Proteomics,Multiomics

Dataset Information

0

Protrusion vs Cell-body pulsed-SILAC proteomics


ABSTRACT: In mesenchymal-like cell migration, cells need to polarise into a protrusive front and a retracting cell body. Apart from proteins, several mRNAs are known to be localised to cell protrusions but to what extent RNA localisation and local translation contributes toward front-back assymetry is unknown. We set out to quantify the relative translation rates of cellular proteins between protrusions and cell-body by Pulsed-SILAC proteomics, using MDA-MB-231 cells, a highly invasive breast cancer cell-line. We utilised a transwell filter based fractionation method in conjugation with pulsed-SILAC. In this method, cells are seeded on top of 3μm transwell filters to enable protrusions to form through the pores of the filters 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, which can then be lysed and prepared separately. Prepration of protrusion and cell-body fractions from SILAC heavy vs. medium label pulsed cells then allows for reciprocal mixing and quantification of nascent proteins between protrusions and cell-body. We determined the relative local translation rates of 1150 proteins between protrusions and cell-body from two pulsed-SILAC mixes.

OTHER RELATED OMICS DATASETS IN: MSV000079570

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Faraz Mardakheh  

LAB HEAD: Christopher J Marshall

PROVIDER: PXD002649 | Pride | 2015-10-09

REPOSITORIES: Pride

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Publications

Global Analysis of mRNA, Translation, and Protein Localization: Local Translation Is a Key Regulator of Cell Protrusions.

Mardakheh Faraz K FK   Paul Angela A   Kümper Sandra S   Sadok Amine A   Paterson Hugh H   Mccarthy Afshan A   Yuan Yinyin Y   Marshall Christopher J CJ  

Developmental cell 20151101 3


Polarization of cells into a protrusive front and a retracting cell body is the hallmark of mesenchymal-like cell migration. Many mRNAs are localized to protrusions, but it is unclear to what degree mRNA localization contributes toward protrusion formation. We performed global quantitative analysis of the distributions of mRNAs, proteins, and translation rates between protrusions and the cell body by RNA sequencing (RNA-seq) and quantitative proteomics. Our results reveal local translation as a  ...[more]

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