of mechanical and physical stress on cell and tissue health, including cell-culture conditions with matrigels and other surfaces that alter substrate stiffness and ex vivo tissue models that can apply stress directly to organs like muscle or tendons. Here, we sought to develop a novel method in an in vivo, model organism setting to study the impact of mechanical stress on aging, by increasing substrate stiffness in solid agar medium of C. elegans. To our surprise, we found shockingly limited impact of growth of C. elegans on stiffer substrates, including limited effects on cellular health, gene expression, organismal health, stress resilience, and longevity. Overall, our studies reveal that altering substrate stiffness of growth medium for C. elegans have only mild impact on animal health and longevity; however, these impacts were not nominal and open up important considerations for C. elegans biologists in standardizing agar medium choice for experimental assays.
    Keywords:  Aging; ER; actin; mitochondria; stress
    DOI:  https://doi.org/10.1101/2024.04.11.589121
  13. PNAS Nexus. 2024 Apr;3(4): pgae141
    Alteration of mechanical stresses in the murine brain by age and hemorrhagic stroke.
    Siyi Zheng, Rohin Banerji, Rob LeBourdais, Sue Zhang, Eric DuBois, Timothy O'Shea, Hadi T Nia.
      Residual mechanical stresses, also known as solid stresses, emerge during rapid differential growth or remodeling of tissues, as observed in morphogenesis and tumor growth. While residual stresses typically dissipate in most healthy adult organs, as the growth rate decreases, high residual stresses have been reported in mature, healthy brains. However, the origins and consequences of residual mechanical stresses in the brain across health, aging, and disease remain poorly understood. Here, we utilized and validated a previously developed method to map residual mechanical stresses in the brains of mice across three age groups: 5-7 days, 8-12 weeks, and 22 months. We found that residual solid stress rapidly increases from 5-7 days to 8-12 weeks and remains high in mature 22 months mice brains. Three-dimensional mapping revealed unevenly distributed residual stresses from the anterior to posterior coronal brain sections. Since the brain is rich in negatively charged hyaluronic acid, we evaluated the contribution of charged extracellular matrix (ECM) constituents in maintaining solid stress levels. We found that lower ionic strength leads to elevated solid stresses, consistent with its unshielding effect and the subsequent expansion of charged ECM components. Lastly, we demonstrated that hemorrhagic stroke, accompanied by loss of cellular density, resulted in decreased residual stress in the murine brain. Our findings contribute to a better understanding of spatiotemporal alterations of residual solid stresses in healthy and diseased brains, a crucial step toward uncovering the biological and immunological consequences of this understudied mechanical phenotype in the brain.
    Keywords:  age-related alteration; hemorrhagic stroke; mouse brain; residual solid stress
    DOI:  https://doi.org/10.1093/pnasnexus/pgae141
  14. Soft Robot. 2024 Apr 25.
    Model-Based Design of Variable Stiffness Soft Gripper Actuated by Smart Hydrogels.
    Qianyi Chen, Dingena Schott, Jovana Jovanova.
      Soft grippers have shown their ability to grasp fragile and irregularly shaped objects, but they often require external mechanisms for actuation, limiting their use in large-scale situations. Their limited capacity to handle loads and deformations also restricts their customized grasping capabilities. To address these issues, a model-based soft gripper with adaptable stiffness was proposed. The proposed actuator comprises a silicone chamber with separate units containing hydrogel spheres. These spheres exhibit temperature-triggered swelling and shrinking behaviors. In addition, variable stiffness strips embedded in the units are introduced as the stiffness variation method. The validated finite element method model was used as the model-based design approach to describe the hydrogel behaviors and explore the affected factors on the bending performance. The results demonstrate that the actuator can be programmed to respond in a desired way, and the stiffness variation method enhances bending stiffness significantly. Specifically, a direct correlation exists between the bending angle and hydrogel sphere layers, with a maximum of 128° achieved. In addition, incorporating gap configurations into the chamber membrane results in a maximum threefold increase in the bending angle. Besides, the membrane type minimally impacts the bending angle from 21.3° to 24.6°. In addition, the embedded variable stiffness strips substantially increase stiffness, resulting in a 30-fold rise in bending stiffness. In conclusion, the novel soft gripper actuator enables substantial bending and stiffness control through active actuation, showcasing the potential for enhancing soft gripper performance in complex and multiscale grasping scenarios.
    Keywords:  FEM; model-based design; soft gripper; stiffness variation; temperature-sensitive hydrogel
    DOI:  https://doi.org/10.1089/soro.2023.0185
  15. Open Biol. 2024 Apr;14(4): 240001
    Optogenetically engineered calcium oscillations promote autophagy-mediated cell death via AMPK activation.
    Yi-Shyun Lai, Meng-Ru Hsieh, Thi My Hang Nguyen, Ying-Chi Chen, Hsueh-Chun Wang, Wen-Tai Chiu.
      Autophagy is a double-edged sword for cells; it can lead to both cell survival and death. Calcium (Ca2+) signalling plays a crucial role in regulating various cellular behaviours, including cell migration, proliferation and death. In this study, we investigated the effects of modulating cytosolic Ca2+ levels on autophagy using chemical and optogenetic methods. Our findings revealed that ionomycin and thapsigargin induce Ca2+ influx to promote autophagy, whereas the Ca2+ chelator BAPTA-AM induces Ca2+ depletion and inhibits autophagy. Furthermore, the optogenetic platform allows the manipulation of illumination parameters, including density, frequency, duty cycle and duration, to create different patterns of Ca2+ oscillations. We used the optogenetic tool Ca2+-translocating channelrhodopsin, which is activated and opened by 470 nm blue light to induce Ca2+ influx. These results demonstrated that high-frequency Ca2+ oscillations induce autophagy. In addition, autophagy induction may involve Ca2+-activated adenosine monophosphate (AMP)-activated protein kinases. In conclusion, high-frequency optogenetic Ca2+ oscillations led to cell death mediated by AMP-activated protein kinase-induced autophagy.
    Keywords:  AMPK; autophagy; calcium; cell death; optogenetics
    DOI:  https://doi.org/10.1098/rsob.240001
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