Modeling microtubule-based transport and anchoring of mRNA.

Localization of messenger RNA (mRNA) at the vegetal cortex plays an important role in the early development of Xenopus laevis oocytes. While it is known that molecular motors are responsible for the transport of mRNA cargo along microtubules to the cortex, the mechanisms of localization remain unclear. We model cargo transport along microtubules using partial differential equations with spatially-dependent rates. A theoretical analysis of reduced versions of our model predicts effective velocity and diffusion rates for the cargo and shows that randomness of microtubule networks enhances effective transport. A more complex model using parameters estimated from fluorescence microscopy data reproduces the spatial and timescales of mRNA localization observed in Xenopus oocytes, corroborates experimental hypotheses that anchoring may be necessary to achieve complete localization, and shows that anchoring of mRNA complexes actively transported to the cortex is most effective in achieving robust accumulation at the cortex.

L-bodies are RNA-protein condensates driving RNA localization in Xenopus oocytes.

Ribonucleoprotein (RNP) granules are membraneless compartments within cells, formed by phase separation, that function as regulatory hubs for diverse biological processes. However, the mechanisms by which RNAs and proteins interact to promote RNP granule structure and function in vivo remain unclear. In Xenopus laevis oocytes, maternal mRNAs are localized as large RNPs to the vegetal hemisphere of the developing oocyte, where local translation is critical for proper embryonic patterning. Here we demonstrate that RNPs containing vegetally localized RNAs represent a new class of cytoplasmic RNP granule, termed localization-bodies (L-bodies). We show that L-bodies contain a dynamic protein-containing phase surrounding a nondynamic RNA-containing phase. Our results support a role for RNA as a critical component within these RNP granules and suggest that cis-elements within localized mRNAs may drive subcellular RNA localization through control over phase behavior.

Whole-Mount Immunofluorescence for Visualizing Endogenous Protein and Injected RNA in Xenopus Oocytes.

Asymmetric distribution of mRNA and protein is a hallmark of cell polarity in many systems. The Xenopus laevis oocyte provides many technical advantages to studying such polarity. Thousands of oocytes at different stages of maturity can be harvested from a single ovary and, owing to their relatively large size, even the youngest oocytes can be manually microinjected. Microinjection of fluorescently labeled RNA combined with immunofluorescence of endogenous proteins can provide insight into the cytoplasmic interactions contributing to polarity. Here, we present an updated method to image endogenous protein and microinjected RNA in X. laevis oocytes.