RESUMEN
The importance of mechanical force in biology is evident across diverse length scales, ranging from tissue morphogenesis during embryo development to mechanotransduction across single adhesion proteins at the cell surface. Consequently, many force measurement techniques rely on optical microscopy to measure forces being applied by cells on their environment, to visualize specimen deformations due to external forces, or even to directly apply a physical perturbation to the sample via photoablation or optogenetic tools. Recent developments in advanced microscopy offer improved approaches to enhance spatiotemporal resolution, imaging depth, and sample viability. These advances can be coupled with already existing force measurement methods to improve sensitivity, duration and speed, amongst other parameters. However, gaining access to advanced microscopy instrumentation and the expertise necessary to extract meaningful insights from these techniques is an unavoidable hurdle. In this Live Cell Imaging special issue Review, we survey common microscopy-based force measurement techniques and examine how they can be bolstered by emerging microscopy methods. We further explore challenges related to the accompanying data analysis in biomechanical studies and discuss the various resources available to tackle the global issue of technology dissemination, an important avenue for biologists to gain access to pre-commercial instruments that can be leveraged for biomechanical studies.
RESUMEN
Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.
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Regulación de la Expresión Génica/fisiología , Uniones Intercelulares/fisiología , Neutrófilos/fisiología , Animales , Línea Celular , Proteínas Fluorescentes Verdes , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Músculo Esquelético/fisiología , Músculo Esquelético/ultraestructuraRESUMEN
The shuttling of transcription factors and transcriptional regulators into and out of the nucleus is central to the regulation of many biological processes. Here we describe a new method for studying the rates of nuclear entry and exit of transcriptional regulators. A photo-responsive LOV (light-oxygen-voltage) domain from Avena sativa is used to sequester fluorescently labelled transcriptional regulators YAP1 and TAZ (also known as WWTR1) on the surface of mitochondria and to reversibly release them upon blue light illumination. After dissociation, fluorescent signals from the mitochondria, cytoplasm and nucleus are extracted by a bespoke app and used to generate rates of nuclear entry and exit. Using this method, we demonstrate that phosphorylation of YAP1 on canonical sites enhances its rate of nuclear export. Moreover, we provide evidence that, despite high intercellular variability, YAP1 import and export rates correlate within the same cell. By simultaneously releasing YAP1 and TAZ from sequestration, we show that their rates of entry and exit are correlated. Furthermore, combining the optogenetic release of YAP1 with lattice light-sheet microscopy reveals high heterogeneity of YAP1 dynamics within different cytoplasmic regions, demonstrating the utility and versatility of our tool to study protein dynamics. This article has an associated First Person interview with Anna M. Dowbaj, joint first author of the paper.
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Núcleo Celular , Optogenética , Transporte Activo de Núcleo Celular , Proteínas Adaptadoras Transductoras de Señales , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Proteínas Señalizadoras YAPRESUMEN
Recent technological advances have made microscopy indispensable in life science research. Its ubiquitous use, in turn, underscores the importance of ensuring that microscopy-based experiments are replicable and that the resulting data comparable. While there has been a wealth of review articles, practical guides and conferences devoted to the topic of maintaining standard instrument operating conditions, the paucity of attention dedicated to properly documenting microscopy experiments is undeniable. This lack of emphasis on accurate reporting extends beyond life science researchers themselves, to the review panels and editorial boards of many journals. Such oversight at the final step of communicating a scientific discovery can unfortunately negate the many valiant efforts made to ensure experimental quality control in the name of scientific reproducibility. This Review aims to enumerate the various parameters that should be reported in an imaging experiment by illustrating how their inconsistent application can lead to irreconcilable results.
