Analysis of mRNA Subcellular Distribution in Collective Cell Migration.

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.

RAB13 mRNA compartmentalisation spatially orients tissue morphogenesis.

Polarised targeting of diverse mRNAs to cellular protrusions is a hallmark of cell migration. Although a widespread phenomenon, definitive functions for endogenous targeted mRNAs and their relevance to modulation of in vivo tissue dynamics remain elusive. Here, using single-molecule analysis, gene editing and zebrafish live-cell imaging, we report that mRNA polarisation acts as a molecular compass that orients motile cell polarity and spatially directs tissue movement. Clustering of protrusion-derived RNAseq datasets defined a core 192-nt localisation element underpinning precise mRNA targeting to sites of filopodia formation. Such targeting of the small GTPase RAB13 generated tight spatial coupling of mRNA localisation, translation and protein activity, achieving precise subcellular compartmentalisation of RAB13 protein function to create a polarised domain of filopodia extension. Consequently, genomic excision of this localisation element and perturbation of RAB13 mRNA targeting-but not translation-depolarised filopodia dynamics in motile endothelial cells and induced mispatterning of blood vessels in zebrafish. Hence, mRNA polarisation, not expression, is the primary determinant of the site of RAB13 action, preventing ectopic functionality at inappropriate subcellular loci and orienting tissue morphogenesis.