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1.
Biomedicines ; 10(1)2022 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-35052817

RESUMO

Calcium (Ca2+) elevation is an essential secondary messenger in many cellular processes, including disease progression and adaptation to external stimuli, e.g., gravitational load. Therefore, mapping and quantifying Ca2+ signaling with a high spatiotemporal resolution is a key challenge. However, particularly on microgravity platforms, experiment time is limited, allowing only a small number of replicates. Furthermore, experiment hardware is exposed to changes in gravity levels, causing experimental artifacts unless appropriately controlled. We introduce a new experimental setup based on the fluorescent Ca2+ reporter CaMPARI2, onboard LED arrays, and subsequent microscopic analysis on the ground. This setup allows for higher throughput and accuracy due to its retrograde nature. The excellent performance of CaMPARI2 was demonstrated with human chondrocytes during the 75th ESA parabolic flight campaign. CaMPARI2 revealed a strong Ca2+ response triggered by histamine but was not affected by the alternating gravitational load of a parabolic flight.

2.
Dis Model Mech ; 5(6): 940-7, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22563063

RESUMO

Nonsense mutations that result in the expression of truncated, N-terminal, fragments of the adenomatous polyposis coli (APC) tumour suppressor protein are found in most sporadic and some hereditary colorectal cancers. These mutations can cause tumorigenesis by eliminating ß-catenin-binding sites from APC, which leads to upregulation of ß-catenin and thereby results in the induction of oncogenes such as MYC. Here we show that, in three distinct experimental model systems, expression of an N-terminal fragment of APC (N-APC) results in loss of directionality, but not speed, of cell motility independently of changes in ß-catenin regulation. We developed a system to culture and fluorescently label live pieces of gut tissue to record high-resolution three-dimensional time-lapse movies of cells in situ. This revealed an unexpected complexity of normal gut cell migration, a key process in gut epithelial maintenance, with cells moving with spatial and temporal discontinuity. Quantitative comparison of gut tissue from wild-type mice and APC heterozygotes (APC(Min/+); multiple intestinal neoplasia model) demonstrated that cells in precancerous epithelia lack directional preference when moving along the crypt-villus axis. This effect was reproduced in diverse experimental systems: in developing chicken embryos, mesoderm cells expressing N-APC failed to migrate normally; in amoeboid Dictyostelium, which lack endogenous APC, expressing an N-APC fragment maintained cell motility, but the cells failed to perform directional chemotaxis; and multicellular Dictyostelium slug aggregates similarly failed to perform phototaxis. We propose that N-terminal fragments of APC represent a gain-of-function mutation that causes cells within tissue to fail to migrate directionally in response to relevant guidance cues. Consistent with this idea, crypts in histologically normal tissues of APC(Min/+) intestines are overpopulated with cells, suggesting that a lack of migration might cause cell accumulation in a precancerous state.


Assuntos
Proteína da Polipose Adenomatosa do Colo/química , Proteína da Polipose Adenomatosa do Colo/metabolismo , Movimento Celular , Transformação Celular Neoplásica/patologia , Genes Dominantes , Modelos Animais , Fragmentos de Peptídeos/metabolismo , Polipose Adenomatosa do Colo/patologia , Animais , Transformação Celular Neoplásica/metabolismo , Embrião de Galinha , Dictyostelium/citologia , Dictyostelium/metabolismo , Enterócitos/metabolismo , Enterócitos/patologia , Feminino , Intestino Delgado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linha Primitiva/metabolismo , Linha Primitiva/patologia
3.
J Cell Biol ; 176(2): 183-95, 2007 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17227893

RESUMO

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene initiate a majority of colorectal cancers. Acquisition of chromosomal instability is an early event in these tumors. We provide evidence that the loss of APC leads to a partial loss of interkinetochore tension at metaphase and alters mitotic progression. Furthermore, we show that inhibition of APC in U2OS cells compromises the mitotic spindle checkpoint. This is accompanied by a decrease in the association of the checkpoint proteins Bub1 and BubR1 with kinetochores. Additionally, APC depletion reduced apoptosis. As expected from this combination of defects, tetraploidy and polyploidy are consequences of APC inhibition in vitro and in vivo. The removal of APC produced the same defects in HCT116 cells that have constitutively active beta-catenin. These data show that the loss of APC immediately induces chromosomal instability as a result of a combination of mitotic and apoptotic defects. We suggest that these defects amplify each other to increase the incidence of tetra- and polyploidy in early stages of tumorigenesis.


Assuntos
Proteína da Polipose Adenomatosa do Colo/deficiência , Apoptose/fisiologia , Mitose/fisiologia , Poliploidia , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Caspase 3/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Ciclina B/metabolismo , Ciclina B1 , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HCT116 , Histonas/análise , Humanos , Mucosa Intestinal/metabolismo , Intestinos/química , Intestinos/patologia , Camundongos , Camundongos Transgênicos , Mitose/efeitos dos fármacos , Mitose/genética , Modelos Biológicos , Nocodazol/farmacologia , Paclitaxel/farmacologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , RNA Interferente Pequeno/genética , Fuso Acromático/metabolismo , Estaurosporina/farmacologia , beta Catenina/análise , beta Catenina/metabolismo
4.
Biotechniques ; 41(2): 199-208, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16925022

RESUMO

Biomedical research and drug development increasingly involve the extraction of quantitative data from digital microscope images, such as those obtained using fluorescence microscopy. Here, we describe a novel approach for both managing and analyzing such images. The Open Microscopy Environment (OME) is a sophisticated open-source scientific image management database that coordinates the organization, storage, and analysis of the large volumes of image data typically generated by modern imaging methods. We describe FindSpots, a powerful image-analysis package integrated in OME that will be of use to those who wish to identify and measure objects within microscope images or time-lapse movies. The algorithm used in FindSpots is in fact only one of many possible segmentation (object detection) algorithms, and the underlying data model used by OME to capture and store its results can also be used to store results from other segmentation algorithms. In this report, we illustrate how image segmentation can be achieved in OME using one such implementation of a segmentation algorithm, and how this output subsequently can be displayed graphically or processed numerically using a spreadsheet.


Assuntos
Biologia Computacional , Interpretação de Imagem Assistida por Computador , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Microscopia de Fluorescência , Algoritmos , Animais , Sistemas de Gerenciamento de Base de Dados , Bases de Dados Factuais , Fibroblastos/metabolismo , Corantes Fluorescentes , Indóis , Armazenamento e Recuperação da Informação , Cinetocoros/metabolismo , Camundongos , Proteínas Quinases/análise , Proteínas Serina-Treonina Quinases , Reprodutibilidade dos Testes , Interface Usuário-Computador
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