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Microscopía , Reproducibilidad de los ResultadosRESUMEN
Symmetric cell division requires the even partitioning of genetic information and cytoplasmic contents between daughter cells. Whereas the mechanisms coordinating the segregation of the genome are well known, the processes that ensure organelle segregation between daughter cells remain less well understood1. Here we identify multiple actin assemblies with distinct but complementary roles in mitochondrial organization and inheritance in mitosis. First, we find a dense meshwork of subcortical actin cables assembled throughout the mitotic cytoplasm. This network scaffolds the endoplasmic reticulum and organizes three-dimensional mitochondrial positioning to ensure the equal segregation of mitochondrial mass at cytokinesis. Second, we identify a dynamic wave of actin filaments reversibly assembling on the surface of mitochondria during mitosis. Mitochondria sampled by this wave are enveloped within actin clouds that can spontaneously break symmetry to form elongated comet tails. Mitochondrial comet tails promote randomly directed bursts of movement that shuffle mitochondrial position within the mother cell to randomize inheritance of healthy and damaged mitochondria between daughter cells. Thus, parallel mechanisms mediated by the actin cytoskeleton ensure both equal and random inheritance of mitochondria in symmetrically dividing cells.
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Actinas/química , Actinas/metabolismo , Mitocondrias/metabolismo , Mitosis , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animales , División Celular , Línea Celular , Citocinesis , Retículo Endoplásmico/metabolismo , Hipocampo/citología , Hipocampo/embriología , Humanos , Mitocondrias/química , Neuronas , RatasRESUMEN
The genetic and metabolic heterogeneity of RAS-driven cancers has confounded therapeutic strategies in the clinic. To address this, rapid and genetically tractable animal models are needed that recapitulate the heterogeneity of RAS-driven cancers in vivo. Here, we generate a Drosophila melanogaster model of Ras/Lkb1 mutant carcinoma. We show that low-level expression of oncogenic Ras (RasLow) promotes the survival of Lkb1 mutant tissue, but results in autonomous cell cycle arrest and non-autonomous overgrowth of wild-type tissue. In contrast, high-level expression of oncogenic Ras (RasHigh) transforms Lkb1 mutant tissue resulting in lethal malignant tumors. Using simultaneous multiview light-sheet microcopy, we have characterized invasion phenotypes of Ras/Lkb1 tumors in living larvae. Our molecular analysis reveals sustained activation of the AMPK pathway in malignant Ras/Lkb1 tumors, and demonstrate the genetic and pharmacologic dependence of these tumors on CaMK-activated Ampk. We further show that LKB1 mutant human lung adenocarcinoma patients with high levels of oncogenic KRAS exhibit worse overall survival and increased AMPK activation. Our results suggest that high levels of oncogenic KRAS is a driving event in the malignant transformation of LKB1 mutant tissue, and uncovers a vulnerability that may be used to target this aggressive genetic subset of RAS-driven tumors.
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Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes ras , Mutación , Neoplasias Experimentales/genética , Proteínas Quinasas/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP/metabolismo , Adenocarcinoma del Pulmón/enzimología , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/mortalidad , Animales , Animales Modificados Genéticamente , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Muerte Celular , Movimiento Celular , Bases de Datos Genéticas , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/enzimología , Activación Enzimática , Predisposición Genética a la Enfermedad , Humanos , Larva/enzimología , Larva/genética , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidad , Invasividad Neoplásica , Neoplasias Experimentales/enzimología , Fenotipo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Filopodia are peripheral F-actin-rich structures that enable cell sensing of the microenvironment. Fascin is an F-actin-bundling protein that plays a key role in stabilizing filopodia to support efficient adhesion and migration. Fascin is also highly up-regulated in human cancers, where it increases invasive cell behavior and correlates with poor patient prognosis. Previous studies have shown that fascin phosphorylation can regulate F-actin bundling, and that this modification can contribute to subcellular fascin localization and function. However, the factors that regulate fascin dynamics within filopodia remain poorly understood. In the current study, we used advanced live-cell imaging techniques and a fascin biosensor to demonstrate that fascin phosphorylation, localization, and binding to F-actin are highly dynamic and dependent on local cytoskeletal architecture in cells in both 2D and 3D environments. Fascin dynamics within filopodia are under the control of formins, and in particular FMNL2, that binds directly to dephosphorylated fascin. Our data provide new insight into control of fascin dynamics at the nanoscale and into the mechanisms governing rapid cytoskeletal adaptation to environmental changes. This filopodia-driven exploration stage may represent an essential regulatory step in the transition from static to migrating cancer cells.
