RESUMO
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.
Assuntos
Actinas/química , Actinas/metabolismo , Mitocôndrias/metabolismo , Mitose , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animais , Divisão Celular , Linhagem Celular , Citocinese , Retículo Endoplasmático/metabolismo , Hipocampo/citologia , Hipocampo/embriologia , Humanos , Mitocôndrias/química , Neurônios , RatosRESUMO
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.
Assuntos
Microscopia , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
Núcleo Celular , Optogenética , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAPRESUMO
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.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Genoma , Histonas/metabolismo , Lisina/metabolismo , Transcrição Gênica , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Zigoto/metabolismo , Acetilação/efeitos dos fármacos , Alfa-Amanitina/farmacologia , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Código das Histonas/efeitos dos fármacos , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Zigoto/efeitos dos fármacosRESUMO
To better assess risks associated with nano-enabled products including multiwalled carbon nanotubes (MWCNT) within polymer matrices, it is important to understand how MWCNT are dispersed throughout the composite. The current study presents a method which employs imaging X-ray photoelectron spectroscopy (XPS) to chemically detect spatially segregated MWCNT rich regions at an epoxy composites surface by exploiting differential charging. MWCNT do not charge due to high conductivity and have previously been shown to energetically separate from their insulating surroundings when characterized by XPS. XPS in imaging mode revealed that these conductive regions were spatially separated due to micrometer-scale MWCNT aggregation and poor dispersion during the formation of the composite. Three MWCNT concentrations were studied; (1, 4 and 5) % by mass MWCNT within an epoxy matrix. Images acquired in periodic energy intervals were processed using custom algorithms designed to efficiently extract spectra from regions of interest. As a result, chemical and electrical information on aggregate and non-aggregate portions of the composite was extracted. Raman imaging and scanning electron microscopy were employed as orthogonal techniques for validating this XPS-based methodology. Results demonstrate that XPS imaging of differentially charging MWCNT composite samples is an effective means for assessing dispersion quality.
RESUMO
Length fractionation of colloidal single-wall carbon nanotube (SWCNT) dispersions is required for many studies. Size-exclusion chromatography (SEC) has been developed as a reliable method for high-resolution length fractionation of DNA-dispersed SWCNTs but has not been applied to surfactant-dispersed SWCNTs due to their lower dispersion stability and tendency to adsorb onto SEC stationary phases. Here, we report that SEC length fractionation can be achieved for bile salt dispersed SWCNTs by using porous silica-based beads as the stationary phase and bile salt solution as the mobile phase. We demonstrate that the SEC length sorting method can be combined with existing ultracentrifugation SWCNT sorting methods to produce "orthogonally sorted" samples, including length sorted semiconducting SWCNTs, which are important for electronics applications as well as length sorted empty-core SWCNTs. Importantly, we show that unlike simple length fractionation by SEC or any other method, orthogonal sorting produces samples of consistent quality for different length fractions, with similar UV-vis-nearIR absorption and Raman spectral features.
RESUMO
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.
RESUMO
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.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes ras , Mutação , Neoplasias Experimentais/genética , Proteínas Quinases/genética , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP/metabolismo , Adenocarcinoma de Pulmão/enzimologia , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/mortalidade , Animais , Animais Geneticamente Modificados , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Morte Celular , Movimento Celular , Bases de Dados Genéticas , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/enzimologia , Ativação Enzimática , Predisposição Genética para Doença , Humanos , Larva/enzimologia , Larva/genética , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Invasividade Neoplásica , Neoplasias Experimentais/enzimologia , Fenótipo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
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.
