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1.
EMBO J ; 42(4): e112030, 2023 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-36594262

RESUMO

B lymphocytes recognize bacterial or viral antigens via different classes of the B cell antigen receptor (BCR). Protrusive structures termed microvilli cover lymphocyte surfaces, and are thought to perform sensory functions in screening antigen-bearing surfaces. Here, we have used lattice light-sheet microscopy in combination with tailored custom-built 4D image analysis to study the cell-surface topography of B cells of the Ramos Burkitt's Lymphoma line and the spatiotemporal organization of the IgM-BCR. Ramos B-cell surfaces were found to form dynamic networks of elevated ridges bridging individual microvilli. A fraction of membrane-localized IgM-BCR was found in clusters, which were mainly associated with the ridges and the microvilli. The dynamic ridge-network organization and the IgM-BCR cluster mobility were linked, and both were controlled by Arp2/3 complex activity. Our results suggest that dynamic topographical features of the cell surface govern the localization and transport of IgM-BCR clusters to facilitate antigen screening by B cells.


Assuntos
Linfoma de Burkitt , Receptores de Antígenos de Linfócitos B , Humanos , Receptores de Antígenos de Linfócitos B/metabolismo , Membrana Celular/metabolismo , Linfócitos B , Linfoma de Burkitt/metabolismo , Imunoglobulina M/metabolismo
2.
Histochem Cell Biol ; 160(3): 199-209, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37341795

RESUMO

Bioimaging has now entered the era of big data with faster-than-ever development of complex microscopy technologies leading to increasingly complex datasets. This enormous increase in data size and informational complexity within those datasets has brought with it several difficulties in terms of common and harmonized data handling, analysis, and management practices, which are currently hampering the full potential of image data being realized. Here, we outline a wide range of efforts and solutions currently being developed by the microscopy community to address these challenges on the path towards FAIR bioimaging data. We also highlight how different actors in the microscopy ecosystem are working together, creating synergies that develop new approaches, and how research infrastructures, such as Euro-BioImaging, are fostering these interactions to shape the field.


Assuntos
Ecossistema , Microscopia
3.
Histochem Cell Biol ; 160(3): 223-251, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37428210

RESUMO

A growing community is constructing a next-generation file format (NGFF) for bioimaging to overcome problems of scalability and heterogeneity. Organized by the Open Microscopy Environment (OME), individuals and institutes across diverse modalities facing these problems have designed a format specification process (OME-NGFF) to address these needs. This paper brings together a wide range of those community members to describe the cloud-optimized format itself-OME-Zarr-along with tools and data resources available today to increase FAIR access and remove barriers in the scientific process. The current momentum offers an opportunity to unify a key component of the bioimaging domain-the file format that underlies so many personal, institutional, and global data management and analysis tasks.


Assuntos
Microscopia , Software , Humanos , Apoio Comunitário
4.
Commun Biol ; 6(1): 61, 2023 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-36650210

RESUMO

Reactive oxygen species (ROS) are constant by-products of aerobic life. In excess, ROS lead to cytotoxic protein aggregates, which are a hallmark of ageing in animals and linked to age-related pathologies in humans. Acylamino acid-releasing enzymes (AARE) are bifunctional serine proteases, acting on oxidized proteins. AARE are found in all domains of life, albeit under different names, such as acylpeptide hydrolase (APEH/ACPH), acylaminoacyl peptidase (AAP), or oxidized protein hydrolase (OPH). In humans, AARE malfunction is associated with age-related pathologies, while their function in plants is less clear. Here, we provide a detailed analysis of AARE genes in the plant lineage and an in-depth analysis of AARE localization and function in the moss Physcomitrella and the angiosperm Arabidopsis. AARE loss-of-function mutants have not been described for any organism so far. We generated and analysed such mutants and describe a connection between AARE function, aggregation of oxidized proteins and plant ageing, including accelerated developmental progression and reduced life span. Our findings complement similar findings in animals and humans, and suggest a unified concept of ageing may exist in different life forms.


