Analysis of RNA distribution between pseudopodia and cell bodies in response to Fibronectin
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE10227 | GEO | 2008/03/18
SECONDARY ACCESSION(S): PRJNA109015
REPOSITORIES: GEO
Similar Datasets
Project description:The goal of the study was to identify on a genome-wide scale RNAs that are enriched at the leading edge of migrating cells. For this, we employed a fractionation method in which cells are plated on a microporous filter whose bottom side only is coated with fibronectin. The cells thus polarize and extend pseudopodial protrusions towards the bottom surface. These protruding pseudopodia can then be physically isolated from the bottom surface of the filter and their contents compared with the remaining cell bodies, which are isolated from the upper surface of the filter. Experiment Overall Design: We analyzed 2 independent pseudopodia samples (samples 06-23_PS_A and 06-23_PS_B) and 2 independent cell body samples (samples 06-23_CB_A and 06-23_CB_B)
2008-04-05 | E-GEOD-10227 | biostudies-arrayexpress
Project description:The goal of the study was to identify on a genome-wide scale RNAs that are enriched at the leading edge of migrating cells. For this, we employed a fractionation method in which serum-starved cells are first plated and allowed to spread on a microporous filter. Addition of LPA at the bottom side of the filter induces the cells to polarize and extend pseudopodial protrusions. These protruding pseudopodia can then be physically isolated from the bottom surface of the filter and their contents compared with the remaining cell bodies, which are isolated from the upper surface of the filter. Experiment Overall Design: We analyzed two independent pseudopodia samples (05-61_PS_A and 06-04_PS_A) and two independent cell body samples (05-61_CB_A and 06-04_CB_A)
2008-04-05 | E-GEOD-10226 | biostudies-arrayexpress
Project description:The goal of the study was to identify on a genome-wide scale RNAs that are enriched at the leading edge of migrating cells. For this, we employed a fractionation method in which serum-starved cells are first plated and allowed to spread on a microporous filter. Addition of LPA at the bottom side of the filter induces the cells to polarize and extend pseudopodial protrusions. These protruding pseudopodia can then be physically isolated from the bottom surface of the filter and their contents compared with the remaining cell bodies, which are isolated from the upper surface of the filter. Keywords: comparative RNA distribution
2008-03-18 | GSE10226 | GEO
Project description:mRNA trafficking and local protein translation are associated with protrusive cellular domains, such as neuronal growth cones, and deregulated control of protein translation is associated with tumor malignancy. We show here that activated RhoA, but not Rac1, is enriched in pseudopodia of MSV-MDCK-INV tumor cells and that Rho, Rho kinase (ROCK) and myosin II regulate the microtubule-independent targeting of RNA to these tumor cell domains. ROCK inhibition does not affect pseudopodial actin turnover but significantly reduces the dynamics of pseudopodial RNA turnover. Gene array analysis shows that 7.3% of the total genes analyzed exhibited a greater than 1.6-fold difference between the pseudopod and cell body fractions. Of these, only 13.2% (261 genes) are enriched in pseudopodia, suggesting that only a limited number of total cellular mRNAs are enriched in tumor cell protrusions. Comparison of the tumor pseudopod mRNA cohort and a cohort of mRNAs enriched in neuronal processes identified tumor pseudopod-specific signaling networks that were defined by expression of M-Ras and the Shp2 protein phosphatase. Pseudopod expression of M-Ras and Shp2 mRNA were diminished by ROCK inhibition linking pseudopodial Rho/ROCK activation to the localized expression of specific mRNAs. Pseudopodial enrichment for mRNAs involved in protein translation and signaling suggests that local mRNA translation regulates pseudopodial expression of less stable signaling molecules as well as the cellular machinery to translate these mRNAs. mRNA trafficking and local protein translation are associated with protrusive cellular domains, such as neuronal growth cones, and deregulated control of protein translation is associated with tumor malignancy. We show here that activated RhoA, but not Rac1, is enriched in pseudopodia of MSV-MDCK-INV tumor cells and that Rho, Rho kinase (ROCK) and myosin II regulate the microtubule-independent targeting of RNA to these tumor cell domains. ROCK inhibition does not affect pseudopodial actin turnover but significantly reduces the dynamics of pseudopodial RNA turnover. Gene array analysis shows that 7.3% of the total genes analyzed exhibited a greater than 1.6-fold difference between the pseudopod and cell body fractions. Of these, only 13.2% (261 genes) are enriched in pseudopodia, suggesting that only a limited number of total cellular mRNAs are enriched in tumor cell protrusions. Comparison of the tumor pseudopod mRNA cohort and a cohort of mRNAs enriched in neuronal processes identified tumor pseudopod-specific signaling networks that were defined by expression of M-Ras and the Shp2 protein phosphatase. Pseudopod expression of M-Ras and Shp2 mRNA were diminished by ROCK inhibition linking pseudopodial Rho/ROCK activation to the localized expression of specific mRNAs. Pseudopodial enrichment for mRNAs involved in protein translation and signaling suggests that local mRNA translation regulates pseudopodial expression of less stable signaling molecules as well as the cellular machinery to translate these mRNAs. Pseudopodial Rho/ROCK activation may impact on tumor cell migration and metastasis by stimulating the pseudopodial translocation of mRNAs and thereby regulating the expression of local signaling cascades. Keywords: tumor cells, protrusive cellular domains, pseudopodia vs cell body, RNA trafficking Pseudopod purification- Pseudopodia purification was performed as previously described (Jia et al, J. Biol. Chem. 280, 30564-73, 2005). Briefly, 107 MSV-MDCK-INV cells were plated on 100 mm 1um pore filters mounted between two 3-3.5â custom-made washers (Boulons Plus, Anjou, QC) in a 100 mm Falcon Petri dish and the exterior sealed with agarose to prevent cell leakage to the bottom of the filter. After 24 hours culture, the filter was washed 4 times with cold PBS containing 0.1 mM Ca2+ and 1 mM Mg2+ and the top (cell bodies) and bottom (pseudopodia) of the filter scraped with a glass cover slip in ice-cold lysis buffer (20 mM Tris-HCl pH 7.6, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA containing freshly added 2 mM DTT, 0.5 mM PMSF, 1 mM sodium vanadate, 2.5 mM sodium fluoride and 1uM leupeptin). Affymetrix gene array- GeneChips were processed at the London Regional Genomics Centre (Robarts Research Institute, London, Ontario, Canada; http://www.lrge.ca). Six Affymetrix canine arrays (3 pseudopodia, 3 cell body) were scanned with the Affymetrix GeneChip Scanner 3000 and signal intensities for genes generated with GCOS1.2 (Affymetrix Inc., Santa Clara, CA) using default values for statistical expression algorithm parameters, a target signal of 150 for all probe sets and a normalization value of 1. To determine significant differences in gene expression levels between pseudopodia and cell body samples, 1 way-ANOVA was performed using Partek Pro (St. Louis, MO). Significantly modulated genes were defined as those with absolute log2 fold change greater than 0.7. Genes were summarized together using hierarchical clustering by Z score normalized data crossing all 6 samples.
