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  1. Evaluation Summary:

    This work traces at the protein domain level the associations made within droplets containing RNA-binding protein FUS and how they change as a function of time (and maturity), in the presence and absence of the small heat-shock protein HSPB8, by chemical cross-linking coupled to mass spectrometry. This work is an important step forward in our general understanding of the macromolecular interactions within liquid-liquid phase-separated protein droplets, and how they are regulated by small heat-shock protein molecular chaperones.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, #2, and #3 agreed to shares their name with the authors.)

  2. Reviewer #1 (Public Review):

    Boczek and colleagues have investigated the phase-transition properties of the RNA-binding protein FUS and its ability to form condensates in the presence of HspB8. The phenomenon of phase transition of proteins that contain structurally disordered regions or so-called low-complexity domains (LCDs) leads to condensates of increased local protein concentration that are also observed in a variety of (stress) granules, or other intracellular assemblages/"bodies" in a cell. The ability of a certain class of proteins to form such condensates provides a novel concept in molecular biology that might explain the local action of proteins in essential cellular pathways. Recently, several studies have addressed this phenomenon and found mechanisms, e.g. through protein-protein interactions and protein modifications that lead to, or disrupt, condensation of proteins.
    In the work presented here, Boczek et al. introduce an interesting approach to monitor the properties of proteins in condensates and during protein ageing. The methodological novelty of this approach is the use of protein-protein crosslinking to study the conformation of the RNA-binding domain of FUS in condensates in the absence and presence of HspB8. In the past, such studies of proteins that undergo phase transition/condensation were performed mainly by using NMR.
    I consider the approach of Boczek and colleagues to be an interesting one, which might make possible an alternative view of how protein properties change in phase transition.

    Strengths of the work:

    1. The presence of Hsp8B in stress granules containing FUS or hnRNPA1 protein has been observed in cells in previous studies, and the authors here provide an idea of the molecular basis of how the FUS and HspB8 protein conformations and their interactions change in condensates. Crosslinking might indeed be used as an alternative assay method to monitor changes in protein conformations and interactions in a time-resolved manner, and the results can moreover be quantified.

    2. The authors provide a model of how HspB8 interacts with FUS, i.e. through interaction of the aCD domain with the RRM of FUS, and they show that the binding of FUS to RNA probably competes with its binding to Hsp8B.

    3. The authors performed studies with the mutated HspB8 present in Charcot-Marie-Tooth (CMT) disease and found, by crosslinking and FRAP, that the interaction between FUS and mtHspB8 is strengthened by the mutation, indicating that this interaction is not reversed in the disease.

    Weaknesses of the work:

    This is almost entirely an in vitro study, lacking in vivo data to support the model.

  3. Reviewer #2 (Public Review):

    In this work, the authors aim to understand the nature of protein-protein and protein-RNA interactions within stress granules by means of time-resolved chemical cross linking coupled to mass spectrometry.

    Through careful control of conditions through reconstituting stress granules in vitro using the archetypal phase-separating model protein, FUS, the authors develop an excellent system to allow detailed structural and dynamical interrogation of the interactions within droplets.

    A particular focus is examining the role that the small heat-shock protein HSPB8 - known to be associated with stress granules - might have in preventing maturation of the FUS-rich droplets.

    The authors demonstrate convincingly that HSPB8 associates with FUS, with arginines in the HSPB8 N-terminal region targeting it to the droplets, and the alpha-crystallin domain associating with the FUS RNA-recognition motif (in competition with RNA).

    Excitingly, the authors observe that a pathogenic variant of HSPB8 associated with Charcot-Marie-Tooth Disease does not display the same effect as the WT molecular chaperone in stabilising FUS.

    In sum, this study provides a clear mechanism by why HSPB8 interacts with FUS, leading to a general hypothesis for how small heat-shock proteins associated with phase-separation play a protective role in vivo.

  4. Reviewer #3 (Public Review):

    This manuscript describes a detailed investigation of the effect of aging in Fus-based droplets. These droplets can change from a liquid-like phase to a solid-like phase which is correlated with the transition from a normal cellular state as seen for example in stress granules to a disease state with fibrils being the end point. The authors use cross-linking techniques to characterize this aging process in structural detail and investigate the effect of adding small heat shock proteins. These chaperones can partition into the droplets based on their own unfolded domain and interact with the RNA binding domain of FUS via their a-crystalline domain. Through a series of clever mutation experiments the authors demonstrate the relevance of these interactions for preventing the transition into the disease, fibrillar state.