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Analysis of mRNA Subcellular Distribution in Collective Cell Migration.

Joshua J Bradbury1, Holly E Lovegrove1, Marta Giralt-Pujol1

  • 1Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK.

Methods in Molecular Biology (Clifton, N.J.)
|January 18, 2023
PubMed
Summary

Collective cell migration involves specialized cell roles driven by gene expression control. This study details methods using single-molecule fluorescent in situ hybridization (smFISH) and immunofluorescence in angiogenesis models to visualize RNA and protein localization.

Keywords:
AngiogenesisCollective cell migrationEndothelial cellFibrin gel bead assayImmunofluorescenceRNA localizationScratch wound assaysmFISH

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Developmental Biology

Background:

  • Collective cell migration requires cell specialization and division of labor.
  • Spatiotemporal control of gene expression is crucial for establishing distinct cell states.
  • Sprouting angiogenesis serves as a model for collective cell migration.

Purpose of the Study:

  • To describe methods for investigating RNA and protein localization during collective cell migration.
  • To enable high-resolution imaging of gene expression in angiogenesis models.
  • To understand the relationship between RNA and protein distribution in migrating endothelial cells.

Main Methods:

  • Utilizing in vitro angiogenesis models.
  • Employing single-molecule fluorescent in situ hybridization (smFISH) for RNA quantification and localization.
  • Combining smFISH with immunofluorescence microscopy for simultaneous RNA and protein analysis.

Main Results:

  • Demonstration of smFISH and immunofluorescence applicability in angiogenesis models.
  • Visualization of RNA and protein spatial distribution during endothelial collective cell migration.
  • Method allows for quantification of RNA number and spatial distribution relative to protein.

Conclusions:

  • The described methods facilitate detailed investigation of gene expression dynamics in collective cell migration.
  • This approach enhances understanding of cellular mechanisms underlying angiogenesis.
  • Precise spatiotemporal analysis of RNA and protein is key to unraveling cell state acquisition during migration.