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1.
J Cell Sci ; 137(7)2024 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-38629499

RESUMO

Expansion microscopy (ExM) is a revolutionary novel approach to increase resolution in light microscopy. In contrast to super-resolution microscopy methods that rely on sophisticated technological advances, including novel instrumentation, ExM instead is entirely based on sample preparation. In ExM, labeled target molecules in fixed cells are anchored in a hydrogel, which is then physically enlarged by osmotic swelling. The isotropic swelling of the hydrogel pulls the labels apart from one another, and their relative organization can thus be resolved using conventional microscopes even if it was below the diffraction limit of light beforehand. As ExM can additionally benefit from the technical resolution enhancements achieved by super-resolution microscopy, it can reach into the nanometer range of resolution with an astoundingly low degree of error induced by distortion during the physical expansion process. Because the underlying chemistry is well understood and the technique is based on a relatively simple procedure, ExM is easily reproducible in non-expert laboratories and has quickly been adopted to address an ever-expanding spectrum of problems across the life sciences. In this Review, we provide an overview of this rapidly expanding new field, summarize the most important insights gained so far and attempt to offer an outlook on future developments.


Assuntos
Hidrogéis , Microscopia de Fluorescência/métodos
2.
Angew Chem Int Ed Engl ; 62(28): e202302318, 2023 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-37158034

RESUMO

Expansion microscopy (ExM) is a recently developed technique that allows for the resolution of structures below the diffraction limit by physically enlarging a hydrogel-embedded facsimile of the biological sample. The target structure is labeled and this label must be retained in a relative position true to the original, smaller state before expansion by linking it into the gel. However, gel formation and digestion lead to a significant loss in target-delivered label, resulting in weak signal. To overcome this problem, we have here developed an agent combining targeting, fluorescent labeling and gel linkage in a single small molecule. Similar approaches in the past have still suffered from significant loss of label. Here we show that this loss is due to insufficient surface grafting of fluorophores into the hydrogel and develop a solution by increasing the amount of target-bound monomers. Overall, we obtain a significant improvement in fluorescence signal retention and our new dye allows the resolution of nuclear pores as ring-like structures, similar to STED microscopy. We furthermore provide mechanistic insight into dye retention in ExM.


Assuntos
Corantes Fluorescentes , Hidrogéis , Microscopia de Fluorescência/métodos , Corantes Fluorescentes/química , Hidrogéis/química
3.
Am J Hum Genet ; 104(6): 1060-1072, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31104773

RESUMO

The developmental and epileptic encephalopathies (DEEs) are heterogeneous disorders with a strong genetic contribution, but the underlying genetic etiology remains unknown in a significant proportion of individuals. To explore whether statistical support for genetic etiologies can be generated on the basis of phenotypic features, we analyzed whole-exome sequencing data and phenotypic similarities by using Human Phenotype Ontology (HPO) in 314 individuals with DEEs. We identified a de novo c.508C>T (p.Arg170Trp) variant in AP2M1 in two individuals with a phenotypic similarity that was higher than expected by chance (p = 0.003) and a phenotype related to epilepsy with myoclonic-atonic seizures. We subsequently found the same de novo variant in two individuals with neurodevelopmental disorders and generalized epilepsy in a cohort of 2,310 individuals who underwent diagnostic whole-exome sequencing. AP2M1 encodes the µ-subunit of the adaptor protein complex 2 (AP-2), which is involved in clathrin-mediated endocytosis (CME) and synaptic vesicle recycling. Modeling of protein dynamics indicated that the p.Arg170Trp variant impairs the conformational activation and thermodynamic entropy of the AP-2 complex. Functional complementation of both the µ-subunit carrying the p.Arg170Trp variant in human cells and astrocytes derived from AP-2µ conditional knockout mice revealed a significant impairment of CME of transferrin. In contrast, stability, expression levels, membrane recruitment, and localization were not impaired, suggesting a functional alteration of the AP-2 complex as the underlying disease mechanism. We establish a recurrent pathogenic variant in AP2M1 as a cause of DEEs with distinct phenotypic features, and we implicate dysfunction of the early steps of endocytosis as a disease mechanism in epilepsy.


Assuntos
Complexo 2 de Proteínas Adaptadoras/genética , Subunidades mu do Complexo de Proteínas Adaptadoras/genética , Encefalopatias/etiologia , Clatrina/metabolismo , Endocitose , Epilepsia/etiologia , Mutação de Sentido Incorreto , Transtornos do Neurodesenvolvimento/etiologia , Adolescente , Animais , Encefalopatias/patologia , Criança , Pré-Escolar , Clatrina/genética , Epilepsia/patologia , Feminino , Humanos , Lactente , Camundongos , Camundongos Knockout , Transtornos do Neurodesenvolvimento/patologia , Sequenciamento do Exoma
4.
Mol Biol Cell ; 33(8): ar76, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35594179

RESUMO

The combination of image analysis and superresolution microscopy methods allows for unprecedented insight into the organization of macromolecular assemblies in cells. Advances in deep learning (DL)-based object recognition enable the automated processing of large amounts of data, resulting in high accuracy through averaging. However, while the analysis of highly symmetric structures of constant size allows for a resolution approaching the dimensions of structural biology, DL-based image recognition may introduce bias. This prohibits the development of readouts for processes that involve significant changes in size or shape of amorphous macromolecular complexes. Here we address this problem by using changes of septin ring structures in single molecule localization-based superresolution microscopy data as a paradigm. We identify potential sources of bias resulting from different training approaches by rigorous testing of trained models using real or simulated data covering a wide range of possible results. In a quantitative comparison of our models, we find that a trade-off exists between measurement accuracy and the range of recognized phenotypes. Using our thus verified models, we find that septin ring size can be explained by the number of subunits they are assembled from alone. Furthermore, we provide a new experimental system for the investigation of septin polymerization.


Assuntos
Aprendizado Profundo , Microscopia , Citoesqueleto/química , Substâncias Macromoleculares , Microscopia/métodos , Septinas/química , Imagem Individual de Molécula/métodos
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