The recruitment of biomolecules into liquid-like condensates is a strategy employed by cells to regulate biochemical reactions, organize their content, and sequester unwanted molecules. This process, known as liquid-liquid phase separation (LLPS), involves the partitioning of biomolecules, specifically proteins and RNA, into two distinct liquid-like phases: a dilute phase and a dense phase (collectively referred to as condensates). Under certain conditions, these liquid-like structures can aberrantly transition into solid-like structures with distinct material properties, which are strongly associated with cytotoxicity. Investigating the mechanisms behind this liquid-to-solid transition is thereby crucial. Research on phase-separated systems has primarily focused on the micron-scale dense phase, often overlooking the heterogeneity at the nanoscale. Although LLPS can also occur at the nanoscale through the formation of nano-complexes, a lack of appropriate methodologies has restricted the investigation of these structures, particularly in living cells. This work employs photon-resolved microscopy to overcome this limitation. By integrating a single-photon detector array into a confocal laser-scanning microscope, photon-resolved microscopy accesses information typically discarded by conventional methods. Specifically, we combined photon-resolved microscopy with fluorescence fluctuation spectroscopy (FFS) to investigate the dynamics and interactions of biomolecules undergoing LLPS. FFS techniques analyse fluctuations of fluorescent molecules within a confocal volume to determine their diffusion, interactions, and nano-environment. Conventional FFS techniques average the signal from the entire confocal volume, masking inherent heterogeneity of biological systems. In contrast, photon-resolved microscopy collects the signal photon-by-photon, avoiding averaging in both time and space and thus revealing heterogeneities. The method is applied to the phase separation of the protein alpha-synuclein into an in vitro reconstituted system, as well as the phase separation of the RNA NEAT1 in living cells. For alpha-synuclein, the dynamic states in both liquid-like phases are characterized, particularly focusing on how RNA affects the material properties of the dense phase. Results indicate that RNA has a weak impact on the dilute phase but drastically alters the material properties of the dense phase toward solid-like structures. For NEAT1, fluorescent labeling of RNA in living cells represents a technical challenge. The issue was approached by labeling it with a fluorescent light-up aptamer, which represents the most promising tool currently available for this purpose. The labeling was successfully validated and allowed for the quantification of its dynamics in physiological condensates. By integrating biophysics and molecular biology, the research presented here is relevant across multiple research fields and paves the way for advancements in FFS method development as well as a deeper exploration of protein-RNA interactions during phase transitions.

Photon-resolved microscopy to study protein and RNA dynamics in biomolecular condensates

ZAPPONE, SABRINA
2025-06-12

Abstract

The recruitment of biomolecules into liquid-like condensates is a strategy employed by cells to regulate biochemical reactions, organize their content, and sequester unwanted molecules. This process, known as liquid-liquid phase separation (LLPS), involves the partitioning of biomolecules, specifically proteins and RNA, into two distinct liquid-like phases: a dilute phase and a dense phase (collectively referred to as condensates). Under certain conditions, these liquid-like structures can aberrantly transition into solid-like structures with distinct material properties, which are strongly associated with cytotoxicity. Investigating the mechanisms behind this liquid-to-solid transition is thereby crucial. Research on phase-separated systems has primarily focused on the micron-scale dense phase, often overlooking the heterogeneity at the nanoscale. Although LLPS can also occur at the nanoscale through the formation of nano-complexes, a lack of appropriate methodologies has restricted the investigation of these structures, particularly in living cells. This work employs photon-resolved microscopy to overcome this limitation. By integrating a single-photon detector array into a confocal laser-scanning microscope, photon-resolved microscopy accesses information typically discarded by conventional methods. Specifically, we combined photon-resolved microscopy with fluorescence fluctuation spectroscopy (FFS) to investigate the dynamics and interactions of biomolecules undergoing LLPS. FFS techniques analyse fluctuations of fluorescent molecules within a confocal volume to determine their diffusion, interactions, and nano-environment. Conventional FFS techniques average the signal from the entire confocal volume, masking inherent heterogeneity of biological systems. In contrast, photon-resolved microscopy collects the signal photon-by-photon, avoiding averaging in both time and space and thus revealing heterogeneities. The method is applied to the phase separation of the protein alpha-synuclein into an in vitro reconstituted system, as well as the phase separation of the RNA NEAT1 in living cells. For alpha-synuclein, the dynamic states in both liquid-like phases are characterized, particularly focusing on how RNA affects the material properties of the dense phase. Results indicate that RNA has a weak impact on the dilute phase but drastically alters the material properties of the dense phase toward solid-like structures. For NEAT1, fluorescent labeling of RNA in living cells represents a technical challenge. The issue was approached by labeling it with a fluorescent light-up aptamer, which represents the most promising tool currently available for this purpose. The labeling was successfully validated and allowed for the quantification of its dynamics in physiological condensates. By integrating biophysics and molecular biology, the research presented here is relevant across multiple research fields and paves the way for advancements in FFS method development as well as a deeper exploration of protein-RNA interactions during phase transitions.
12-giu-2025
liquid-liquid phase separation
biomolecular condensates
alpha-synuclein
NEAT1
fluorescence fluctuation spectroscopy
super-resolution microscopy
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11567/1249581
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