RESUMO
While some people pore over the textbook and train through the classics of the field, many scientists come to immunology when they discover it intersecting with their "first love" interests. Five of these "accidental immunologists" tell us how they found their way to a fascination with the immune system.
Assuntos
Alergia e Imunologia , Humanos , História do Século XX , História do Século XXI , Animais , Sistema ImunitárioRESUMO
The proteins of the BCL-2 family are key regulators of mitochondrial apoptosis, acting as either promoters or inhibitors of cell death. The functional interplay and balance between the opposing BCL-2 family members control permeabilization of the outer mitochondrial membrane, leading to the release of activators of the caspase cascade into the cytosol and ultimately resulting in cell death. Despite considerable research, our knowledge about the mechanisms of the BCL-2 family of proteins remains insufficient, which complicates cell fate predictions and does not allow us to fully exploit these proteins as targets for drug discovery. Detailed understanding of the formation and molecular architecture of the apoptotic pore in the outer mitochondrial membrane remains a holy grail in the field, but new studies allow us to begin constructing a structural model of its arrangement. Recent literature has also revealed unexpected activities for several BCL-2 family members that challenge established concepts of how they regulate mitochondrial permeabilization. In this Review, we revisit the most important advances in the field and integrate them into a new structure-function-based classification of the BCL-2 family members that intends to provide a comprehensive model for BCL-2 action in apoptosis. We close this Review by discussing the potential of drugging the BCL-2 family in diseases characterized by aberrant apoptosis.
Assuntos
Apoptose , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Apoptose/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Caspases/metabolismoRESUMO
Mitochondria are cellular organelles with a major role in many cellular processes, including not only energy production, metabolism, and calcium homeostasis but also regulated cell death and innate immunity. Their proteobacterial origin makes them a rich source of potent immune agonists, normally hidden within the mitochondrial membrane barriers. Alteration of mitochondrial permeability through mitochondrial pores thus provides efficient mechanisms not only to communicate mitochondrial stress to the cell but also as a key event in the integration of cellular responses. In this regard, eukaryotic cells have developed diverse signaling networks that sense and respond to the release of mitochondrial components into the cytosol and play a key role in controlling cell death and inflammatory pathways. Modulating pore formation at mitochondria through direct or indirect mechanisms may thus open new opportunities for therapy. In this review, we discuss the current understanding of the structure and molecular mechanisms of mitochondrial pores and how they function at the interface between cell death and inflammatory signaling to regulate cellular outcomes.
Assuntos
Mitocôndrias , Membranas Mitocondriais , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Apoptose/fisiologia , Morte Celular , Transdução de SinaisRESUMO
Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.
Assuntos
Linfoma de Burkitt , Desidrocolesteróis , Ferroptose , Neuroblastoma , Animais , Humanos , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Sobrevivência Celular , Desidrocolesteróis/metabolismo , Peroxidação de Lipídeos , Transplante de Neoplasias , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Oxirredução , Fenótipo , Reprodutibilidade dos TestesRESUMO
BAX and BAK are key apoptosis regulators that mediate the decisive step of mitochondrial outer membrane permeabilization. However, the mechanism by which they assemble the apoptotic pore remains obscure. Here, we report that BAX and BAK present distinct oligomerization properties, with BAK organizing into smaller structures with faster kinetics than BAX. BAK recruits and accelerates BAX assembly into oligomers that continue to grow during apoptosis. As a result, BAX and BAK regulate each other as they co-assemble into the same apoptotic pores, which we visualize. The relative availability of BAX and BAK molecules thereby determines the growth rate of the apoptotic pore and the relative kinetics by which mitochondrial contents, most notably mtDNA, are released. This feature of BAX and BAK results in distinct activation kinetics of the cGAS/STING pathway with implications for mtDNA-mediated paracrine inflammatory signaling.
