Abstract
<jats:p>Spatiotemporal regulation of mRNA localisation is fundamental to cell identity specification and function, yet tracking transcript dynamics in living differentiating cells remains technically challenging. Here, we establish a robust pipeline for MS2 tagging of endogenous transcripts in human induced pluripotent stem cells (iPSCs), coupled with single-particle tracking and Hidden Markov Modelling to map mRNA mobility landscapes during differentiation and cell state transitions. Applying this approach to different cytoskeleton-encoding transcripts in diverse contexts - neural organoids, directly programmed neurons, and vascular organoids - we reveal a conserved principle: Both, β-actin and β2b-tubulin particle dynamics progressively shift towards constrained, compartmentalised patterns as cells acquire cell type identity. Perturbation experiments demonstrate that microtubule-dependent tethering is a common, conserved mechanism controlling β-actin mRNA localisation in all cell types studied, whereas translation-dependent anchoring and actin filaments contribute in a context-dependent manner. Analysis of particle dynamics in migrating blood vessel progenitors further showed that β-actin mRNAs accumulating at cell edges are highly diffusive, while those in perinuclear regions show constrained movement. Together, our integrated framework provides a scalable foundation for mechanistic dissection of mRNA targeting in human developmental and disease models.</jats:p>