Nuclear bodies protect phase separated proteins from degradation in stressed proteome
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Curated by eLife
eLife assessment
This study presents a novel fluorescence based imaging strategy to investigate the folding status of RNA-binding proteins (RBPs) and their association with molecular chaperones under stress. It provides fundamental findings that will potentially advance our understanding in the folding and aggregation status of RBPs in nuclear stress bodies in a significant manner. However, there is also the concern that the evidence regarding protein fate is incomplete and additional controls are needed to fully support the conclusion. The imaging methodology can be adapted to study many other proteins that undergo liquid-liquid phase separation under specific cellular conditions.
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Abstract
RNA-binding proteins (RBPs) containing intrinsically disordered domains undergo liquid-liquid phase separation to form nuclear bodies under stress conditions. This process is also connected to the misfolding and aggregation of RBPs, which are associated with a series of neurodegenerative diseases. However, it remains elusive how folding states of RBPs changes upon the formation and maturation of nuclear bodies. Here, we describe SNAP-tag based imaging methods to visualize the folding states of RBPs in live cells via time-resolved quantitative microscopic analyses of their micropolarity and microviscosity. Using these imaging methods in conjunction with immunofluorescence imaging, we demonstrate that RBPs, represented by TDP-43, initially enters the PML nuclear bodies in its native state upon transient proteostasis stress, albeit it begins to misfolded during prolonged stress. Furthermore, we show that heat shock protein 70 co-enters the PML nuclear bodies to prevent the degradation of TDP-43 from the proteotoxic stress, thus revealing a previously unappreciated protective role of the PML nuclear bodies in the prevention of stress-induced degradation of TDP-43. In summary, our imaging methods described in the manuscript, for the first time, reveal the folding states of RBPs, which were previously challenging to study with conventional methods in nuclear bodies of live cells. This study uncovers the mechanistic correlations between the folding states of a protein and functions of nuclear bodies, in particular PML bodies. We envision that the imaging methods can be generally applied to elucidating the structural aspects of other proteins that exhibit granular structures under biological stimulus.
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eLife assessment
This study presents a novel fluorescence based imaging strategy to investigate the folding status of RNA-binding proteins (RBPs) and their association with molecular chaperones under stress. It provides fundamental findings that will potentially advance our understanding in the folding and aggregation status of RBPs in nuclear stress bodies in a significant manner. However, there is also the concern that the evidence regarding protein fate is incomplete and additional controls are needed to fully support the conclusion. The imaging methodology can be adapted to study many other proteins that undergo liquid-liquid phase separation under specific cellular conditions.
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Reviewer #1 (Public Review):
This manuscript sets out to implement a multi-stage fluorescence imaging essay to test two working models in understanding the folding states of RNA-binding proteins (RBPs) in stress-induced nuclear bodies. In conjunction with live-cell fluorescence lifetime imaging, the authors revealed and conformed a previously unclear phenomenon that the RBPs investigated in this work initially enter the nuclear bodies in native state in transient stress and then begin to misfold after prolonged stress. Comparing to conventional methods, the imaging strategy reported in this work is unique, comprehensive, and effective in surveying all three-stages (native, soluble oligomer, aggregates) of folding states for RBPs in one shot. Using this strategy, the authors then found that the heat shock protein 70 may protects RBPs …
Reviewer #1 (Public Review):
This manuscript sets out to implement a multi-stage fluorescence imaging essay to test two working models in understanding the folding states of RNA-binding proteins (RBPs) in stress-induced nuclear bodies. In conjunction with live-cell fluorescence lifetime imaging, the authors revealed and conformed a previously unclear phenomenon that the RBPs investigated in this work initially enter the nuclear bodies in native state in transient stress and then begin to misfold after prolonged stress. Comparing to conventional methods, the imaging strategy reported in this work is unique, comprehensive, and effective in surveying all three-stages (native, soluble oligomer, aggregates) of folding states for RBPs in one shot. Using this strategy, the authors then found that the heat shock protein 70 may protects RBPs from being degraded under stress. The manuscript is very well-written.
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Reviewer #2 (Public Review):
The authors combine the use of fluorogenic tools with fluorescence bioimaging to visualize how changes in the folding states of the RBPs TDP-43, FUS and TAF15 affect their subcellular localization and recruitment inside nuclear bodies, as well as protein fate. While the development of SNAP-tag substrates coupled with confocal microscopy in living cells (including FLIM) to monitor changes in protein folding states represents an important conceptual and technical advance for the field, I am not convinced that the authors fully achieved their aim. The authors cannot conclude on protein fate only based on the experiments performed here. Showing a correlation between a decrease in TDP-43 levels upon Hsp70 inhibition and colocalization at nuclear bodies with Hsp70 and DNAJA2 is not supporting their conclusion …
Reviewer #2 (Public Review):
The authors combine the use of fluorogenic tools with fluorescence bioimaging to visualize how changes in the folding states of the RBPs TDP-43, FUS and TAF15 affect their subcellular localization and recruitment inside nuclear bodies, as well as protein fate. While the development of SNAP-tag substrates coupled with confocal microscopy in living cells (including FLIM) to monitor changes in protein folding states represents an important conceptual and technical advance for the field, I am not convinced that the authors fully achieved their aim. The authors cannot conclude on protein fate only based on the experiments performed here. Showing a correlation between a decrease in TDP-43 levels upon Hsp70 inhibition and colocalization at nuclear bodies with Hsp70 and DNAJA2 is not supporting their conclusion about protein degradation. A number of additional control experiments are needed to support their claims.
Yet, the optimization of these methods has unlimited potential since it may provide new ways to visualize and monitor a large variety of fundamental intracellular processes, including protein aggregation and fate.
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Reviewer #3 (Public Review):
This manuscript presents a novel fluorescence toolkit designed for investigating the folding states of RNA-binding proteins (RBPs) and their association with molecular chaperones during liquid-liquid phase separation (LLPS) in the formation of nuclear bodies under stress. The strategy is to use SNAP-tag technology including cell lines stably expressing three model proteins fused with SNAP tag and a series of environmentally sensitive fluorophores that can selectively label on the SNAP proteins. The changes in the microenvironment such as microviscosity and micropolarity can be well characterized by these fluorophores to reflect the conformational states of the RBPs.
The strength of this method is that the SNAP protein is smaller than classic fluorescent proteins like GFP and thus its impact on the …
Reviewer #3 (Public Review):
This manuscript presents a novel fluorescence toolkit designed for investigating the folding states of RNA-binding proteins (RBPs) and their association with molecular chaperones during liquid-liquid phase separation (LLPS) in the formation of nuclear bodies under stress. The strategy is to use SNAP-tag technology including cell lines stably expressing three model proteins fused with SNAP tag and a series of environmentally sensitive fluorophores that can selectively label on the SNAP proteins. The changes in the microenvironment such as microviscosity and micropolarity can be well characterized by these fluorophores to reflect the conformational states of the RBPs.
The strength of this method is that the SNAP protein is smaller than classic fluorescent proteins like GFP and thus its impact on the conformation and behavior of the targeted proteins is much smaller. The experiment is carefully designed and well thought-out. Overall, this work is of very high quality.
This method can thus be adapted by other protein systems to study the LLPS process and thus I believe it will be of great interest to cell biologists and biophysicists.
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