Endogenous oligomer formation underlies DVL2 condensates and promotes Wnt/β-catenin signaling

Reviewer #1 (Public Review):

Summary:

The study "Endogenous oligomer formation underlies DVL2 condensates and promotes Wnt/β-catenin signaling" by Senem Ntourmas et al. contributes to the understanding of phase separation in Dishevelled (DVL) proteins, specifically focusing on DVL2. It builds upon existing research by investigating the endogenous complexes of DVL2 using ultracentrifugation and contrasting them with DVL1 and DVL3 behavior. The study identifies a DVL2-specific region involved in condensate formation and introduces the "two-step" concept of DVL2 condensate formation, enriching the field's knowledge.

Strengths:

A notable strength of this study is the validation of endogenous DVL2 complexes, providing insights into its behavior compared to DVL1 and DVL3. The functional validation of the DVL C-terminus (here termed conserved domain 2 (CD2) and the identification of DVL2-specific regions (here termed LCR4) involved in condensate formation are significant contributions that complement the current knowledge on the importance of DVL DIX domain, DEP domain and intrinsically disordered regions between DIX and PDZ domains. Additionally, the introduction of the concept where oligomerization (step 1) precedes condensate formation (step 2) is an interesting hypothesis, which can be further experimentally challenged in the future.

Weaknesses:

However, the applicability of the findings to full-length DVL2 protein, hence the physiological relevance, is limited. This is mostly due to the fact that the authors almost completely depend on the set of DVL2 mutants, which lack the (i) DEP domain and (ii) nuclear export signal (NES). These variants fail to establish DEP domain-mediated interactions, including those with FZD receptors. Of note, the DEP domain itself represents a dimerization/tetramerization interface, which could affect the protein condensate formation of these mutants. Possibly even more importantly, the used mutants localize into the nucleus, which has different biochemical & biophysical properties than a cytoplasm, where DVL typically reside, which in turn affects the condensate formation. On top, in the nucleus, most of the DVL binding partners, including relevant kinases, which were reported to affect protein condensate formation, are missing.

Second, the use of an overexpression system, while suitable for comparing DVL2 protein condensate features, falls short in functional assays. The study could benefit from employing established "rescue systems" using DVL1/2/3 knockout cells and re-expression of DVL variants for more robust functional assessments.

Furthermore, the discussion and introduction overlook some essential aspects of DVL biology. One such example is the importance of the open/close conformation of DVL and its effects on DVL phase separation and activity. In the context of this study, it is important to say that this conformational plasticity is mediated by DVL C-terminus (CD2 in this study). The second example is the reported roles of DVL1 and DVL3, which can both mediate the Wnt3a signal. How this can be interpreted when DVL1 and DVL3 lack LCR4 and still form condensates?

In order to increase the physiological relevance of the study, I would recommend analyzing several key mutants in the context of the full-length DVL2 protein using the rescue/complementation system. Further, a more thorough discussion and connections with the existing literature on DVL protein condensates/puncta/LLPS can improve the impact of the study.

Reviewer #2 (Public Review):

Summary:

The authors aimed to identify which regions of DVL2 contribute to its endogenous/basal clustering, as well as the relevance of such domains to condensate/phase separation and WNT activation.

Strengths:

A strength of the study is the focus on endogenous DVL2 to set up the research questions, as well as the incorporation of various techniques to tackle it. I found also quite interesting that DVL2-CFR addition to DVL1 increased its MW in density gradients.

Weaknesses:

I think that several of the approaches of the manuscript are subpar to achieve the goals and/or support several of the conclusions. For example:

(1) Although endogenous DVL2 indeed seems to form complexes (Figure 1A), neither the number of proteins involved nor whether those are homo-complexes can be determined with a density gradient. Super-resolution imaging or structural analyses are needed to support these claims.

(2) Follow-up analyses of the relevance of the DVL2 domains solely rely on overexpressed proteins. However, there were previous questions arising from o/e studies that prompted the focus on endogenous, physiologically relevant DVL interactions, clustering, and condensate formation. Although the title, conclusions, and relevance all point to the importance of this study for understanding endogenous complexes, only Figures 1A and B deal with endogenous DVL2.

(3) Mutants lacking activity/complex formation, e.g. DVL2_1-418, may need further validation. For instance, DVL2_1-506 (same mutant but with DEP) seems to form condensates and it is functional in WNT signalling (King et al., 20223). These differences could be caused by the lack of DEP domain in this particular construct and/or folding differences.

(4) The key mutants, DeltaCFR and VV/FF only show mild phenotypes. The authors' results suggest that these regions contribute but are not necessary for 1) complex formation (Density gradient Figures 7A and B), condensate formation (Figures 7C and D), and WNT activity (Figure 7E). Of note Figure 7C shows examples for the mutants with no condensates while the qualification indicates that 50% of the cells do have condensates.

(5) Most of the o/e analyses (including all reporter assays) should be performed in DVL1-3 KO cells in order to explore specifically the behaviour of the investigated mutants.

(6) How comparable are condensates found in the cytoplasm (usually for wt DVL) with those located in the nucleus (DEP mutants)?

Several studies in the last two decades have analysed the relevance of DVL homo - and hetero-clustering by relying on overexpressed proteins. Recent studies also explored the possibility of DVL undergoing liquid-liquid phase separation following similar principles. As highlighted by the authors in the introduction, there is a need to understand DVL dynamics under endogenous/physiological conditions. Recent super-resolution studies aimed at that question by characterising endogenously edited DVL2. The authors seemed to aim in the same direction with their initial findings (Figure 1A) but quickly moved to o/e proteins without going back to the initial question. This reviewer thinks that to support their conclusions and advance in this important question, the authors should introduce the relevant mutations in the endogenous locus (e.g. by Cas9+ donor template encoding the required 3' exons, as done by others before for WNT components, including DVL2) and determine their impact in the above-indicated processes.