Assuntos
DNA Mitocondrial , Mitocôndrias , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Apoptose/genética , Linhagem Celular Tumoral , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Multimerização Proteica , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
The apoptotic executioner protein BAX and the dynamin-like protein DRP1 co-localize at mitochondria during apoptosis to mediate mitochondrial permeabilization and fragmentation. However, the molecular basis and functional consequences of this interplay remain unknown. Here, we show that BAX and DRP1 physically interact, and that this interaction is enhanced during apoptosis. Complex formation between BAX and DRP1 occurs exclusively in the membrane environment and requires the BAX N-terminal region, but also involves several other BAX surfaces. Furthermore, the association between BAX and DRP1 enhances the membrane activity of both proteins. Forced dimerization of BAX and DRP1 triggers their activation and translocation to mitochondria, where they induce mitochondrial remodeling and permeabilization to cause apoptosis even in the absence of apoptotic triggers. Based on this, we propose that DRP1 can promote apoptosis by acting as noncanonical direct activator of BAX through physical contacts with its N-terminal region.
Assuntos
Apoptose , Dinaminas , Apoptose/fisiologia , Dinaminas/genética , Dinaminas/metabolismo , Mitocôndrias/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
During apoptosis, the BCL-2-family protein tBID promotes mitochondrial permeabilization by activating BAX and BAK and by blocking anti-apoptotic BCL-2 members. Here, we report that tBID can also mediate mitochondrial permeabilization by itself, resulting in release of cytochrome c and mitochondrial DNA, caspase activation and apoptosis even in absence of BAX and BAK. This previously unrecognized activity of tBID depends on helix 6, homologous to the pore-forming regions of BAX and BAK, and can be blocked by pro-survival BCL-2 proteins. Importantly, tBID-mediated mitochondrial permeabilization independent of BAX and BAK is physiologically relevant for SMAC release in the immune response against Shigella infection. Furthermore, it can be exploited to kill leukaemia cells with acquired venetoclax resistance due to lack of active BAX and BAK. Our findings define tBID as an effector of mitochondrial permeabilization in apoptosis and provide a new paradigm for BCL-2 proteins, with implications for anti-bacterial immunity and cancer therapy.
Assuntos
Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Células HCT116 , Células HeLa , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Domínios Proteicos , Proteólise , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
The proteins of the BCL-2 family are known as key regulators of apoptosis, with interactions between family members determining permeabilisation of the mitochondrial outer membrane (MOM) and subsequent cell death. However, the exact mechanism through which they form the apoptotic pore responsible for MOM permeabilisation (MOMP), the structure and specific components of this pore, and what roles BCL-2 proteins play outside of directly regulating MOMP are incompletely understood. Owing to the link between apoptosis dysregulation and disease, the BCL-2 proteins are important targets for drug development. With the development and clinical use of drugs targeting BCL-2 proteins showing success in multiple haematological malignancies, enhancing the efficacy of these drugs, or indeed developing novel drugs targeting BCL-2 proteins is of great interest to treat cancer patients who have developed resistance or who suffer other disease types. Here, we review our current understanding of the molecular mechanism of MOMP, with a particular focus on recently discovered roles of BCL-2 proteins in apoptosis and beyond, and discuss what implications these functions might have in both healthy tissues and disease.
Assuntos
Membranas Mitocondriais , Proteínas Proto-Oncogênicas c-bcl-2 , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína X Associada a bcl-2/química , Membranas Mitocondriais/metabolismo , Apoptose/fisiologiaRESUMO
The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy.
Assuntos
Apoptose/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Dineínas/metabolismo , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Animais , Proteína 11 Semelhante a Bcl-2/genética , Células CACO-2 , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Células HeLa , Humanos , Células MCF-7 , Camundongos , Ligação Proteica , Multimerização Proteica/genética , Estabilidade Proteica , Interferência de RNA , Proteína X Associada a bcl-2/genéticaRESUMO
The discovery of alternative signaling pathways that regulate cell death has revealed multiple strategies for promoting cell death with diverse consequences at the tissue and organism level. Despite the divergence in the molecular components involved, membrane permeabilization is a common theme in the execution of regulated cell death. In apoptosis, the permeabilization of the outer mitochondrial membrane by BAX and BAK releases apoptotic factors that initiate the caspase cascade and is considered the point of no return in cell death commitment. Pyroptosis and necroptosis also require the perforation of the plasma membrane at the execution step, which involves Gasdermins in pyroptosis, and MLKL in the case of necroptosis. Although BAX/BAK, Gasdermins and MLKL share certain molecular features like oligomerization, they form pores in different cellular membranes via distinct mechanisms. Here, we compare and contrast how BAX/BAK, Gasdermins, and MLKL alter membrane permeability from a structural and biophysical perspective and discuss the general principles of membrane permeabilization in the execution of regulated cell death.
