Components and Mechanisms of Nuclear Mechanotransduction.

The cell nucleus is best known as the container of the genome. Its envelope provides a barrier for passive macromolecule diffusion, which enhances the control of gene expression. As its largest and stiffest organelle, the nucleus also defines the minimal space requirements of a cell. Internal or external pressures that deform a cell to its physical limits cause a corresponding nuclear deformation. Evidence is consolidating that the nucleus, in addition to its genetic functions, serves as a physical sensing device for critical cell body deformation. Nuclear mechanotransduction allows cells to adapt their acute behaviors, mechanical stability, paracrine signaling, and fate to their physical surroundings. This review summarizes the basic chemical and mechanical properties of nuclear components, and how these properties are thought to be utilized for mechanosensing.

The Cell Nucleus Serves as a Mechanotransducer of Tissue Damage-Induced Inflammation.

Tissue damage activates cytosolic phospholipase A2 (cPLA2), releasing arachidonic acid (AA), which is oxidized to proinflammatory eicosanoids by 5-lipoxygenase (5-LOX) on the nuclear envelope. How tissue damage is sensed to activate cPLA2 is unknown. We investigated this by live imaging in wounded zebrafish larvae, where damage of the fin tissue causes osmotic cell swelling at the wound margin and the generation of a chemotactic eicosanoid signal. Osmotic swelling of cells and their nuclei activates cPla2 by translocating it from the nucleoplasm to the nuclear envelope. Elevated cytosolic Ca(2+) was necessary but not sufficient for cPla2 translocation, and nuclear swelling was required in parallel. cPla2 translocation upon nuclear swelling was reconstituted in isolated nuclei and appears to be a simple physical process mediated by tension in the nuclear envelope. Our data suggest that the nucleus plays a mechanosensory role in inflammation by transducing cell swelling and lysis into proinflammatory eicosanoid signaling.

Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish.

Tissue damage and infection are deemed likewise triggers of innate immune responses. But whereas neutrophil responses to microbes are generally protective, neutrophil recruitment into damaged tissues without infection is deleterious. Why neutrophils respond to tissue damage and not just to microbes is unknown. Is it a flaw of the innate immune system that persists because evolution did not select against it, or does it provide a selective advantage? Here we dissect the contribution of tissue damage signaling to antimicrobial immune responses in a live vertebrate. By intravital imaging of zebrafish larvae, a powerful model for innate immunity, we show that prevention of tissue damage signaling upon microbial ear infection abrogates leukocyte chemotaxis and reduces animal survival, at least in part, through suppression of cytosolic phospholipase A2 (cPla2), which integrates tissue damage- and microbe-derived cues. Thus, microbial cues are insufficient, and damage signaling is essential for antimicrobial neutrophil responses in zebrafish.

A synergy between mechanosensitive calcium- and membrane-binding mediates tension-sensing by C2-like domains.

When nuclear membranes are stretched, the peripheral membrane enzyme cytosolic phospholipase A2 (cPLA2) binds via its calcium-dependent C2 domain (cPLA2-C2) and initiates bioactive lipid signaling and tissue inflammation. More than 150 C2-like domains are encoded in vertebrate genomes. How many of them are mechanosensors and quantitative relationships between tension and membrane recruitment remain unexplored, leaving a knowledge gap in the mechanotransduction field. In this study, we imaged the mechanosensitive adsorption of cPLA2 and its C2 domain to nuclear membranes and artificial lipid bilayers, comparing it to related C2-like motifs. Stretch increased the Ca2+ sensitivity of all tested domains, promoting half-maximal binding of cPLA2 at cytoplasmic resting-Ca2+ concentrations. cPLA2-C2 bound up to 50 times tighter to stretched than to unstretched membranes. Our data suggest that a synergy of mechanosensitive Ca2+ interactions and deep, hydrophobic membrane insertion enables cPLA2-C2 to detect stretched membranes with antibody-like affinity, providing a quantitative basis for understanding mechanotransduction by C2-like domains.

Wound redox gradients revisited.

Evidence emerges that redox gradients regulate morphogenesis, inflammation, regeneration, and healing of tissues. At the example of redox signaling during the zebrafish wound response, I briefly discuss current ideas on how such patterns might be sensed and spatially regulated to guide physiological processes over distances in animals.

Do not scratch that mole!

Nevi harbor some of the same oncogene mutations that also drive malign melanoma. Further tumor promoting events are required to unleash their carcinogenic potential. Using zebrafish whose melanocytes overexpress an HRAS-oncogene, a new study reports that injury induces melanoma, possibly through recruitment of neutrophils that trigger proliferation of preneoplastic melanocytes.

Image-Based Measurement of H2O2 Reaction-Diffusion in Wounded Zebrafish Larvae.