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Actinas/genética , Proteínas Portadoras/genética , Forminas/genética , Proteínas de Microfilamentos/genética , Neoplasias/genética , Seudópodos/genética , Técnicas Biosensibles , Proteínas Portadoras/aislamiento & purificación , Adhesión Celular/genética , Movimiento Celular/genética , Microambiente Celular/genética , Células HeLa , Humanos , Proteínas de Microfilamentos/aislamiento & purificación , Imagen Molecular , Neoplasias/patología , Fosforilación , Unión Proteica/genética , Seudópodos/metabolismoRESUMEN
Neutrophil and macrophage (MÏ) migration underpin the inflammatory response. However, the fast velocity, multidirectional instantaneous movement, and plastic, ever-changing shape of phagocytes confound high-resolution intravital imaging. Lattice lightsheet microscopy (LLSM) captures highly dynamic cell morphology at exceptional spatiotemporal resolution. We demonstrate the first extensive application of LLSM to leukocytes in vivo, utilizing optically transparent zebrafish, leukocyte-specific reporter lines that highlighted subcellular structure, and a wounding assay for leukocyte migration. LLSM revealed details of migrating leukocyte morphology, and permitted intricate, volumetric interrogation of highly dynamic activities within their native physiological setting. Very thin, recurrent uropod extensions must now be considered a characteristic feature of migrating neutrophils. LLSM resolved trailing uropod extensions, demonstrating their surprising length, and permitting quantitative assessment of cytoskeletal contributions to their evanescent form. Imaging leukocytes in blood vessel microenvironments at LLSM's spatiotemporal resolution displayed blood-flow-induced neutrophil dynamics and demonstrated unexpected leukocyte-endothelial interactions such as leukocyte-induced endothelial deformation against the intravascular pressure. LLSM of phagocytosis and cell death provided subcellular insights and uncovered novel behaviors. Collectively, we provide high-resolution LLSM examples of leukocyte structures (filopodia lamellipodia, uropod extensions, vesicles), and activities (interstitial and intravascular migration, leukocyte rolling, phagocytosis, cell death, and cytoplasmic ballooning). Application of LLSM to intravital leukocyte imaging sets the stage for transformative studies into the cellular and subcellular complexities of phagocyte biology.
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Quimiotaxis de Leucocito/fisiología , Microscopía Intravital , Leucocitos/citología , Leucocitos/fisiología , Animales , Animales Modificados Genéticamente , Biomarcadores , Adhesión Celular , Muerte Celular , Endotelio Vascular/metabolismo , Técnica del Anticuerpo Fluorescente , Microscopía Intravital/métodos , Macrófagos/citología , Macrófagos/fisiología , Modelos Biológicos , Neutrófilos/citología , Neutrófilos/fisiología , Fagocitosis , Pez CebraRESUMEN
Histone post-translational modifications are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We applied a Fab-based live endogenous modification labeling technique to monitor the changes in histone modification levels during zygotic genome activation (ZGA) in living zebrafish embryos. Among various histone modifications, H3 Lys27 acetylation (H3K27ac) exhibited most drastic changes, accumulating in two nuclear foci in the 64- to 1k-cell-stage embryos. The elongating form of RNA polymerase II, which is phosphorylated at Ser2 in heptad repeats within the C-terminal domain (RNAP2 Ser2ph), and miR-430 transcripts were also concentrated in foci closely associated with H3K27ac. When treated with α-amanitin to inhibit transcription or JQ-1 to inhibit binding of acetyl-reader proteins, H3K27ac foci still appeared but RNAP2 Ser2ph and miR-430 morpholino were not concentrated in foci, suggesting that H3K27ac precedes active transcription during ZGA. We anticipate that the method presented here could be applied to a variety of developmental processes in any model and non-model organisms.
