RESUMEN
Single-molecule localisation microscopy (SMLM) has the potential to reveal the underlying organisation of specific molecules within supramolecular complexes and their conformations, which is not possible with conventional microscope resolution. However, the detection efficiency for fluorescent molecules in cells can be limited in SMLM, even to below 1% in thick and dense samples. Segmentation of individual complexes can also be challenging. To overcome these problems, we have developed a software package termed PERPL: Pattern Extraction from Relative Positions of Localisations. This software assesses the relative likelihoods of models for underlying patterns behind incomplete SMLM data, based on the relative positions of pairs of localisations. We review its principles and demonstrate its use on the 3D lattice of Z-disk proteins in mammalian cardiomyocytes. We find known and novel features at ~20 nm with localisations of less than 1% of the target proteins, using mEos fluorescent protein constructs.
RESUMEN
Inferring the organization of fluorescently labeled nanosized structures from single molecule localization microscopy (SMLM) data, typically obscured by stochastic noise and background, remains challenging. To overcome this, we developed a method to extract high-resolution ordered features from SMLM data that requires only a low fraction of targets to be localized with high precision. First, experimentally measured localizations are analyzed to produce relative position distributions (RPDs). Next, model RPDs are constructed using hypotheses of how the molecule is organized. Finally, a statistical comparison is used to select the most likely model. This approach allows pattern recognition at sub-1% detection efficiencies for target molecules, in large and heterogeneous samples and in 2D and 3D data sets. As a proof-of-concept, we infer ultrastructure of Nup107 within the nuclear pore, DNA origami structures, and α-actinin-2 within the cardiomyocyte Z-disc and assess the quality of images of centrioles to improve the averaged single-particle reconstruction.
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ADN , Imagen Individual de MoléculaRESUMEN
The Polyneuropathy And Treatment with Hizentra (PATH) study required subjects with chronic inflammatory demyelinating polyneuropathy (CIDP) to show dependency on immunoglobulin G (IgG) and then be restabilized on IgG before being randomized to placebo or one of two doses of subcutaneous immunoglobulin (SCIG). Nineteen of the 51 subjects (37%) randomized to placebo did not relapse over the next 24 weeks. This article explores the reasons for this effect. A post-hoc analysis of the PATH placebo group was undertaken. A literature search identified other placebo-controlled CIDP trials for review and comparison. In PATH, subjects randomized to placebo who did not relapse were significantly older, had more severe disease, and took longer to deteriorate in the IgG dependency period compared with those who relapsed. Published trials in CIDP, whose primary endpoint was stability or deterioration, had a mean non-deterioration (placebo effect) of 43%, while trials with a primary endpoint of improvement had a placebo response of only 11%. Placebo is an important variable in the design of CIDP trials. Trials designed to show clinical improvement will have a significantly lower effect of this phenomenon than those designed to show stability or deterioration.
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Factores Inmunológicos , Evaluación de Resultado en la Atención de Salud , Efecto Placebo , Polirradiculoneuropatía Crónica Inflamatoria Desmielinizante/tratamiento farmacológico , Ensayos Clínicos Controlados Aleatorios como Asunto , Proyectos de Investigación , HumanosRESUMEN
A challenge in biological imaging is to capture high-resolution images at fast frame rates in live cells. The "instant structured illumination microscope" (iSIM) is a system designed for this purpose. Similarly to standard structured illumination microscopy (SIM), an iSIM provides a twofold improvement over widefield microscopy, in x, y and z, but also allows much faster image acquisition, with real-time display of super-resolution images. The assembly of an iSIM is reasonably complex, involving the combination and alignment of many optical components, including three micro-optics arrays (two lenslet arrays and an array of pinholes, all with a pitch of 222 µm) and a double-sided scanning mirror. In addition, a number of electronic components must be correctly controlled. Construction of the system is therefore not trivial, but is highly desirable, particularly for live-cell imaging. We report, and provide instructions for, the construction of an iSIM, including minor modifications to a previous design in both hardware and software. The final instrument allows us to rapidly acquire fluorescence images at rates faster than 100 fps, with approximately twofold improvement in resolution in both x-y and z; sub-diffractive biological features have an apparent size (full width at half maximum) of 145 nm (lateral) and 320 nm (axial), using a 1.49 NA objective and 488 nm excitation.
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Aumento de la Imagen , Imagenología Tridimensional/instrumentación , Microscopía Fluorescente/instrumentación , Programas Informáticos , Límite de DetecciónRESUMEN
Compartmentalisation of cellular processes is fundamental to regulation of metabolism in Eukaryotic organisms and is primarily provided by membrane-bound organelles. These organelles are dynamic structures whose membrane barriers are continually shaped, remodelled and scaffolded by a rich variety of highly sophisticated protein complexes. Towards the goal of bottom-up assembly of compartmentalised protocells in synthetic biology, we believe it will be important to harness and reconstitute the membrane shaping and sculpting characteristics of natural cells. We review different in vitro membrane models and how biophysical investigations of minimal systems combined with appropriate theoretical modelling have been used to gain new insights into the intricate mechanisms of these membrane nanomachines, paying particular attention to proteins involved in membrane fusion, fission and cytoskeletal scaffolding processes. We argue that minimal machineries need to be developed and optimised for employment in artificial protocell systems rather than the complex environs of a living organism. Thus, well-characterised minimal components might be predictably combined into functional, compartmentalised protocellular materials that can be engineered for wide-ranging applications.
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Células Artificiales/metabolismo , Membrana Celular/metabolismo , Nanotecnología , Células Artificiales/química , Membrana Celular/química , Humanos , Proteínas/química , Proteínas/metabolismoRESUMEN
Micronuclei represent the cellular attempt to compartmentalize DNA to maintain genomic integrity threatened by mitotic errors and genotoxic events. Some micronuclei show aberrant nuclear envelopes (NEs) that collapse, generating damaged DNA that can promote complex genome alterations. However, ruptured micronuclei also provide a pool of cytosolic DNA that can stimulate antitumor immunity, revealing the complexity of micronuclear impact on tumor progression. The ESCRT-III (Endosomal Sorting Complex Required for Transport-III) complex ensures NE reseals during late mitosis and is repaired in interphase. Therefore, ESCRT-III activity maybe crucial for maintaining the integrity of other genomic structures enclosed by a NE. ESCRT-III activity at the NE is coordinated by the subunit CHMP7. We show that CHMP7 and ESCRT-III protect against the genomic instability associated with micronuclei formation. Loss of ESCRT-III activity increases the population of micronuclei with ruptured NEs, revealing that its NE repair activity is also necessary to maintain micronuclei integrity. Surprisingly, aberrant accumulation of ESCRT-III are found at the envelope of most acentric collapsed micronuclei, suggesting that ESCRT-III is not recycled efficiently from these structures. Moreover, CHMP7 depletion relieves micronuclei from the aberrant accumulations of ESCRT-III. CHMP7-depleted cells display a reduction in micronuclei containing the DNA damage marker RPA and a sensor of cytosolic DNA. Thus, ESCRT-III activity appears to protect from the consequence of genomic instability in a dichotomous fashion: ESCRT-III membrane repair activity prevents the occurrence of micronuclei with weak envelopes, but the aberrant accumulation of ESCRT-III on a subset of micronuclei appears to exacerbate DNA damage and sustain proinflammatory pathways.