Epithelial injury induces rapid recruitment of antimicrobial leukocytes to the wound site. In zebrafish larvae, activation of the epithelial NADPH oxidase Duox at the wound margin is required early during this response. Before injury, leukocytes are near the vascular region, that is, ∼100-300 µm away from the injury site. How Duox establishes long-range signaling to leukocytes is unclear. We conceived that extracellular hydrogen peroxide (H2O2) generated by Duox diffuses through the tissue to directly regulate chemotactic signaling in these cells. But before it can oxidize cellular proteins, H2O2 must get past the antioxidant barriers that protect the cellular proteome. To test whether, or on which length scales this occurs during physiological wound signaling, we developed a computational method based on reaction-diffusion principles that infers H2O2 degradation rates from intravital H2O2-biosensor imaging data. Our results indicate that at high tissue H2O2 levels the peroxiredoxin-thioredoxin antioxidant chain becomes overwhelmed, and H2O2 degradation stalls or ceases. Although the wound H2O2 gradient reaches deep into the tissue, it likely overcomes antioxidant barriers only within ∼30 µm of the wound margin. Thus, Duox-mediated long-range signaling may require other spatial relay mechanisms besides extracellular H2O2 diffusion.

A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish.

Barrier structures (for example, epithelia around tissues and plasma membranes around cells) are required for internal homeostasis and protection from pathogens. Wound detection and healing represent a dormant morphogenetic program that can be rapidly executed to restore barrier integrity and tissue homeostasis. In animals, initial steps include recruitment of leukocytes to the site of injury across distances of hundreds of micrometres within minutes of wounding. The spatial signals that direct this immediate tissue response are unknown. Owing to their fast diffusion and versatile biological activities, reactive oxygen species, including hydrogen peroxide (H(2)O(2)), are interesting candidates for wound-to-leukocyte signalling. Here we probe the role of H(2)O(2) during the early events of wound responses in zebrafish larvae expressing a genetically encoded H(2)O(2) sensor. This reporter revealed a sustained rise in H(2)O(2) concentration at the wound margin, starting approximately 3 min after wounding and peaking at approximately 20 min, which extended approximately 100-200 microm into the tail-fin epithelium as a decreasing concentration gradient. Using pharmacological and genetic inhibition, we show that this gradient is created by dual oxidase (Duox), and that it is required for rapid recruitment of leukocytes to the wound. This is the first observation, to our knowledge, of a tissue-scale H(2)O(2) pattern, and the first evidence that H(2)O(2) signals to leukocytes in tissues, in addition to its known antiseptic role.

Maintenance of mitochondrial oxygen homeostasis by cosubstrate compensation.

Mitochondria maintain a constant rate of aerobic respiration over a wide range of oxygen levels. However, the control strategies underlying oxygen homeostasis are still unclear. Using mathematical modeling, we found that the mitochondrial electron transport chain (ETC) responds to oxygen level changes by undergoing compensatory changes in reduced electron carrier levels. This emergent behavior, which we named cosubstrate compensation (CSC), enables the ETC to maintain homeostasis over a wide of oxygen levels. When performing CSC, our ETC models recapitulated a classic scaling relationship discovered by Chance [Chance B (1965) J. Gen. Physiol. 49:163-165] relating the extent of oxygen homeostasis to the kinetics of mitochondrial electron transport. Analysis of an in silico mitochondrial respiratory system further showed evidence that CSC constitutes the dominant control strategy for mitochondrial oxygen homeostasis during active respiration. Our findings indicate that CSC constitutes a robust control strategy for homeostasis and adaptation in cellular biochemical networks.

Oxoeicosanoid signaling mediates early antimicrobial defense in zebrafish.

5-oxoETE is a bioactive lipid derived from arachidonic acid generated when phospholipase A2 activation coincides with oxidative stress. Through its G protein-coupled receptor OXER1, pure 5-oxoETE is a potent leukocyte chemoattractant. Yet, its physiological function has remained elusive owing to the unusual OXER1 conservation pattern. OXER1 is conserved from fish to primates but not in rodents, precluding genetic loss-of-function studies in mouse. To determine its physiological role, we combine transcriptomic, lipidomic, and intravital imaging assays with genetic perturbations of the OXER1 ortholog hcar1-4 in zebrafish. Pseudomonas aeruginosa infection induces the synthesis of 5-oxoETE and its receptor, along with other inflammatory pathways. Hcar1-4 deletion attenuates neutrophil recruitment and decreases post-infection survival, which could be rescued by ectopic expression of hcar1-4 or human OXER1. By revealing 5-oxoETE as dominant lipid regulator of the early antimicrobial response in a non-rodent vertebrate, our work expands the current, rodent-centric view of early inflammation.