Assuntos
Arabidopsis , Bryopsida , Peptídeo Hidrolases , Animais , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Bryopsida/enzimologia , Bryopsida/genética , Peptídeo Hidrolases/metabolismo , Espécies Reativas de Oxigênio/metabolismo
5.
bioRxiv ; 2023 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-36865282

RESUMO

A growing community is constructing a next-generation file format (NGFF) for bioimaging to overcome problems of scalability and heterogeneity. Organized by the Open Microscopy Environment (OME), individuals and institutes across diverse modalities facing these problems have designed a format specification process (OME-NGFF) to address these needs. This paper brings together a wide range of those community members to describe the cloud-optimized format itself -- OME-Zarr -- along with tools and data resources available today to increase FAIR access and remove barriers in the scientific process. The current momentum offers an opportunity to unify a key component of the bioimaging domain -- the file format that underlies so many personal, institutional, and global data management and analysis tasks.

6.
Comput Struct Biotechnol J ; 19: 2106-2120, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995906

RESUMO

Cytoskeletal filaments are structures of utmost importance to biological cells and organisms due to their versatility and the significant functions they perform. These biopolymers are most often organised into network-like scaffolds with a complex morphology. Understanding the geometrical and topological organisation of these networks provides key insights into their functional roles. However, this non-trivial task requires a combination of high-resolution microscopy and sophisticated image processing/analysis software. The correct analysis of the network structure and connectivity needs precise segmentation of microscopic images. While segmentation of filament-like objects is a well-studied concept in biomedical imaging, where tracing of neurons and blood vessels is routine, there are comparatively fewer studies focusing on the segmentation of cytoskeletal filaments and networks from microscopic images. The developments in the fields of microscopy, computer vision and deep learning, however, began to facilitate the task, as reflected by an increase in the recent literature on the topic. Here, we aim to provide a short summary of the research on the (semi-)automated enhancement, segmentation and tracing methods that are particularly designed and developed for microscopic images of cytoskeletal networks. In addition to providing an overview of the conventional methods, we cover the recently introduced, deep-learning-assisted methods alongside the advantages they offer over classical methods.

7.
Commun Biol ; 4(1): 964, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385580

RESUMO

Production of biopharmaceuticals relies on the expression of mammalian cDNAs in host organisms. Here we show that the expression of a human cDNA in the moss Physcomitrium patens generates the expected full-length and four additional transcripts due to unexpected splicing. This mRNA splicing results in non-functional protein isoforms, cellular misallocation of the proteins and low product yields. We integrated these results together with the results of our analysis of all 32,926 protein-encoding Physcomitrella genes and their 87,533 annotated transcripts in a web application, physCO, for automatized optimization. A thus optimized cDNA results in about twelve times more protein, which correctly localizes to the ER. An analysis of codon preferences of different production hosts suggests that similar effects occur also in non-plant hosts. We anticipate that the use of our methodology will prevent so far undetected mRNA heterosplicing resulting in maximized functional protein amounts for basic biology and biotechnology.


Assuntos
Bryopsida/genética , DNA Complementar/genética , Plantas Geneticamente Modificadas/genética , Splicing de RNA , RNA Mensageiro/química , Bryopsida/química , DNA Complementar/química , Humanos , Plantas Geneticamente Modificadas/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética
8.
Comput Struct Biotechnol J ; 18: 2774-2788, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33101614

