RESUMO
Glioblastomas exhibit remarkable heterogeneity at various levels, including motility modes and mechanoproperties that contribute to tumor resistance and recurrence. In a recent study using gridded micropatterns mimicking the brain vasculature, glioblastoma cell motility modes, mechanical properties, formin content, and substrate chemistry are linked. Now is presented, SP2G (SPheroid SPreading on Grids), an analytic platform designed to identify the migratory modes of patient-derived glioblastoma cells and rapidly pinpoint the most invasive sub-populations. Tumorspheres are imaged as they spread on gridded micropatterns and analyzed by this semi-automated, open-source, Fiji macro suite that characterizes migration modes accurately. SP2G can reveal intra-patient motility heterogeneity with molecular correlations to specific integrins and EMT markers. This system presents a versatile and potentially pan-cancer workflow to detect diverse invasive tumor sub-populations in patient-derived specimens and offers a valuable tool for therapeutic evaluations at the individual patient level.
RESUMO
Cellular movement is essential for many vital biological functions where it plays a pivotal role both at the single cell level, such as during division or differentiation, and at the macroscopic level within tissues, where coordinated migration is crucial for proper morphogenesis. It also has an impact on various pathological processes, one for all, cancer spreading. Cell migration is a complex phenomenon and diverse experimental methods have been developed aimed at dissecting and analysing its distinct facets independently. In parallel, corresponding analytical procedures and tools have been devised to gain deep insight and interpret experimental results. Here we review established experimental techniques designed to investigate specific aspects of cell migration and present a broad collection of historical as well as cutting-edge computational tools used in quantitative analysis of cell motion.
RESUMO
Long non-coding RNAs (lncRNAs) constitute the largest and most diverse class of non-coding RNAs. They localize to the nucleus, cytoplasm, or both compartments, and regulate gene expression through various mechanisms at multiple levels. LncRNAs tend to evolve faster and present higher tissue- and developmental stage-specific expression than protein-coding genes. Initially considered byproducts of erroneous transcription without biological function, lncRNAs are now recognized for their involvement in numerous biological processes, such as immune response, apoptosis, pluripotency, reprogramming, and differentiation. In this study, we focused on Heart Brake lncRNA 1 (HBL1), a lncRNA recently reported to modulate the process of pluripotent stem cell differentiation toward cardiomyocytes. We employed RT-qPCR and high-resolution RNA FISH to monitor the expression and localization of HBL1 during the differentiation progression. Our findings indicate a significant increase in HBL1 expression during mesodermal and cardiac mesodermal stages, preceding an anticipated decrease in differentiated cells. We detected the RNA in discrete foci in both the nucleus and in the cytoplasm. In the latter compartment, we observed colocalization of HBL1 with Y-box binding protein 1 (YB-1), which likely results from an interaction between the RNA and the protein, as the two were found to be coimmunoprecipitated in RNP-IP experiments. Finally, we provide evidence that HBL1, initially reported as an independent lncRNA gene, is part of the LINC00458 (also known as lncRNA-ES3 or ES3) gene, forming the last exon of some LINC00458 splice isoforms.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , RNA Longo não Codificante , Miócitos Cardíacos/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação Celular/genética , Células-Tronco Pluripotentes/metabolismoRESUMO
Amphiphysin 2 (BIN1) is a membrane and actin remodeling protein mutated in congenital and adult centronuclear myopathies. Here, we report an unexpected function of this N-BAR domain protein BIN1 in filopodia formation. We demonstrated that BIN1 expression is necessary and sufficient to induce filopodia formation. BIN1 is present at the base of forming filopodia and all along filopodia, where it colocalizes with F-actin. We identify that BIN1-mediated filopodia formation requires IRSp53, which allows its localization at negatively-curved membrane topologies. Our results show that BIN1 bundles actin in vitro. Finally, we identify that BIN1 regulates the membrane-to-cortex architecture and functions as a molecular platform to recruit actin-binding proteins, dynamin and ezrin, to promote filopodia formation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas do Tecido Nervoso , Proteínas Nucleares , Pseudópodes , Proteínas Supressoras de Tumor , Humanos , Animais , Células HeLa , Linhagem Celular , Actinas/metabolismo , Pseudópodes/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Membrana Celular/metabolismoRESUMO
In eukaryotes, cytoplasmic and nuclear volumes are tightly regulated to ensure proper cell homeostasis. However, current methods to measure cytoplasmic and nuclear volumes, including confocal 3D reconstruction, have limitations, such as relying on two-dimensional projections or poor vertical resolution. Here, to overcome these limitations, we describe a method, N2FXm, to jointly measure cytoplasmic and nuclear volumes in single cultured adhering human cells, in real time, and across cell cycles. We find that this method accurately provides joint size over dynamic measurements and at different time resolutions. Moreover, by combining several experimental perturbations and analyzing a mathematical model including osmotic effects and tension, we show that N2FXm can give relevant insights on how mechanical forces exerted by the cytoskeleton on the nuclear envelope can affect the growth of nucleus volume by biasing nuclear import. Our method, by allowing for accurate joint nuclear and cytoplasmic volume dynamic measurements at different time resolutions, highlights the non-constancy of the nucleus/cytoplasm ratio along the cell cycle.