Artificial Condensates Functionalized with Motor Proteins Enable Spatial Manipulation of RNA Localization

Core Concepts

Artificial condensates functionalized with motor proteins can be used to spatially manipulate the localization of specific RNAs in living cells.

Abstract

The study describes the development of an engineered system to control the spatial positioning of biomolecular condensates in cells. The authors created artificial condensates by fusing a multivalent protein domain that drives liquid-liquid phase separation (LLPS) with either plus-end directed kinesin motors (KIF1A, KIF5B) or minus-end directed motors/adaptors (KIFC1, BICD2).
The key findings are:

Plus-end motor condensates localized at the cell periphery, while minus-end motor/adaptor condensates localized at the centrosome. This positioning was determined by the nucleation of condensates at the final destination sites rather than by transport of pre-formed condensates.

The timing of non-functionalized LLPS scaffold enrichment into the motor condensates depended on their subcellular localization, with faster enrichment for minus-end motor/adaptor condensates near the nucleus compared to plus-end motor condensates at the cell periphery.

The authors developed a chemically-inducible system to trigger the interaction between dispersed condensates and motor proteins, allowing for rapid repositioning of condensates to the cell periphery or centrosome.

By incorporating an RNA-binding protein (MCP) into the motor condensates, the authors were able to recruit specific MS2-tagged RNAs and delocalize an endogenous ASPM mRNA away from the centrosome towards the cell membrane.

Overall, this engineered system provides a versatile tool to spatially manipulate the localization of RNAs in living cells, with potential applications in studying the role of RNA localization in cellular processes.

Stats

The mean number of condensates per cell was 4.0 ± 3.0 for non-functionalized condensates, 1.4 ± 0.9 for KIFC1 condensates, and 1.2 ± 0.6 for BICD2 condensates.
The fraction of mCherry fluorescence in the peripheral 25% of the cell (I25) was 20% ± 9% for non-functionalized condensates, 34% ± 9% for KIF1A condensates, and 32% ± 8% for KIF5B condensates.
The delay between nucleation of motor condensates and first enrichment of non-functionalized scaffolds was 83 min (CV 40%) for KIF1A, 74 min (CV 39%) for KIF5B, 8 min (CV 163%) for KIFC1, and 18 min (CV 89%) for BICD2.

Quotes

"Whereas motor-driven transport of RNP condensates plays a prominent role in RNA localization in cells, their studies remain limited by the scarcity of available tools allowing to manipulate condensates in a spatial manner."
"Our method opens novel perspectives to examine the role of RNA localization as a driver of cellular functions."
"Artificial condensates functionalized with motor proteins can efficiently delocalize ASPM-MS2 mRNAs away from centrosomes towards the cell membrane across mitosis."

Key Insights Distilled From

Condensate functionalization with motors directs their nucleation in space and allows manipulating RNA localization

by Cochard,A., ... at www.biorxiv.org 07-10-2022

https://www.biorxiv.org/content/10.1101/2022.07.10.499452v1

Deeper Inquiries

How could this engineered system be further extended to study the functional consequences of altering RNA localization in different cellular contexts?

The engineered system described in the context can be further extended to study the functional consequences of altering RNA localization in different cellular contexts by incorporating additional elements and experimental designs. Here are some ways this system could be expanded:

Cell Type Specificity: The system can be applied to different cell types to investigate how altering RNA localization impacts cellular functions in a cell-type-specific manner. By using cell-specific promoters to drive the expression of the motor-condensates and RNA targets, researchers can explore how RNA localization influences cell behavior in various cellular contexts.

Dynamic Imaging: Implementing live-cell imaging techniques can provide insights into the real-time dynamics of RNA localization upon manipulation. Time-lapse microscopy can capture the movement of RNA molecules in response to changes in condensate positioning, allowing for a detailed analysis of the temporal aspects of RNA localization.

Functional Assays: Introducing functional assays to assess the consequences of altered RNA localization can provide valuable information about the impact on cellular processes. For example, measuring protein expression levels or cellular responses to specific stimuli after manipulating RNA localization can reveal the functional outcomes of these changes.

High-Throughput Screening: Utilizing high-throughput screening approaches can enable the systematic analysis of the effects of altered RNA localization on a wide range of cellular functions. By screening for specific phenotypic changes or molecular signatures associated with manipulated RNA localization, researchers can identify key pathways and processes affected by these alterations.

Integration with Omics Technologies: Integrating the engineered system with omics technologies such as transcriptomics, proteomics, and metabolomics can provide a comprehensive view of the molecular changes resulting from altered RNA localization. This multi-omics approach can uncover novel regulatory networks and signaling pathways influenced by RNA positioning.

How could this engineered system be further extended to study the functional consequences of altering RNA localization in different cellular contexts?

One potential limitation or challenge in using this approach to manipulate the localization of endogenous, non-tagged RNAs is the specificity and efficiency of RNA recruitment to the motor-condensates. Since endogenous RNAs are not artificially tagged with MS2 sequences, the recruitment of these non-tagged RNAs to the condensates may be less efficient and specific compared to the recruitment of MS2-tagged RNAs. This could result in lower signal-to-noise ratios and difficulties in distinguishing between specific RNA recruitment and non-specific interactions.
Additionally, the natural variability in RNA expression levels, turnover rates, and subcellular localization patterns among different endogenous RNAs may pose challenges in standardizing the recruitment of non-tagged RNAs to the motor-condensates. Ensuring the selective recruitment of specific endogenous RNAs to the condensates without affecting the localization of other RNAs or cellular components may require optimization of the system and careful validation of the results.
Furthermore, the potential impact of the artificial recruitment of endogenous RNAs to motor-condensates on normal cellular processes and RNA metabolism should be considered. Altering the localization of endogenous RNAs through artificial recruitment may disrupt normal RNA trafficking, translation, and regulatory mechanisms, leading to unintended consequences on cellular functions.

Could a similar strategy be applied to control the spatial organization of other types of biomolecular condensates, such as stress granules or P-bodies, and investigate their role in cellular processes?

Yes, a similar strategy could be applied to control the spatial organization of other types of biomolecular condensates, such as stress granules or P-bodies, to investigate their role in cellular processes. By engineering artificial condensates functionalized with specific proteins or domains involved in stress granule or P-body formation, researchers can manipulate the localization and dynamics of these condensates within cells.
For stress granules, which are dynamic ribonucleoprotein complexes that form in response to cellular stress, the engineered system could be used to study how altering the spatial organization of stress granules affects stress response pathways, RNA metabolism, and cell survival under stress conditions. By recruiting stress granule components to specific subcellular locations using motor-condensates, researchers can explore the functional consequences of modulating stress granule dynamics.
Similarly, for P-bodies, which are involved in mRNA degradation and storage, the engineered system could be employed to investigate the impact of altered P-body localization on mRNA turnover, translation regulation, and cellular homeostasis. By manipulating the positioning of P-body components using motor-condensates, researchers can elucidate the role of P-bodies in mRNA metabolism and cellular processes.
Overall, applying this strategy to study stress granules, P-bodies, and other biomolecular condensates can provide valuable insights into the functional significance of their spatial organization and dynamics in cellular processes.