30 research outputs found
Purification and quantitative proteomic analysis of cell bodies and protrusions
No full textSummary: Actin-rich protrusions are membrane extensions generated by actin polymerization that drive mesenchymal-like cell migration. Characterization of protrusions proteome is crucial for understanding their function. We present a complete step-by-step protocol based on microporous filter-based fractionation of protrusive cellular domains coupled with sample preparation for quantitative proteomics, mass spectrometric data acquisition, and data analysis. This protocol enables purification, quantification, and analysis of the distribution of proteins present in protrusions and cell bodies.For complete details on the use and execution of this protocol, please refer to Dermit et al. (2020)
Subcellular mRNA localization regulates ribosome biogenesis in migrating cells
No full textDermit et al. reveal that ribosomal protein (RP)-mRNAs localize to the protrusive fronts of migratory cells, where their translation is locally increased, leading to upregulation of ribosome biogenesis and protein synthesis. In aggressive carcinomas, this pathway is upregulated in order to support the high anabolic demands of invasive cancer cells
Understanding the biochemical alterations in cancer cells chronically treated with PI3K/mTOR inhibitors
No full textPhDThe PI3K/mTOR signalling pathway plays a major role in biology and disease. Therefore, effective
inhibitors that target proteins of this pathway have been developed. However, acquired resistance of
cancer cells is a prevalent phenomenon that limits the durable response of these compounds.
It is becoming apparent that experimental approaches for comprehensive biochemical analysis
contribute to understand the complex mechanisms that confer drug resistance, and advances in largescale
technologies including genomic sequencing and proteomics allow unprecedented molecular
coverage without being biased for specific genes/cellular pathways.
Initially, the phenotypic response of sensitive and resistant cells to the absence or presence of a
PI3K inhibitor (PI3Ki), as well as other kinases, was examined. This study revealed that PI3Ki-resistant
cells experience extensive phenotypic changes upon withdrawal of the PI3Ki from the culture media.
The regulation of the proteome and phosphoproteome of sensitive and PI3Ki-resistant cells grown
with or without the PI3Ki was analysed by shotgun mass spectrometry-based label-free quantitative
technology. This analysis demonstrated that the proteomes and phosphoproteomes of drug-resistant
cells are remodelled conditional to the presence of PI3Ki, and that the levels of enzymes with
metabolic roles are modulated in resistant cells. Functional analysis of the metabolism of cells capable
to survive in absence of PI3K/mTOR activity demonstrated that the bioenergetic activity of these cells
is contingent on the presence of the selection drug.
The complete set of protein-coding regions of the genome (exome) of sensitive and PI3Ki-resistant
cells was then sequenced. This study unveiled common alterations in exome regions across PI3Kiresistant
cell lines, as well as a degree of genomic heterogeneity between them.
Lastly, the impact of lactic acid, a metabolic product, on a defined signalling network of the MCF7
breast cancer cell line was analysed. This study described the capacity of this metabolite to change
the activity of signalling network branches
Methods for monitoring and measurement of protein translation in time and space
No full textRegulation of protein translation constitutes a crucial step in control of gene expression. In comparison
to transcriptional regulation, however, translational control has remained a significantly under-studied
layer of gene expression. This trend is now beginning to shift thanks to recent advances in nextgeneration sequencing, proteomics, and microscopy based methodologies which allow accurate
monitoring of protein translation rates, from single target messenger RNA molecules to genome-wide
scale studies. In this review, we summarize these recent advances, and discuss how they are enabling
researchers to study translational regulation in a wide variety of in vitro and in vivo biological systems,
with unprecedented depth and spatiotemporal resolution.The authors are funded by a Medical Research Council (MRC) Career Development Award to F. K. M. (MR/P009417/1)
Years Teachers' Late Concerns and Perceived Needs in Science: An Exploratory Study’, European
No full textScience in early childhood is of great importance to many aspects of children's development. Planning for improvement of science instruction should take into consideration the problems teachers encounter in their performance of their task. However, exposition of problems of early years teachers, especially those with many years of experience, is somewhat rare in science education literature. The purpose of the present work was to investigate those factors that hinder experienced early years teachers in the performance of their task in science, and their needs, as perceived by the teachers themselves. The study was carried out in Greece. One take-home written task and two group interviews were used for data collection. Qualitative approaches were employed for the analysis of the data. The emergent themes related to teachers' problems, teachers' worries induced by these problems, problem consequences and teachers' perceived needs in science. La science sur la précoce enfance est très importante pour beaucoup