2010-05-18 | E-GEOD-13253 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series:; GSE10226: Analysis of RNA distribution between pseudopodia and cell bodies in response to LPA stimulation; GSE10227: Analysis of RNA distribution between pseudopodia and cell bodies in response to Fibronectin Experiment Overall Design: Refer to individual Series
2008-04-05 | E-GEOD-10230 | biostudies-arrayexpress
Project description:This upload concerns 2 independent but related experiments that were searched together: a) To reveal proteins that consistently localise to actin-rich protrusions across different human migratory cell-lines, we profiled the distribution of cellular proteins between protrusions and cell-bodies in a panel of 5 established human cell-lines (see experimental design). Cell lines were seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to the filter overnight. The next day, the media on top of the cells was refreshed, and media was also added to the bottom chamber in order to open the pores and allow formation of protrusions. Cells were allowed to form protrusions for 2 hrs before being fixed with ice-cold methanol. Cells were then rehydrated in PBS, followed by independent lysis of protrusions and cell-bodies on opposite sides of the filters in 2% SDS, 100mM Tris pH 7.5 b) To analyse the temporal dynamics of protein localisation to cell protrusions, we carried out a time-course spatial proteomics analysis of protrusions and cell-bodies in MDA-MB231 cells. Cells were seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to filter overnight. The next day, the media on top of cells was refreshed, and the filters were either left with no media in the bottom chamber (closed pores), or media added to the bottom chamber (open pores) for different lengths of times (1, 2, 4 , & 8 hrs) to induce protrusion formation. Cells were then fixed with ice-cold methanol, rehydrated in PBS, followed by independent lysis of protrusions and cell-bodies on opposite sides of each filter by 2% SDS, 100mM Tris pH 7.5.
2020-10-20 | PXD021239 | Pride
Project description:We quantified the distribution of cellular mRNAs between protrusions and cell-body of MDA-MB-231 breast carcinoma cells by RNA-seq, using a filter based fractionation method. In this method, cells are seeded on top of 3micrometer transwell filters to enable protrusions to form but to prevent the cell-bodies passing through due to the small size of the pores, thus resulting in separation of protrusions and cell-bodies on opposite sides of the filter, from which RNA can be prepared separately. RNA samples for Illumina sequencing were generated in duplicates from two biological replicate experiments (two protrusions and two cell-body).
2015-11-08 | E-MTAB-2546 | biostudies-arrayexpress
Project description:To assess the impact of protrusion induction on the proteome of mesenchymal-like migratory cells, MDA-MB231 breast cancer cells were seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to filter overnight. The next day, the media on top of cells was refreshed, and the filters were either left with no media in the bottom chamber (closed pores), or with media also added to the bottom chamber (open pores) in order to allow formation of protrusions. Cells were allowed to form protrusions for 2 or 24 hrs, before being lysed and analysed by mass spectrometry, using TMT mediated quantitative proteomics.
2020-10-20 | PXD021206 | Pride
Project description:To assess if LARP6 is required for upregulation of ribosomal proteins upon protrusion induction, MDA-MB231 breast cancer cells were treated with non-targeting (NT) or LARP6 siRNA for 48 hrs, before being seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to filter overnight. The next day, the media on top of cells was refreshed, and the filters were either left with no media in the bottom chamber (closed pores), or with media also added to the bottom chamber (open pores) in order to allow formation of protrusions. Cells were allowed to form protrusions for 12 hrs before being lysed and analysed by mass spectrometry.
2020-10-20 | PXD021180 | Pride
Project description:To assess the impact of protrusion formation on protein synthesis rates on a global scale, we carried out a pulsed-SILAC (pSILAC) analysis in MDA-MB231 cells grown with or without protrusions for 1, 2, 4, and 8 hrs. Briefly, light SILAC labelled cells were seeded on top of 2x 3 micron transwell filters without any media added to the bottom chamber. Cells were allowed to attach to the filter overnight, and the next day the media on the top was changed to medium (M) or heavy(H) SILAC DMEM. At the same time, the H SILAC media was also added to the bottom chamber of the transwell with H media on the top to induce protrusions. Cells were then allowed to form protrusions for 1, 2, 4, or 8 hrs, before being lysed and mixed with the M labelled lysates (cells without protrusions) from the same timepoint. The experiment was then repeated with switched SILAC labelling.
2020-10-20 | PXD021203 | Pride