Assuntos
Morte Celular/imunologia , Morte Celular/fisiologia , Morte Celular Regulada/imunologia , Morte Celular Regulada/fisiologia , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Caspases/metabolismo , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Necroptose/fisiologia , Proteínas Quinases/metabolismo , Piroptose/fisiologia , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: No study has focused on left atrial (LA) function assessed by echocardiography in adult patients with simple D-TGA after arterial switch operation (ASO). We aimed to describe LA strain parameters in these patients. METHODS: A prospective cohort study including 42 adult patients with simple D-TGA after ASO and 33 aged-matched controls. Phasic LA and LV global longitudinal strain (GLS) were obtained by transthoracic 2D-speckle tracking echocardiography (STE). Volumetric and functional analysis of LA and LV were also evaluated by 2D and 3D analysis. A multivariable model was performed to investigate the variables that best differentiate patients with D-TGA from healthy controls. RESULTS: LA strain parameters in D-TGA patients were within the normal range described for healthy subjects. However, the three LA strain parameters (Reservoir, Conduit, and Contraction) were lower in patients (LASr: 31.13 ± 7.67 vs. 49.71 ± 8.38; LAS cd: -22.91 ± 5.69 vs. -34.55 ± 6.54; LASct: -8.14 ± 4.93 vs. -15.15 ± 6.07, p < .001 for all three comparisons). LA volumes were similar between patients and controls. LV-GLS remained significantly lower in the D-TGA group than in controls (-17.29 ± 2.68 vs. -21.98 ± 1.84, p < .001). D-TGA patients had evidence of worse LV ejection fraction measured by the Teichholz method (63.38 ± 8.23 vs. 69.28 ± 5.92, p = .001) and 3D analysis (57.97% ± 4.16 vs. 60.67 ± 3.39, p = .011) and diastolic dysfunction as compared to healthy controls. LV-GLS and conduit LAS were the variables best differentiating patients with D-TGA from healthy controls. CONCLUSIONS: LA strain is impaired in young adults with simple D-TGA late after the ASO, probably in agreement with some degree of LV dysfunction previously described.
Assuntos
Transposição das Grandes Artérias , Transposição dos Grandes Vasos , Disfunção Ventricular Esquerda , Adulto Jovem , Humanos , Idoso , Transposição dos Grandes Vasos/diagnóstico por imagem , Transposição dos Grandes Vasos/cirurgia , Estudos Prospectivos , Átrios do Coração/diagnóstico por imagem , Artérias , Função Ventricular EsquerdaRESUMO
The minimum requirement for mitochondrial apoptosis has been controversial ever since the discovery of BCL-2 as a cell death regulator. In this issue ofGenes&Development, O'Neill and colleagues (pp. 973-988) end a long-standing debate by creating a cellular system free of BCL-2 family proteins, thereby identifying the outer mitochondrial membrane rather than BH3-only proteins as the only requirement for BAX/BAK activation and mitochondrial outer membrane permeabilization (MOMP).
Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genética , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Sistemas CRISPR-Cas , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Biológicos , Permeabilidade , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Perinatal loss is a traumatic and stressful experience with a significant emotional toll on the physical and psychological wellbeing of the healthcare professionals. We included 216 healthcare professionals working in an obstetrics-gynecology service or neonatal intensive care unit in a cross-sectional study and aimed to analyze the possible association between the healthcare professionals' level of professional quality of life, their coping with death competence, and their personal and work-related characteristics. Compassion fatigue and burnout did not correlate significantly with healthcare professionals' personal and work-related characteristics. Formal training was strongly associated with high levels of compassion satisfaction and coping with death competence. A low level of coping with death competence was found in women and in younger healthcare professionals, those who are single, and with little professional experience. Self-care activities and hospital support systems can be effective resources in coping with death.