Assuntos
Regulação da Expressão Gênica/fisiologia , Junções Intercelulares/fisiologia , Neutrófilos/fisiologia , Animais , Linhagem Celular , Proteínas de Fluorescência Verde , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Músculo Esquelético/fisiologia , Músculo Esquelético/ultraestruturaRESUMO
Glioblastomas are the most common and most lethal primary brain tumor. Recent studies implicate an important role for a restricted population of neoplastic cells (glioma stem cells (GSCs)) in glioma maintenance and recurrence. We now demonstrate that GSCs preferentially express two interleukin 6 (IL6) receptors: IL6 receptor alpha (IL6R alpha) and glycoprotein 130 (gp130). Targeting IL6R alpha or IL6 ligand expression in GSCs with the use of short hairpin RNAs (shRNAs) significantly reduces growth and neurosphere formation capacity while increasing apoptosis. Perturbation of IL6 signaling in GSCs attenuates signal transducers and activators of transcription three (STAT3) activation, and small molecule inhibitors of STAT3 potently induce GSC apoptosis. These data indicate that STAT3 is a downstream mediator of prosurvival IL6 signals in GSCs. Targeting of IL6R alpha or IL6 expression in GSCs increases the survival of mice bearing intracranial human glioma xenografts. IL6 is clinically significant because elevated IL6 ligand and receptor expression are associated with poor glioma patient survival. The potential utility of anti-IL6 therapies is demonstrated by decreased growth of subcutaneous human GSC-derived xenografts treated with IL6 antibody. Together, our data indicate that IL6 signaling contributes to glioma malignancy through the promotion of GSC growth and survival, and that targeting IL6 may offer benefit for glioma patients.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Interleucina-6/antagonistas & inibidores , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Apoptose/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Receptor gp130 de Citocina/efeitos dos fármacos , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Glioma/genética , Glioma/imunologia , Sobrevivência de Enxerto/efeitos dos fármacos , Sobrevivência de Enxerto/fisiologia , Inibidores do Crescimento/farmacologia , Humanos , Interleucina-6/metabolismo , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Interferência de RNA/fisiologia , RNA Interferente Pequeno/farmacologia , Receptores de Interleucina-6/efeitos dos fármacos , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Microscopia Intravital , Leucócitos/citologia , Leucócitos/fisiologia , Animais , Animais Geneticamente Modificados , Biomarcadores , Adesão Celular , Morte Celular , Endotélio Vascular/metabolismo , Imunofluorescência , Microscopia Intravital/métodos , Macrófagos/citologia , Macrófagos/fisiologia , Modelos Biológicos , Neutrófilos/citologia , Neutrófilos/fisiologia , Fagocitose , Peixe-ZebraRESUMO
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.
Assuntos
Actinas/genética , Proteínas de Transporte/genética , Forminas/genética , Proteínas dos Microfilamentos/genética , Neoplasias/genética , Pseudópodes/genética , Técnicas Biossensoriais , Proteínas de Transporte/isolamento & purificação , Adesão Celular/genética , Movimento Celular/genética , Microambiente Celular/genética , Células HeLa , Humanos , Proteínas dos Microfilamentos/isolamento & purificação , Imagem Molecular , Neoplasias/patologia , Fosforilação , Ligação Proteica/genética , Pseudópodes/metabolismoRESUMO
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.
Assuntos
Lesões Encefálicas/diagnóstico por imagem , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Neuritos/patologia , Neurônios/patologia , Citoesqueleto de Actina/patologia , Lesões Encefálicas/patologia , Humanos , Células-Tronco Pluripotentes InduzidasRESUMO
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.
Assuntos
Actinas/metabolismo , Macrófagos/metabolismo , Podossomos/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Comunicação Celular , Movimento Celular , Citocalasina B/metabolismo , Feminino , Masculino , Fusão de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/métodos , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
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.
Assuntos
Sinapses Imunológicas/imunologia , Perforina/imunologia , Linfócitos T Citotóxicos/imunologia , Complexo 2-3 de Proteínas Relacionadas à Actina/imunologia , Actinas/imunologia , Animais , Camundongos , Proteína da Síndrome de Wiskott-Aldrich/imunologiaRESUMO
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.
Assuntos
Actinas/metabolismo , Adesão Celular/fisiologia , Miosina Tipo I/metabolismo , Miosinas/metabolismo , Fagocitose/fisiologia , Actinas/ultraestrutura , Animais , Células da Medula Óssea , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Feminino , Microscopia Intravital , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Microscopia de Fluorescência , Miosina Tipo I/genética , Miosinas/genética , Cultura Primária de Células , Células RAW 264.7 , Receptores Fc/metabolismo , Receptores Fc/ultraestrutura , Imagem com Lapso de TempoRESUMO
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.
Assuntos
Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Células Endoteliais da Veia Umbilical Humana/metabolismo , Processamento de Imagem Assistida por Computador , Transdução de Sinais , Software , Proteínas de Ligação a Telômeros/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Microscopia de Fluorescência , Complexo Shelterina , Proteínas de Ligação a Telômeros/genéticaRESUMO
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.
Assuntos
Actinas/metabolismo , Estruturas da Membrana Celular/metabolismo , Macrófagos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/genética , Animais , Sistemas CRISPR-Cas , Estruturas da Membrana Celular/genética , Deleção de Genes , Lipopolissacarídeos/farmacologia , Camundongos , Células RAW 264.7 , Proteínas rab de Ligação ao GTP/genéticaRESUMO
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.