Analysis of mRNA Subcellular Distribution in Collective Cell Migration

Joshua Bradbury, Holly Lovegrove, Marta Giralt-Pujol, Shane Herbert

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review


The movement of groups of cells by collective cell migration requires division of labor between group members. Therefore, distinct cell identities, unique cell behaviors, and specific cellular roles are acquired by cells undergoing collective movement. A key driving force behind the acquisition of discrete cell states is the precise control of where, when, and how genes are expressed, both at the subcellular and supracellular level. Unraveling the mechanisms underpinning the spatiotemporal control of gene expression in collective cell migration requires not only suitable experimental models but also high-resolution imaging of messenger RNA and protein localization during this process. In recent times, the highly stereotyped growth of new blood vessels by sprouting angiogenesis has become a paradigm for understanding collective cell migration, and consequently this has led to the development of numerous user-friendly in vitro models of angiogenesis. In parallel, single-molecule fluorescent in situ hybridization (smFISH) has come to the fore as a powerful technique that allows quantification of both RNA number and RNA spatial distribution in cells and tissues. Moreover, smFISH can be combined with immunofluorescence to understand the precise interrelationship between RNA and protein distribution. Here, we describe methods for use of smFISH and immunofluorescence microscopy in in vitro angiogenesis models to enable the investigation of RNA and protein expression and localization during endothelial collective cell migration.
Original languageEnglish
Title of host publicationCell Migration in Three Dimensions
EditorsCoert Margadant
Place of PublicationNew York, USA
PublisherHumana Press, Inc
Number of pages19
ISBN (Electronic)9781071628874
ISBN (Print)9781071628867, 9781071628898
Publication statusPublished - 5 Feb 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • angiogenesis
  • endothelial cell
  • collective cell migration
  • RNA localization
  • smFISH
  • scratch wound assay
  • fibrin gel bead assay
  • immunofluorescence


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