Phosphofructokinase relocalizes into subcellular compartments with liquid-like properties in vivo
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Reviewer #1:
**Summary**
Jang et al., address the important question of spatially localized or compartmentalized metabolic enzymes with a focus on the glycolytic enzyme PFK1. Using a good strategy of inserting a fluorescent tag at the endogenous PFK1 locus with tissue-specific inducible expression in C. elegans, combined with strong quantitative longitudinal imaging and innovative bioengineered microfluidic-hydrogels to control oxygen availability as well as optogenetic approaches, they show PFK1 condensates, which are not stress granules and not seen in normoxia, assemble with hypoxia. PFK1 condensates are dynamic, reversible, localized at the synapse in …
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Reply to the reviewers
Reviewer #1:
**Summary**
Jang et al., address the important question of spatially localized or compartmentalized metabolic enzymes with a focus on the glycolytic enzyme PFK1. Using a good strategy of inserting a fluorescent tag at the endogenous PFK1 locus with tissue-specific inducible expression in C. elegans, combined with strong quantitative longitudinal imaging and innovative bioengineered microfluidic-hydrogels to control oxygen availability as well as optogenetic approaches, they show PFK1 condensates, which are not stress granules and not seen in normoxia, assemble with hypoxia. PFK1 condensates are dynamic, reversible, localized at the synapse in neurons, and recruit aldolase, another glycolytic enzyme. Although glycolytic proteins were previously shown to compartmentalize near the plasma membrane, and PFK1 was previously shown to assemble into filaments in vitro and be punctate at the plasma membrane in mammalian cells, evidence for cellular localized PFK1 condensates in animals is highly significant. The work includes strong biophysical characterization of PFK1 phase-separated condensates, but no clear indication of the composition of condensates. More significantly, the findings lack functional significance related to PFK1 activity or glycolytic flux with hypoxia vs normoxia. Despite previous work by this group showing that disrupting subcellular localization of glycolytic enzymes impairs neuronal activity in response with hypoxia, the reader is left with questions on the importance of localized and PFK1 condensates and their make-up .
**Major comments:**
Key conclusions are convincing, and most experimental approaches, biophysical characterization including thermodynamic principles, and data analysis are exemplary and well described. However, as indicated above, the work is limited to a descriptive analysis of cellular localization of PFK1 condensates and their biophysical properties without insights on functional significance relative to enzyme activity - or at least glycolytic flux or metabolic reprogramming with hypoxia. At best, only correlations can be drawn from hypoxia-induced localized PFK1 condensates and the authors' previous report (Jang et al., 2016) on hypoxia-regulated neuronal activity. Some insight or at least prediction in the discussion on the differences in spatially localized PFK1 in muscle vs neurons with regard to metabolic or energy distinctions should be included.
We have added additional discussions on the differences of the spatially localized PFK-1.1 in muscles versus neurons, explaining that in both tissues the cellular enrichment appears to be at sites predicted to have high ATP consumption (lines 128-133; 482-484).
Despite the strong biophysical analysis of condensates, several important features are not determined. First is at best a rudimentary analysis of the composition of condensates and also how PFK1 is assembled into these structures. For the former, is the core of the condensate predominantly PFK1 with perhaps aldolase only recruited to the periphery or is aldolase an integral component of the structure. Hence, is it a PFK1 condensate or a glycolytic condensate? For the latter question, is there a particular orientation for PFK1 in condensates, i.e a collection of filaments as previously reported, which might provide insight on assembly? Finally, and less critical but also important is the criterion for spherical, which is not well defined, and at least some idea or speculation on determinants for a spherical morphology - compared with filaments that have been reported for other non-glycolytic metabolic enzymes.
We have now co-expressed PFK-1.1 and ALDO-1 and examined their dynamic formation during hypoxic conditions. We observe PFK-1.1 and ALDO-1 form condensates simultaneously, with gradual enrichment of both molecules. We now include this new data in Figure 7E and Video 8; lines 422-441, 964-989). We also include genetic data demonstrating the ALDO-1 requires *pfk-1.1 *to form condensates, and that PFK-1.1 requires aldo-1 as well. Therefore, the enzymes are interdependent on each other to form condensates (Figures 7G, 7H, S7B, and S7C).
The spheroid geometry reflects liquid-like properties, which arises from surface tension of molecules loosely held together via multi-valent interactions. Filamentous arrangements reflect crystalline-like structures resulting from more stable interactions between molecules into solid-like states. While we did not perform high resolution studies, like Cryo-EM, to resolve this question, the spheroid geometry of PFK-1.1 condensates, along with its fluid-like properties, suggest the condensates are liquid-like compartment distinct to filamentous structures. We now add this discussion in lines 467-470.
