An improved molecular crowding sensor CRONOS for detection of crowding changes in membrane-less organelles under pathological conditions

Abstract

Membrane-less organelles (MLOs) formed by liquid-liquid phase separation (LLPS) play pivotal roles in biological processes. During LLPS, proteins and nucleotides are extremely condensed, resulting in changes of their conformation and biological functions. Disturbed LLPS homeostasis in MLOs cause fatal diseases such as amyotrophic lateral sclerosis. Therefore, it is important to detect changes of the degree of crowding in MLOs. However, it has not been investigated well due to lack of an appropriate method. To address this, we developed a genetically-encoded molecular crowding sensor CRONOS that senses the degree of macromolecular crowding in MLOs using fluorescence resonance energy transfer (FRET) system. CRONOS is a very bright biosensor with wider dynamic range and detect changes in the macromolecular volume fraction better than the previously reported mCer-mCit sensor in solution. By fusing to scaffold protein of each MLO, we successfully delivered CRONOS to MLO of interest and detected previously undescribed difference of the degree of crowding in each MLO. If not tagged, CRONOS localized to interstitial space of MLOs, giving us the crowding information of inspace. CRONOS also detected changes of degree of macromolecular crowding in nucleolus induced by environmental stress or inhibition of transcription. These findings suggest that CRONOS can be a useful tool for determination of molecular crowding and detection of pathological changes in MLOs in live cells.

Investigating molecular crowding during cell division and hyperosmotic stress in budding yeast with FRET

“…However, an increase to e.g., 40 ms would result in better sampling of the system as well as brighter foci for improved tracking and signal to noise ratios. Similarly, using the even brighter CRONOS sensor ( Miyagi et al, 2021 ) which uses mNeonGreen in place of mEGFP would improve sampling efficiency. We note that this explanation of the NFRET similarity is supported by the higher number of high FRET outliers in the 1M NaCl data in Figure 12c – with greater sampling we may observe a greater difference between conditions.…”