Assuntos
Esgotamento Profissional , Qualidade de Vida , Recém-Nascido , Humanos , Feminino , Estudos Transversais , Esgotamento Profissional/psicologia , Capacidades de Enfrentamento , Empatia , Atenção à Saúde , Inquéritos e QuestionáriosRESUMO
Membrane proteins are an essential part of signaling and transport processes and are targeted by multiple drugs. To isolate and investigate them in their native state, polymer-bounded nanodiscs have become valuable tools. In this study, we investigate the lipid model system dimyristoyl-phosphocholine (DMPC) with the nanodisc-forming copolymers styrene maleic acid (SMA) and diisobutylene maleic acid (DIBMA). Using small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS), we studied the influence of polymer concentration and temperature on the nanodisc structure. In Tris buffer, the size of nanodiscs formed with SMA is smaller compared to DIBMA at the same polymer ratio. In both cases, the size decreases monotonically with increasing polymer concentration, and this effect is more pronounced when using SMA. Measurements at temperatures (T) between 5 and 30 °C in phosphate buffer showed an incomplete solubilization at high T even at polymer/lipid ratios above that required for complete lipid solubilization. For DIBMA, the nanodiscs developed at lower temperatures are stable and the net repulsion increases, while for SMA, the individual nanodiscs possess smaller sizes and are less affected by T. However, using DLS, one can observe SMA agglomerates at low T. Interestingly, for both polymers, no drastic changes of the observable parameters (radius and bilayer thickness) are seen upon cooling, which would indicate a sharp (first-order) phase transition from liquid-crystalline to gel, but only gradual changes. Hence, we conclude that the transition from a gel toward a liquid-crystalline lipid phase proceeds over a broad T-range compared to a continuous lipid bilayer. These results can pave the way toward the development of better protocols for studying membrane proteins stabilized in this type of membrane mimics.
Assuntos
Nanoestruturas , Nanoestruturas/química , Polímeros/química , Espalhamento a Baixo Ângulo , Difração de Raios X , Bicamadas Lipídicas/química , Maleatos/química , Proteínas de Membrana/química , Estireno/químicaRESUMO
Single molecule fluorescence microscopy has the unique advantage to provide real-time information on the spatiotemporal assembly of individual protein complexes in cellular membranes. This includes the assembly of proteins into oligomer species of numerous copy numbers. However, there is a need for improved tracing analysis of the real-time growth kinetics of these assemblies in cells with single molecule resolution. Here, we present an automated analysis software to accurately measure the real-time kinetics of assembly of individual high-order oligomer complexes. Our software comes with a simple Graphical User Interface (GUI), is available as a source code and an executable, and can analyze a full data set of several hundred to thousand molecules in less than 2 minutes. Importantly, this software is suitable for the analysis of intracellular protein oligomers, whose stoichiometry is usually more difficult to quantify due to variability in signal detection in the different areas of the cell. We validated our method with simulated ground-truth data and time-lapse images of diffraction-limited oligomeric assemblies of BAX and BAK proteins on mitochondria of cells undergoing apoptosis. Our approach provides the broad community of biologists with a fast, user-friendly tool to trace the compositional evolution of macromolecular assemblies, and potentially model their growth for a deeper understanding of the structural and biophysical mechanisms underlying their functions.