The work is an important advance in our understanding on the self-assembly of metabolic enzymes by showing hypoxia-induced PFK1 condensates in vivo, their spatially-restricted subcellular localization in muscle cells and neurons, and their biophysical properties, the latter being distinct from those of stress granules. Taken together, these findings are more extensive than many previous reports on the assembly of metabolic enzymes into filaments or condensates, but fall short for new insights on functional significance.
We focus this study on the biophysical characterization of the condensates, and how that results in compartmentalized enrichment of glycolytic proteins. Examination of the functional significance of the phase separation to the enzymatic reactions *in vivo *is not currently possible because we lack probes we can use *in vivo *to measure the metabolites resulting from the reaction. We have now added discussion acknowledging this and framing its significance in the context of what has been published in the field (lines 484-492). For example, a recent manuscript in ChemRxiv demonstrated, in vitro, that the enzymatic activity of glycolytic proteins, hexokinase and glucose-6 phosphate dehydrogenase, promote these enzymes condensing into liquid droplets. The authors further found that the condensation accelerated the glycolytic reactions (Ura et al., 2020). This raises the question whether glycolytic proteins compartmentalize, and form condensates, in vivo, which we address in this manuscript. We capture this point in (lines 444-464) where we explain that, while it has long been hypothesized that glycolytic proteins like PFK-1 could be compartmentalized, this remained controversial due to lack of dynamic *in vivo * imaging. In our study, and through a systematic examination of endogenous PFK-1.1 via the use of a hybrid microfluidic-hydrogel device, we conclusively determine that PFK-1.1 indeed displays distinct patterns of subcellular localization in specific tissues in vivo.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
This paper reports on the condensation of the glycolytic enzyme PFK-1 in response to hypoxic conditions in neurons of C. elegans. The authors employ a microfluidic-hydrogel device to dynamically monitor the relocalisation of PFK-1 from a mostly diffuse state to clusters in response to hypoxia and show that PFK-1 can undergo multiple rounds of PFK-1 clustering and dissolution. The authors work through the key features of a liquid-like compartment (sphericity, fusion, fast internal rearrangements) and give evidence that PFK-1 may have all three. Finally, the authors tag PFK-1 with the light-inducible multimerization domain Cry2 and find that even without light PFK-1 will constitutively form clusters that sequestrate endogenous PFK-1 as well as other glycolytic proteins. The strength of this work is that it is characterizing what appears to likely be phase separation in the context of a whole animal experiencing a stress that it could encounter in the natural world. A limitation of the work is that it is unclear what the functional implications are of condensates of PFK-1 at the molecular or cell scale.
**Major comments:**
-All experiments were performed using fluorescently tagged PFK-1 expressed from endogenous promoter or from the native genetic locus which is important for excluding overexpression artifacts. However, there is still risk that the GFP tag is driving the assembly process. In order to exclude tag-specific effects that may cause aggregation of the tetrameric PFK-1, ideally a control would be done in which PFK-1 is visualized through immunofluorescence experiments of WT cells. Alternatively, a short tag (e.g HA, His) as epitope for is an alternative .
We used fluorescent tags to observe the dynamic relocalization in vivo. While in the study we have not performed immunofluorescence, we established the validity of the labeling method by: 1) using monomeric versions of GFP; 2) using different fluorophores to show the same condensation phenomenon; 3) performing CRISPR for single copy insertions; 4) Demonstrating that different glycolytic proteins form condensates; 5) demonstrating the GFP-tagged versions of the protein are capable of rescuing the loss-of-function alleles and 6) Now adding new data demonstrating the observed localization specifically depend on the presence of other glycolytic proteins. This last result supports that GFP tag is not driving the assembly process of glycolytic condensate and that the glycolytic condensate formation requires the presence of specific molecules in the pathway. I add that we routinely use fluorophore markers to over a dozen distinct proteins that label subcellular compartments, and we have never observed the dynamic relocalization reported here, with the exception of other glycolytic proteins that interact with PFK, suggesting this is a property specific to glycolytic proteins, and, based on the genetic studies, dependent on the glycolytic reaction. We add and discuss these findings in Figures 7G, 7H, S7B, and S7C; lines 422-441, 964-989.
-For the Cry2-section, the complementation of the pfk-1 mutant supports functionality of the synaptic clustering phenotype. Are there other features of function that can be evaluated or could you look at how Cry-2 vs wt worms recover from different durations of stress or frequencies. Could you see if the Cry-2-fusion will rescue function to a partial-loss-of-function allele or a tetramerization deficient allele? A detailed analysis of the effects of constitutive presence of PFK-1-Cry2 clusters would be necessary to bolster claims that this is fully functional construct. Can enzyme activity be somehow monitored?