des aspects de la développe-ment des enfants. Il faut que le croquis de l'instruction de la science aille considérer les problèmes que les instituteurs affrontent. Mais, dans la littérature de l'instruction de la science, il n'y a pas beaucoup des instituteurs, spécialement des expérimentes, qui exposent leurs problèmes. Le but de présent travail était l'investigation de ces facteurs qui empêchent les instituteurs expérimentes de travailler dans le secteur de la science sur la précoce enfance; et, aussi, leurs besoins, conformément a eux-mêmes. Le travail était réalisé en Grèce. Un travail, écrit à la maison, et deux interviews des groups étaient utilisées pour la collection des éléments. les résultats concernaient les problèmes des instituteurs, les inquiétudes des instituteurs à cause de ces problèmes, les conséquences des ces problèmes et les besoins des instituteurs de la science sur la précoce enfance. La ciencia en la primera infancia es de gran importancia en muchos aspectos del desarrollo de los niñ os. La planificación para mejorar la ciencia de la educación debe tomar en consideración los problemas que encuentran los profesores en la realización de su tarea. Sin embargo, la exposición de problemas de las profesores de primera infancia, especialmente la de los que tienen muchos anos de experiencia, es algo rara en la literatura de la ciencia de la educación. El propó sito del actual trabajo era investigar esos factores que obstaculizan a profesores experimentados de primera infancia en la realización de su tarea en la ciencia y de sus necesidades, segú n lo percibido por los mismos profesores. El estudio se llevo a cabo en Grecia. Una tarea escrita para realizar en casa y dos entrevistas del grupo se utilizaron para la recogida de datos. Se emplearon aproximaciones cualitativas para el análisis de los datos. Los temas que surgieron se relacionan 148 M. Kallery con los problemas de los profesores, las preocupaciones de los profesores inducidos por estos problemas, las consecuencias del problema y las necesidades percibidas por los profesores en la ciencia. Die Naturwissenschaften sind von großer Bedeutung im frü heren Kindesalter bei vielen Aspekten der Kinderentwicklung. BeimPlanen derUnterrichtsvorbereitung mü ssen die Probleme, mit denen sich die Lehrer auseinandersetzen, mitberü cksichtigt werden. Doch die Bekanntgebung der Probleme der Vorschulerzieher, vorallem dermit langer Berufserfahrung kommt selten in der Erziehungsbibliographie der Naturwissenschaften vor. Diese Studie hatte als Ziel die Faktoren zu erforschen, die die Erzieher bei ihrer Arbeit hindern und ihre Bedü rfnisse wie sie von den Erziehern selbst wahrgenommen werden. Die studie wurde in Griechenland durchgefü hrt. Ein schriftliches Referat und zwei Gruppeninterviews dienten der Angabensammlung. Qualitative Annäherungen wurden bei der Angabenanalyse verwendet. Die Themen, die sich ergaben, beziehen sich auf die Probleme der Erzieher, ihre Besorgnis wegen der Probleme, auf die Problemkonsequenzen und auf die Bedü rfnisse der Erzieher in den Naturwissenschaften, wi sie von ihnen selbst wahrgenommen werden
TREX reveals proteins that bind to specific RNA regions in living cells.
No full textDifferent regions of RNA molecules can often engage in specific interactions with distinct RNA-binding proteins (RBPs), giving rise to diverse modalities of RNA regulation and function. However, there are currently no methods for unbiased identification of RBPs that interact with specific RNA regions in living cells and under endogenous settings. Here we introduce TREX (targeted RNase H-mediated extraction of crosslinked RBPs)-a highly sensitive approach for identifying proteins that directly bind to specific RNA regions in living cells. We demonstrate that TREX outperforms existing methods in identifying known interactors of U1 snRNA, and reveals endogenous region-specific interactors of NORAD long noncoding RNA. Using TREX, we generated a comprehensive region-by-region interactome for 45S rRNA, uncovering both established and previously unknown interactions that regulate ribosome biogenesis. With its applicability to different cell types, TREX is an RNA-centric tool for unbiased positional mapping of endogenous RNA-protein interactions in living cells
An ERK1/2-driven RNA-binding switch in nucleolin drives ribosome biogenesis and pancreatic tumorigenesis downstream of RAS oncogene
No full textOncogenic RAS signaling reprograms gene expression through both transcriptional and post-transcriptional mechanisms. While transcriptional regulation downstream of RAS is relatively well characterized, how RAS post-transcriptionally modulates gene expression to promote malignancy remains largely unclear. Using quantitative RNA interactome capture analysis, we here reveal that oncogenic RAS signaling reshapes the RNA-bound proteomic landscape of pancreatic cancer cells, with a network of nuclear proteins centered around nucleolin displaying enhanced RNA-binding activity. We show that nucleolin is phosphorylated downstream of RAS, which increases its binding to pre-ribosomal RNA (rRNA), boosts rRNA production, and promotes ribosome biogenesis. This nucleolin-dependent enhancement of ribosome biogenesis is crucial for RAS-induced pancreatic cancer cell proliferation and can be targeted therapeutically to inhibit tumor growth. Our results reveal that oncogenic RAS signaling drives ribosome biogenesis by regulating the RNA-binding activity of nucleolin and highlight a crucial role for this mechanism in RAS-mediated tumorigenesis