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Regulación del Desarrollo de la Expresión Génica , Genoma , Histonas/metabolismo , Lisina/metabolismo , Transcripción Genética , Pez Cebra/embriología , Pez Cebra/genética , Cigoto/metabolismo , Acetilación/efectos de los fármacos , Alfa-Amanitina/farmacología , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Código de Histonas/efectos de los fármacos , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Cigoto/efectos de los fármacosRESUMEN
Lattice light-sheet microscopy (LLSM) is valuable for its combination of reduced photobleaching and outstanding spatiotemporal resolution in 3D. Using LLSM to image biosensors in living cells could provide unprecedented visualization of rapid, localized changes in protein conformation or posttranslational modification. However, computational manipulations required for biosensor imaging with LLSM are challenging for many software packages. The calculations require processing large amounts of data even for simple changes such as reorientation of cell renderings or testing the effects of user-selectable settings, and lattice imaging poses unique challenges in thresholding and ratio imaging. We describe here a new software package, named ImageTank, that is specifically designed for practical imaging of biosensors using LLSM. To demonstrate its capabilities, we use a new biosensor to study the rapid 3D dynamics of the small GTPase Rap1 in vesicles and cell protrusions.
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Técnicas Biosensibles , Transferencia Resonante de Energía de Fluorescencia , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Procesamiento de Imagen Asistido por Computador , Transducción de Señal , Programas Informáticos , Proteínas de Unión a Telómeros/metabolismo , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Microscopía Fluorescente , Complejo Shelterina , Proteínas de Unión a Telómeros/genéticaRESUMEN
Macrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for initiating this process is unknown. Here, we used live cell imaging to show that actin-based protrusions at the leading edge initiate macrophage fusion. Phase-contrast video microscopy demonstrated that in the majority of events, short protrusions (â¼3 µm) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (â¼12 µm). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent protrusions formed at sites enriched in podosomes. Inducing fusion in mixed populations of GFP- and mRFP-LifeAct macrophages showed rapid spatial overlap between GFP and RFP signal at the site of fusion. Cytochalasin B strongly reduced fusion and when rare fusion events occurred, protrusions were not observed. Fusion of macrophages deficient in Wiskott-Aldrich syndrome protein and Cdc42, key molecules involved in the formation of actin-based protrusions and podosomes, was also impaired both in vitro and in vivo. Finally, inhibiting the activity of the Arp2/3 complex decreased fusion and podosome formation. Together these data suggest that an actin-based protrusion formed at the leading edge initiates macrophage fusion.
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Actinas/metabolismo , Macrófagos/metabolismo , Podosomas/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Animales , Comunicación Celular , Movimiento Celular , Citocalasina B/metabolismo , Femenino , Masculino , Fusión de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente/métodos , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Phagocytosis of invading pathogens or cellular debris requires a dramatic change in cell shape driven by actin polymerization. For antibody-covered targets, phagocytosis is thought to proceed through the sequential engagement of Fc-receptors on the phagocyte with antibodies on the target surface, leading to the extension and closure of the phagocytic cup around the target. We find that two actin-dependent molecular motors, class 1 myosins myosin 1e and myosin 1f, are specifically localized to Fc-receptor adhesions and required for efficient phagocytosis of antibody-opsonized targets. Using primary macrophages lacking both myosin 1e and myosin 1f, we find that without the actin-membrane linkage mediated by these myosins, the organization of individual adhesions is compromised, leading to excessive actin polymerization, slower adhesion turnover, and deficient phagocytic internalization. This work identifies a role for class 1 myosins in coordinated adhesion turnover during phagocytosis and supports a mechanism involving membrane-cytoskeletal crosstalk for phagocytic cup closure.