We did not observe any difference between wild-type worms and CRY2-expressing worms with regards to their development, survival, locomotive behavior or synaptic phenotype. While we can not discard the possibility that this is not a full rescue, with available tools, we can not distinguish the recue with PFK-1-Cry2 from that of just PFK-1.
-The analysis of the sphericity of clusters (4A) is limited due to the diffraction limit of light which limits an analysis of a compartment of this size. While this is a limitation of the live organism, this should be more clearly acknowledged.
We have included in the Methods section our criteria for quantifying condensates and avoiding diffraction limit artifacts. Briefly, “Considering the resolution limit of a spinning disc confocal (approximately 300nm), any structure with a diameter less than 500nm and an area smaller than 0.2 µm2 was excluded from the analyses”. To better clarify this point, we also now add a description of the criteria used in the main text (lines 242-243).
In addition, we observed that PFK-1.1 condensates are not perfect spheres, but constrained spheroids (which can not be explained by diffraction-limited point spread functions). We can explain the observed spheroid shapes based on liquid-like properties of the condensates, and the constrains of the diameter of the neurite. To better highlight this finding, we have now moved Figure S4E into the main figure (Figure 4B’).
-Fusion experiments (4C) do not fully exclude that clusters overlap instead of merging. It would be beneficial to show the foci for several subsequent frames. One would expect that upon fusion, the condensate size would increase, but video 3 suggests the opposite. It would be useful to quantify condensate size before and after fusion for several separate fusion events. -an alternative possible experiment would be the tagging of PFK-1 with a photoconvertible fluorophore (e.g. Dendra2) and subsequent analysis of fusion events
To better show the fusion events in Figure 4C, we now include all xy, yz, and zx plane views of before and after fusion events of Figure 4C (Figure S5B). We also added a quantification of four independent fusion events in which we compare the sum of the areas of the two puncta before fusion and the size of the area of the single punctum after fusion (Figure S5C). These data support that we are observing fusions events.
-4D). It is unclear if foci are indeed undergoing fission or if two clusters next to each other are moving apart.
For Figure 4D, in all the frames we had recorded, a single structure maintains a continuous signal until fission occurs and splits into two structures. To better present this event, we now include an unabridged version of figure of 4D in the supplement that shows all the frames captured (Figure S5D).
-The analysis of side-by-side growth and dissolution kinetics are interesting and a novel view into the non-equilibrium aspects of phase separation in cells.
-Purification of PFK-1 and in vitro reconstitution of condensates would be supportive of liquid-like characteristics although I don't think it is necessary however it would add a lot to the relevance to show enzyme activity is different +/- condensate state but I am not sure if an easy enzymatic assay exists in vitro.
We agree. But the significance of this particular paper, specifically in the context of the in vitro enzymatic work on glycolytic proteins, is to examine the dynamic in vivo localization and the biophysical characteristics of the condensates. To better underscore this in the context of the field, we add discussion of a recent in vitro manuscript demonstrating that liquid droplet formation of glycolytic proteins affect their enzymatic activity (Ura et al., 2020) (lines 444-464; 484-492). While we see the value of future studies reconstituting the glycolytic particles, we believe that is beyond the scope of this particular in vivo study.
**Minor comments:**
-Stress granules in other organisms (yeast paper) have different composition depending on stress type. To make the claim that the PFK-1 compartments are independent of SGs one would ideally test multiple different SG markers.
We selected the stress granule protein TIAR-1 because it is one of the most studied stress granule markers in C. elegans and it is reportedly one of the core proteins and universal components of stress granules irrespective of a stress type (Buchan et al., 2011; Gilks et al., 2004; Huelgas-Morales et al., 2016; Kedersha et al., 1999). Although we did not include images in the manuscript, we had tested a total of three stress granule markers: TIAR-1, TDP-43, and G3BP1 with similar results. We now added that as data not shown (lines 193-194).
-it should be stated in the main text that the microfluidic-hydrogel device was fabricated following previously published protocols
We have added the reference in the main text (line 170) to supplement what we had written in the Methods section: “A reusable microfluidic PDMS device was fabricated to deliver gases through a channel adjacent to immobilized animals, following protocols as previously described (Lagoy and Albrecht, 2015)”.
-Figure 4b: Y-axis should be changed from probability to fraction of occurrence
We have corrected this in both the figure and the figure legends (Figure 4B).
-The discussion should be less speculative concerning any effects seen in PFK1-Cry2 expressing C. elegans
We have modified the discussion as suggested.