RESUMO

Sub-cellular mechanics plays a crucial role in a variety of biological functions and dysfunctions. Due to the strong structure-function relationship in cytoskeletal protein networks, light can be shed on their mechanical functionality by investigating their structures. Here, we present a data-driven approach employing a combination of confocal live imaging of fluorescent tagged protein networks, in silico mechanical experiments and machine learning to investigate this relationship. Our designed image processing and nanoFE mechanical simulation framework resolves the structure and mechanical behaviour of cytoskeletal networks and the developed gradient boosting surrogate models linking network structure to its functionality. In this study, for the first time, we perform mechanical simulations of Filamentous Temperature Sensitive Z (FtsZ) complex protein networks with realistic network geometry depicting its skeletal functionality inside organelles (here, chloroplasts) of the moss Physcomitrella patens. Training on synthetically produced simulation data enables predicting the mechanical characteristics of FtsZ network purely based on its structural features ( R 2 ⩾ 0.93 ), therefore allowing to extract structural principles enabling specific mechanical traits of FtsZ, such as load bearing and resistance to buckling failure in case of large network deformation.

9.
Acta Biomater ; 69: 206-217, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29378323

RESUMO

Traditionally, protein structures have been described by the secondary structure architecture and fold arrangement. However, the relatively novel method of 3D confocal microscopy of fluorescent-protein-tagged networks in living cells allows resolving the detailed spatial organization of these networks. This provides new possibilities to predict network functionality, as structure and function seem to be linked at various scales. Here, we propose a quantitative approach using 3D confocal microscopy image data to describe protein networks based on their nano-structural characteristics. This analysis is constructed in four steps: (i) Segmentation of the microscopic raw data into a volume model and extraction of a spatial graph representing the protein network. (ii) Quantifying protein network gross morphology using the volume model. (iii) Quantifying protein network components using the spatial graph. (iv) Linking these two scales to obtain insights into network assembly. Here, we quantitatively describe the filamentous temperature sensitive Z protein network of the moss Physcomitrella patens and elucidate relations between network size and assembly details. Future applications will link network structure and functionality by tracking dynamic structural changes over time and comparing different states or types of networks, possibly allowing more precise identification of (mal) functions or the design of protein-engineered biomaterials for applications in regenerative medicine. STATEMENT OF SIGNIFICANCE: Protein networks are highly complex and dynamic structures that play various roles in biological environments. Analyzing the detailed spatial structure of these networks may lead to new insight into biological functions and malfunctions. Here, we propose a tool set that extracts structural information at two scales of the protein network and allows therefore to address questions such as "how is the network built?" or "how networks grow?".


Assuntos
Bryopsida/metabolismo , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Imageamento Tridimensional
10.
Sci Rep ; 8(1): 11165, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-30042487

RESUMO

Although the concept of the cytoskeleton as a cell-shape-determining scaffold is well established, it remains enigmatic how eukaryotic organelles adopt and maintain a specific morphology. The Filamentous Temperature Sensitive Z (FtsZ) protein family, an ancient tubulin, generates complex polymer networks, with striking similarity to the cytoskeleton, in the chloroplasts of the moss Physcomitrella patens. Certain members of this protein family are essential for structural integrity and shaping of chloroplasts, while others are not, illustrating the functional diversity within the FtsZ protein family. Here, we apply a combination of confocal laser scanning microscopy and a self-developed semi-automatic computational image analysis method for the quantitative characterisation and comparison of network morphologies and connectivity features for two selected, functionally dissimilar FtsZ isoforms, FtsZ1-2 and FtsZ2-1. We show that FtsZ1-2 and FtsZ2-1 networks are significantly different for 8 out of 25 structural descriptors. Therefore, our results demonstrate that different FtsZ isoforms are capable of generating polymer networks with distinctive morphological and connectivity features which might be linked to the functional differences between the two isoforms. To our knowledge, this is the first study to employ computational algorithms in the quantitative comparison of different classes of protein networks in living cells.


Assuntos
Bryopsida/citologia , Bryopsida/metabolismo , Proteínas de Plantas/metabolismo , Mapas de Interação de Proteínas , Algoritmos , Cloroplastos/metabolismo , Biologia Computacional/métodos , Citoesqueleto/metabolismo , Expressão Gênica , Técnicas de Inativação de Genes , Genes de Plantas , Microscopia Confocal , Fenótipo , Proteínas de Plantas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Protoplastos
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