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Actinas/metabolismo , Adhesión Celular/fisiología , Miosina Tipo I/metabolismo , Miosinas/metabolismo , Fagocitosis/fisiología , Actinas/ultraestructura , Animales , Células de la Médula Ósea , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Femenino , Microscopía Intravital , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Microscopía Fluorescente , Miosina Tipo I/genética , Miosinas/genética , Cultivo Primario de Células , Células RAW 264.7 , Receptores Fc/metabolismo , Receptores Fc/ultraestructura , Imagen de Lapso de TiempoRESUMEN
Cytotoxic T lymphocytes (CTLs) kill by forming immunological synapses with target cells and secreting toxic proteases and the pore-forming protein perforin into the intercellular space. Immunological synapses are highly dynamic structures that boost perforin activity by applying mechanical force against the target cell. Here, we used high-resolution imaging and microfabrication to investigate how CTLs exert synaptic forces and coordinate their mechanical output with perforin secretion. Using micropatterned stimulatory substrates that enable synapse growth in three dimensions, we found that perforin release occurs at the base of actin-rich protrusions that extend from central and intermediate locations within the synapse. These protrusions, which depended on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, were required for synaptic force exertion and efficient killing. They also mediated physical deformation of the target cell surface during CTL-target cell interactions. Our results reveal the mechanical basis of cellular cytotoxicity and highlight the functional importance of dynamic, three-dimensional architecture in immune cell-cell interfaces.
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Sinapsis Inmunológicas/inmunología , Perforina/inmunología , Linfocitos T Citotóxicos/inmunología , Complejo 2-3 Proteico Relacionado con la Actina/inmunología , Actinas/inmunología , Animales , Ratones , Proteína del Síndrome de Wiskott-Aldrich/inmunologíaRESUMEN
BACKGROUND: Unbiased screening studies have repeatedly identified actin-related proteins as one of the families of proteins most influenced by neurotrauma. Nevertheless, the status quo model of cytoskeletal reorganization after neurotrauma excludes actin and incorporates only changes in microtubules and intermediate filaments. Actin is excluded in part because it is difficult to image with conventional techniques. However, recent innovations in fluorescent microscopy provide an opportunity to image the actin cytoskeleton at super-resolution resolution in living cells. This study applied these innovations to an in vitro model of neurotrauma. NEW METHOD: New methods are introduced for traumatizing neurons before imaging them with high speed structured illumination microscopy or lattice light sheet microscopy. Also, methods for analyzing structured illumination microscopy images to quantify post-traumatic neurite dystrophy are presented. RESULTS: Human induced pluripotent stem cell-derived neurons exhibited actin organization typical of immature neurons. Neurite dystrophy increased after trauma but was not influenced by jasplakinolide treatment. The F-actin content of dystrophies varied greatly from one dystrophy to another. COMPARISON WITH EXISTING METHODS: In contrast to fixation dependent methods, these methods capture the evolution of the actin cytoskeleton over time in a living cell. In contrast to prior methods based on counting dystrophies, this quantification scheme parameterizes the severity of a given dystrophy as it evolves from a local swelling to an almost-perfect spheroid that threatens to transect the neurite. CONCLUSIONS: These methods can be used to investigate genetic factors and therapeutic interventions that modulate the course of neurite dystrophy after trauma.
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Lesiones Encefálicas/diagnóstico por imagen , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Neuritas/patología , Neuronas/patología , Citoesqueleto de Actina/patología , Lesiones Encefálicas/patología , Humanos , Células Madre Pluripotentes InducidasRESUMEN
Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental sampling and receptor internalization and signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure. We offer a new model in which ruffles are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. This process allows for formation of large macropinosomes induced by LPS. We further describe the enrichment of active Rab13 on tent pole ruffles and show that CRISPR deletion of Rab13 results in aberrant tent pole ruffles and blocks the formation of large LPS-induced macropinosomes. Based on the exquisite temporal and spatial resolution of LLSM, we can redefine the ruffling and macropinosome processes that underpin innate immune responses.