The yellow brick road to nuclear membrane mechanotransduction.

The nuclear membrane may function as a mechanosensory surface alongside the plasma membrane. In this Review, we discuss how this idea emerged, where it currently stands, and point out possible implications, without any claim of comprehensiveness.

Spatial patterning of metabolism by mitochondria, oxygen, and energy sinks in a model cytoplasm.

Metabolite gradients might guide mitochondrial localization in cells and angiogenesis in tissues. It is unclear whether they can exist in single cells, because the length scale of most cells is small compared to the expected diffusion times of metabolites. For investigation of metabolic gradients, we need experimental systems in which spatial patterns of metabolism can be systematically measured and manipulated. We used concentrated cytoplasmic extracts from Xenopus eggs as a model cytoplasm, and visualized metabolic gradients formed in response to spatial stimuli. Restriction of oxygen supply to the edge of a drop mimicked distance to the surface of a single cell, or distance from a blood vessel in tissue. We imaged a step-like increase of Nicotinamide adenine dinucleotide (NAD) reduction approximately 600 microm distant from the oxygen source. This oxic-anoxic switch was preceded on the oxic side by a gradual rise of mitochondrial transmembrane potential (Deltapsi) and reactive oxygen species (ROS) production, extending over approximately 600 microm and approximately 300 microm, respectively. Addition of Adenosine triphosphate (ATP)-consuming beads mimicked local energy sinks in the cell. We imaged Deltapsi gradients with a decay length of approximately 50-300 microm around these beads, in the first visualization of an energy demand signaling gradient. Our study demonstrates that mitochondria can pattern the cytoplasm over length scales that are suited to convey morphogenetic information in large cells and tissues and provides a versatile model system for probing of the formation and function of metabolic gradients.

Gradients in the self-organization of the mitotic spindle.

Recent evidence points at a role of protein interaction gradients around chromatin in mitotic spindle morphogenesis in large eukaryotic cells. Here, we explain how gradients can arise over distances of tens of microns around supramolecular structures from mixtures of soluble molecules. We discuss how coupled sets of such reaction diffusion processes generate the spatial information that determines the local dynamics of microtubules required to form a bipolar spindle. We argue that such reaction diffusion processes are involved in the self-organization of supramolecular structures in the cell.

Discrete states of a protein interaction network govern interphase and mitotic microtubule dynamics.

The cytoplasm of eukaryotic cells is thought to adopt discrete "states" corresponding to different steady states of protein networks that govern changes in subcellular organization. For example, in Xenopus eggs, the interphase to mitosis transition is induced solely by activation of cyclin-dependent kinase 1 (CDK1) that phosphorylates many proteins leading to a reorganization of the nucleus and assembly of the mitotic spindle. Among these changes, the large array of stable microtubules that exists in interphase is replaced by short, highly dynamic microtubules in metaphase. Using a new visual immunoprecipitation assay that quantifies pairwise protein interactions in a non-perturbing manner in Xenopus egg extracts, we reveal the existence of a network of interactions between a series of microtubule-associated proteins (MAPs). In interphase, tubulin interacts with XMAP215, which is itself interacting with XKCM1, which connects to APC, EB1, and CLIP170. In mitosis, tubulin interacts with XMAP215, which is connected to EB1. We show that in interphase, microtubules are stable because the catastrophe-promoting activity of XKCM1 is inhibited by its interactions with the other MAPs. In mitosis, microtubules are short and dynamic because XKCM1 is free and has a strong destabilizing activity. In this case, the interaction of XMAP215 with EB1 is required to counteract the strong activity of XKCM1. This provides the beginning of a biochemical description of the notion of "cytoplasmic states" regarding the microtubule system.

Lipid peroxidation regulates long-range wound detection through 5-lipoxygenase in zebrafish.

Rapid wound detection by distant leukocytes is essential for antimicrobial defence and post-infection survival1. The reactive oxygen species hydrogen peroxide and the polyunsaturated fatty acid arachidonic acid are among the earliest known mediators of this process2-4. It is unknown whether or how these highly conserved cues collaborate to achieve wound detection over distances of several hundreds of micrometres within a few minutes. To investigate this, we locally applied arachidonic acid and skin-permeable peroxide by micropipette perfusion to unwounded zebrafish tail fins. As in wounds, arachidonic acid rapidly attracted leukocytes through dual oxidase (Duox) and 5-lipoxygenase (Alox5a). Peroxide promoted chemotaxis to arachidonic acid without being chemotactic on its own. Intravital biosensor imaging showed that wound peroxide and arachidonic acid converged on half-millimetre-long lipid peroxidation gradients that promoted leukocyte attraction. Our data suggest that lipid peroxidation functions as a spatial redox relay that enables long-range detection of early wound cues by immune cells, outlining a beneficial role for this otherwise toxic process.

