Bringing Cellular Condensates to Light

New light-based method reveals the nature of biomolecular condensates through in vivo targeting and manipulation

December 12, 2024

Researchers at the Max Planck Institute of Immunobiology and Epigenetics in Freiburg, Germany, have developed LiTEC, a groundbreaking technique for studying biomolecular condensates - membraneless compartments in cells that regulate key processes such as gene expression. The technology uses light and arbitrary cargoes to enable proteomic analysis in these dynamic molecular clusters. LiTEC provides new insights into the composition and behavior of condensates without disturbing their natural state.

Artistic representation of the principle of method LiTEC by the Youssef Youssef — The image shows an opaque endogenous condensate becomes transparent with blue light exposure, revealing the hidden constituents of the condensates. This reflects a method developed by Lee et al. which targets arbitrary cargo or enzyme into endogenous condensates using blue light, and reveals the components of the condensates by putting markers for proteomics studies.

© Artist: Youssef Youssef

The image shows an opaque endogenous condensate becomes transparent with blue light exposure, revealing the hidden constituents of the condensates. This reflects a method developed by Lee et al. which targets arbitrary cargo or enzyme into endogenous condensates using blue light, and reveals the components of the condensates by putting markers for proteomics studies.

© Artist: Youssef Youssef

In schoolbooks, animal cells are usually depicted as a membrane bubble full of smaller compartments also surrounded by a membrane. The reality is different from this simplified picture. Yes, there are many membrane-bound compartments in living cells, such as mitochondria or lysosomes. But there are also many compartments that are not bound by a membrane. These are called biomolecular condensates, and they are essentially molecular clusters full of proteins, DNA, and RNA that are separated from the rest of the cell by a boundary shaped by biochemical affinity.

“Biomolecular condensates have fascinating physical properties. Cells use these properties to create ideal microenvironments for biochemical reactions,” explains Choongman Lee from the Max Planck Institute of Immunobiology and Epigenetics in Freiburg, Germany and first-author of the study.

How to study condensates in living cells

Cells are hypothesized to use condensates to precisely regulate their internal environment, enabling processes such as gene regulation. Using super-resolution microscopy, the Cissé lab previously discovered transcriptional condensates composed of RNA polymerase II and mediator in mouse embryonic stem cells. Earlier this year (Du et al Cell 2024), the team was able to show that the condensates control gene bursting in a transcriptional “dynamic kissing” process: direct interaction between the condensate and the gene is much more dynamic than previously thought, and results in upregulation of the gene bursting. However, the composition of these condensates are difficult to determine using conventional biological assays such as mass spectrometry, proteomics or genomic approaches.

“These condensates are notoriously difficult to investigate in their natural milieu, in living cells, due to their small size and dynamic nature. Their small size of only a few hundred nanometers and liquid-like properties make them particularly difficult to purify, and cellular fixation often alters their natural composition, making it even more difficult to identify their true components,” explains Ibrahim Cissé, Max Planck Director in Freiburg.

A new technology is needed

To gain a deeper understanding of the biophysical properties and biological functions of cellular condensates, researchers need to study these components as they occur in nature. In a recent study published in the journal Cell, the team of Max Planck researchers from Freiburg, Germany, introduced a novel method called LiTEC (Light-induced Targeting of Endogenous Condensates). Unlike traditional techniques that rely on chemical or mechanical extraction, LiTEC allows scientists to study and manipulate condensates directly in living cells.

A principle of LiTEC system: The Intrinsically disordered region (IDRs with sspB) accumulate in endogenous condensates spontaneously. In the absence of blue light, Cargo (with SunTag and scFv-iLID) is diffused throughout the nucleoplasm. Upon blue light, Cargo accumulates into the condensates.

© MPI of Immunobiology and Epigenetics, Cissé

A principle of LiTEC system: The Intrinsically disordered region (IDRs with sspB) accumulate in endogenous condensates spontaneously. In the absence of blue light, Cargo (with SunTag and scFv-iLID) is diffused throughout the nucleoplasm. Upon blue light, Cargo accumulates into the condensates.

© MPI of Immunobiology and Epigenetics, Cissé

The LiTEC system uses intrinsically disordered regions (IDRs) of specific proteins that naturally accumulate in condensates and serve as molecular “zip codes” to precisely target them. It then uses light-dependent processes to couple the IDRs to arbitrarily cargo molecules, and accumulates the cargo molecules within these condensates at will. This targeted accumulation brings concentrated enzymatic activities into the condensates, allowing researchers to better analyze their composition and behavior using mass spectrometry.

“In our proof-of-concept, we focused on transcription condensates, specifically the difficult-to-purify condensates of RNA Pol II and Mediator in mammalian cells. In principle, however, the same LiTEC approach can be easily applied to other endogenous condensate. By selecting different »zip codes«, other condensates can be targeted. By selecting different cargos, you could in principle enable other genome-wide studies of endogenous condensates” explains Ibrahim Cissé.

It is also possible that LiTEC could be used to manipulate the biophysical properties of condensates by concentrating specific markers to either dissolve or solidify them, thereby influencing their biological functions. Overall, the LiTEC system opens new avenues for proteomics and has significant potential for a wide range of applications in condensate research.

IC/CL/MR