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Actinas/metabolismo , Estructuras de la Membrana Celular/metabolismo , Macrófagos/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Actinas/genética , Animales , Sistemas CRISPR-Cas , Estructuras de la Membrana Celular/genética , Eliminación de Gen , Lipopolisacáridos/farmacología , Ratones , Células RAW 264.7 , Proteínas de Unión al GTP rab/genéticaRESUMEN
Mitochondrial apoptosis is mediated by BAK and BAX, two proteins that induce mitochondrial outer membrane permeabilization, leading to cytochrome c release and activation of apoptotic caspases. In the absence of active caspases, mitochondrial DNA (mtDNA) triggers the innate immune cGAS/STING pathway, causing dying cells to secrete type I interferon. How cGAS gains access to mtDNA remains unclear. We used live-cell lattice light-sheet microscopy to examine the mitochondrial network in mouse embryonic fibroblasts. We found that after BAK/BAX activation and cytochrome c loss, the mitochondrial network broke down and large BAK/BAX pores appeared in the outer membrane. These BAK/BAX macropores allowed the inner mitochondrial membrane to herniate into the cytosol, carrying with it mitochondrial matrix components, including the mitochondrial genome. Apoptotic caspases did not prevent herniation but dismantled the dying cell to suppress mtDNA-induced innate immune signaling.
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Apoptosis , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Citocromos c/metabolismo , ADN Mitocondrial/metabolismo , Fibroblastos , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Membranas Mitocondriales/química , Multimerización de Proteína , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
Emerging applications that exploit the properties of nanoparticles for biotechnology require that the nanoparticles be biocompatible or support biological recognition. These types of particles can be produced through syntheses that involve biologically relevant molecules (proteins or natural extracts, for example). Many of the protocols that rely on these molecules are performed without a clear understanding of the mechanism by which the materials are produced. We have investigated a previously described reaction in which gold nanoparticles are produced from the reaction of chloroauric acid and proteins in solution. We find that modifications to the starting conditions can alter the product from the expected solution-suspended colloids to a product where colloids are formed within a solid, fibrous protein structure. We have interrogated this synthesis, exploiting the change in products to better understand this reaction. We have evaluated the kinetics and products for 7 different proteins over a range of concentrations and temperatures. The key factor that controls the synthetic outcome (colloid or fiber) is the concentration of the protein relative to the gold concentration. We find that the observed fibrous structures are more likely to form at low protein concentrations and when hydrophilic proteins are used. An analysis of the reaction kinetics shows that AuNP formation occurs faster at lower protein (fiber-forming) concentrations than at higher protein (colloid-forming) concentrations. These results contradict traditional expectations for reaction kinetics and protein-fiber formation and are instructive of the manner in which proteins template gold nanoparticle production.
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Oro/química , Nanopartículas del Metal/química , Nanotecnología/métodos , Proteínas/química , Animales , Técnicas de Química Sintética , CinéticaRESUMEN
The basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians [1]; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly [2], this concept suffers from a number of logistical issues when considering its construction in vivo. First, incorporation of BM components appears to be hierarchical [3-5], yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types [6-9], and it is unclear how large, possibly insoluble protein complexes [10] are delivered into the matrix. Here we exploit our ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, we show that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components.
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Membrana Basal/fisiología , Matriz Extracelular/metabolismo , Animales , Membrana Basal/metabolismo , Movimiento Celular/fisiología , Colágeno/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Células Epiteliales/metabolismo , Matriz Extracelular/fisiología , Macrófagos/metabolismoRESUMEN
Calcium carbonate platforms produced by reef-building stony corals over geologic time are pervasive features around the world [1]; however, the mechanism by which these organisms produce the mineral is poorly understood (see review by [2]). It is generally assumed that stony corals precipitate calcium carbonate extracellularly as aragonite in a calcifying medium between the calicoblastic ectoderm and pre-existing skeleton, separated from the overlying seawater [2]. The calicoblastic ectoderm produces extracellular matrix (ECM) proteins, secreted to the calcifying medium [3-6], which appear to provide the nucleation, alteration, elongation, and inhibition mechanisms of the biomineral [7] and remain occluded and preserved in the skeleton [8-10]. Here we show in cell cultures of the stony coral Stylophora pistillata that calcium is concentrated in intracellular pockets that are subsequently exported from the cell where a nucleation process leads to the formation of extracellular aragonite crystals. Analysis of the growing crystals by lattice light-sheet microscopy suggests that the crystals elongate from the cells' surfaces outward.