Assuntos
Software , CinéticaRESUMO
BACKGROUND AND AIMS: Recent evidences highlight a role of the mitochondria calcium homeostasis in the development of colorectal cancer (CRC). To overcome treatment resistance, we aimed to evaluate the role of the mitochondrial sodium-calcium-lithium exchanger (NCLX) and its targeting in CRC. We also identified curcumin as a new inhibitor of NCLX. METHODS: We examined whether curcumin and pharmacological compounds induced the inhibition of NCLX-mediated mitochondrial calcium (mtCa2+) extrusion, the role of redox metabolism in this process. We evaluated their anti-tumorigenic activity in vitro and in a xenograft mouse model. We analyzed NCLX expression and associations with survival in The Cancer Genome Atlas (TCGA) dataset and in tissue microarrays from 381 patients with microsatellite instability (MSI)-driven CRC. RESULTS: In vitro, curcumin exerted strong anti-tumoral activity through its action on NCLX with mtCa2+ and reactive oxygen species overload associated with a mitochondrial membrane depolarization, leading to reduced ATP production and apoptosis. NCLX inhibition with pharmacological and molecular approaches reproduced the effects of curcumin. NCLX inhibitors decreased CRC tumor growth in vivo. Both transcriptomic analysis of TCGA dataset and immunohistochemical analysis of tissue microarrays demonstrated that higher NCLX expression was associated with MSI status, and for the first time, NCLX expression was significantly associated with recurrence-free survival. CONCLUSIONS: Our findings highlight a novel anti-tumoral mechanism of curcumin through its action on NCLX and mitochondria calcium overload that could benefit for therapeutic schedule of patients with MSI CRC.
Assuntos
Neoplasias Colorretais , Curcumina , Instabilidade de Microssatélites , Trocador de Sódio e Cálcio , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Curcumina/farmacologia , Humanos , Camundongos , Repetições de Microssatélites , Proteínas Mitocondriais/metabolismo , Trocador de Sódio e Cálcio/antagonistas & inibidoresRESUMO
TAT-RasGAP317-326 is a cell-penetrating peptide-based construct with anticancer and antimicrobial activities. This peptide kills a subset of cancer cells in a manner that does not involve known programmed cell death pathways. Here we have elucidated the mode of action allowing TAT-RasGAP317-326 to kill cells. This peptide binds and disrupts artificial membranes containing lipids typically enriched in the inner leaflet of the plasma membrane, such as phosphatidylinositol-bisphosphate (PIP2) and phosphatidylserine (PS). Decreasing the amounts of PIP2 in cells renders them more resistant to TAT-RasGAP317-326, while reducing the ability of cells to repair their plasma membrane makes them more sensitive to the peptide. The W317A TAT-RasGAP317-326 point mutant, known to have impaired killing activities, has reduced abilities to bind and permeabilize PIP2- and PS-containing membranes and to translocate through biomembranes, presumably because of a higher propensity to adopt an α-helical state. This work shows that TAT-RasGAP317-326 kills cells via a form of necrosis that relies on the physical disruption of the plasma membrane once the peptide targets specific phospholipids found on the cytosolic side of the plasma membrane.
Assuntos
Morte Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Proteínas Ativadoras de GTPase/farmacologia , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Cricetulus , Proteínas Ativadoras de GTPase/uso terapêutico , Células HeLa , Humanos , Lipossomos/metabolismo , Lipossomos/ultraestrutura , Microscopia Eletrônica , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/uso terapêuticoRESUMO
Self-labeling enzymes (SLE) such as the HaloTag have emerged as powerful tools in high and super-resolution fluorescence microscopy. Newly developed fluorogenic SLE substrates enable imaging in the presence of excess dye. To exploit this feature for reversible labeling, we engineered two variants of HaloTag7 with restored dehalogenase activity. Kinetic studies in vitro showed different turnover kinetics for reHaloTagS (≈0.006â s-1 ) and reHaloTagF (≈0.055â s-1 ). Imaging by confocal and stimulated emission depletion microscopy yielded 3-5-time enhanced photostability of reHaloTag labeling. Prominently, single molecule imaging with reHaloTags enabled controlled and stable labeling density over extended time periods. By combination with structured illumination, simultaneous visualization of single molecule diffusion and organellar dynamics was achieved. These applications highlight the potential of reHaloTag labeling for pushing the limits of advanced fluorescence microscopy techniques.
Assuntos
Corantes Fluorescentes , Corantes Fluorescentes/química , Cinética , Microscopia de Fluorescência/métodosRESUMO
Super-resolution microscopy allows imaging of cellular structures with high throughput and detail. However, the efficient and quantitative analysis of images generated is challenging with existing tools. Here, we develop ASAP (automated structures analysis program) to enable rapid and automated detection, classification and quantification of super-resolved structures. We validate ASAP on ground truth data and demonstrate its broad applicability by analyzing images of nucleoporins, TORC1 complexes, endocytic vesicles and Bax pores.