A Photo-clickable ATP-Mimetic Reveals Nucleotide Interactors in the Membrane Proteome.

ATP is an important energy metabolite and allosteric signal in health and disease. ATP-interacting proteins, such as P2 receptors, control inflammation, cell death, migration, and wound healing. However, identification of allosteric ATP sites remains challenging, and our current inventory of ATP-controlled pathways is likely incomplete. Here, we develop and verify mipATP as a minimally invasive photoaffinity probe for ATP-interacting proteins. Its N6 functionalization allows target enrichment by UV crosslinking and conjugation to reporter tags by "click" chemistry. The additions are compact, allowing mipATP to completely retain the calcium signaling responses of native ATP in vitro and in vivo. mipATP specifically enriched for known nucleotide binders in A549 cell lysates and membrane fractions. In addition, it retrieved unannotated ATP interactors, such as the FAS receptor, CD44, and various SLC transporters. Thus, mipATP is a promising tool to identify allosteric ATP sites in the proteome.

Ferroptosis occurs through an osmotic mechanism and propagates independently of cell rupture.

Ferroptosis is a regulated form of necrotic cell death that is caused by the accumulation of oxidized phospholipids, leading to membrane damage and cell lysis1,2. Although other types of necrotic death such as pyroptosis and necroptosis are mediated by active mechanisms of execution3-6, ferroptosis is thought to result from the accumulation of unrepaired cell damage1. Previous studies have suggested that ferroptosis has the ability to spread through cell populations in a wave-like manner, resulting in a distinct spatiotemporal pattern of cell death7,8. Here we investigate the mechanism of ferroptosis execution and discover that ferroptotic cell rupture is mediated by plasma membrane pores, similarly to cell lysis in pyroptosis and necroptosis3,4. We further find that intercellular propagation of death occurs following treatment with some ferroptosis-inducing agents, including erastin2,9 and C' dot nanoparticles8, but not upon direct inhibition of the ferroptosis-inhibiting enzyme glutathione peroxidase 4 (GPX4)10. Propagation of a ferroptosis-inducing signal occurs upstream of cell rupture and involves the spreading of a cell swelling effect through cell populations in a lipid peroxide- and iron-dependent manner.

Cosignaling of NCAM via lipid rafts and the FGF receptor is required for neuritogenesis.

The neural cell adhesion molecule (NCAM) has been reported to stimulate neuritogenesis either via nonreceptor tyrosine kinases or fibroblast growth factor (FGF) receptor. Here we show that lipid raft association of NCAM is crucial for activation of the nonreceptor tyrosine kinase pathway and induction of neurite outgrowth. Transfection of hippocampal neurons of NCAM-deficient mice revealed that of the three major NCAM isoforms only NCAM140 can act as a homophilic receptor that induces neurite outgrowth. Disruption of NCAM140 raft association either by mutation of NCAM140 palmitoylation sites or by lipid raft destruction attenuates activation of the tyrosine focal adhesion kinase and extracellular signal-regulated kinase 1/2, completely blocking neurite outgrowth. Likewise, NCAM-triggered neurite outgrowth is also completely blocked by a specific FGF receptor inhibitor, indicating that cosignaling via raft-associated kinases and FGF receptor is essential for neuritogenesis.

Quantitative Imaging of Endogenous and Exogenous H2O2 Gradients in Live Zebrafish Larvae.

Quantitative aspects of extracellular H2O2 signaling in animals, such as its spatiotemporal dynamics within tissues, remain little understood. Here we detail an optimized, experimental setup for measuring the dynamics and physiological consequences of extracellular H2O2 application to live tissues by intravital biosensor imaging in zebrafish larvae.

Live imaging of leukocyte recruitment in a zebrafish model of chemical liver injury.

Studying early immune responses to organ damage in situ requires animal models amenable to intravital imaging. Here, we used transparent zebrafish larvae, a powerful animal model for innate immunity, to measure leukocyte recruitment to damaged livers. Bath application of metronidazole (Mtz) to fish expressing nitroreductase (NTR) under a liver-specific promoter damaged the organ within 24 hours causing oxidative stress, distorted liver morphology, accumulation of TUNEL-positive cells, and transcriptional upregulation of apoptotic and antioxidant genes. Inflammatory gene transcription in damaged hepatocytes was attenuated. In line with predominant apoptosis, macrophages were massively recruited into Mtz/NTR-damaged livers. By contrast, neutrophil infiltration was more variable and delayed, consistent with less abundant necrosis and an attenuated inflammatory capacity of damaged hepatocytes.