-it is perplexing that a protein known to tetramerize with no disordered or RNA-binding domains forms condensates like this. Is there anything known from other systems of additional interacting proteins that may have features that promote liquidity and serve to fluidize these assemblies?
Condensates can form via multivalent interactions, which include, but is not exclusive, to disordered or RNA-binding domains. Because glycolytic proteins have dihedral symmetries that can facilitate multivalent interactions, we believe these structural properties, in combination with regulated conformational changes, promote multivalent interactions leading to their condensation. We had a statement in the discussion (__lines 494-519) __now add this more clearly in the results (lines 395-398).
Reviewer #2 (Significance (Required)):
Stimulus-induced phase separation has been observed for dozens of metabolic enzymes from various different pathways (reviewed in Prouteau, 2018). Several studies have published the formation of condensates through PFK-1 in diverse organisms (C. elegans, Yeast, human cancer cells) in response to hypoxia or in some cancer lines also without hypoxia (Jin, 2017, Jang, 2016, Kohnhorst 2017, etc.). A yeast study showed that PFK-1 condensates contain various other glycolytic enzymes and that condensate formation enhances glycolytic rates (Jin, 2017).
This study gives the advance of analyzing the dynamics of PFK-1 condensate formation in vivo in the context of a live animal using a microfluidic-hydrogel device and showing that PFK-1 relocalizes to reversible condensates within minutes of hypoxia. If further appropriate experiments (as mentioned above) are performed, this study would strongly suggest that the underlying process of PFK-1 condensate formation is liquid-liquid phase separation. Ideally, if at all feasible, it would be strengthened if there was some insight into the functional consequences of the condensed assemblies formed in hypoxia. These findings may be interesting to researchers working on glycolysis and metabolism in different cells but particularly in neurons.
Field of expertise
-Phase separation, microscopy, in vitro reconstitution
-no experience with C. elegans biology and do not have a practical handle on ease or difficulties of genetic manipulation of C. elegans or metabolic assays for PFK-1
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
**Summary:**
In this manuscript, the authors focus on the subcellular localization of the key glycolytic enzyme PFK-1.1 in C. elegans, initially in whole animals through GFP tagging of the endogenous locus and subsequently in single cells/tissues using a clever genome editing strategy that permitted tissue-specific expression of GFP-tagged PFK-1.1 from its endogenous locus. They observe that PFK-1.1 localization differs from cell-type to cell-type and can be dynamically reorganized in response to exogenous cues. Focusing on hypoxia, they observe that PFK-1.1 forms foci near synapses in neurons under this stress condition. These foci are not stress granules and they are dissolved upon re-oxygenation. These condensates have properties of liquid droplets and can mature (harden) over time. PFK-1.1 fused to the CRY domain can trigger condensate formation under normoxic conditions, which can co-recruit WT PFK-1.1 as well as aldolase.
**Major comments:**
The conclusions are convincing but the impact could be increased if the authors were able to demonstrate the physiological role that the observed phase separation plays in this stress response. Would it be possible to assess glycolytic flux under hypoxia vs normoxia?
It is currently not possible to assess glycolytic flux *in vivo *in our system, as we lack metabolic sensors (an active area of work we are trying to address, but will take several years to perform correctly). We have added discussion of new in vitro studies examining the consequences of metabolic flux due to glycolytic compartmentation into liquid droplets (Ura et al., 2020), and the significance of those findings in the context of our in vivo studies (lines 444-464; 484-492).
The authors should comment on viability during the hypoxia time course.
C. elegans can survive anoxic condition for a day (Powell-Coffman, 2010). Our hypoxic conditions last minutes, and we can rescue live *C. elegans *upon completion of the assays. We now include a description of this in the Methods (lines 1216-1218).
The co-clustering of ALDO-1 and PFK-1.1::mCh::CRY2 in Figure 7 should be properly quantified/statistically analyzed
We quantified the fraction of animals that displays ALDO-1 clustering in PFK-1.1::mCh::CRY2 co-expressing animals, as suggested (Figure S7C).
A control of mCh::CRY2 + ALDO-1::EGFP is missing from the experiments shown in Figure 7. Is the presence of mCh::CRY2 sufficient to drive ALDO-1::EGFP clustering?
As a control for the CRY2 tag promoting the formation of glycolytic condensates, we had co-expressed mCh::CRY2 with PFK-1.1::EGFP, which is insufficient to cause the formation of the condensate (Figure 7C). We have now added a new data where we show that in pfk-1.1 deletion mutants, ALDO-1 condensate formation is suppressed, which further demonstrates the dependency between PFK-1.1 and ALDO-1 (Figures 7H and S7C).
Does hypoxia trigger co-clustering of ALDO-1 and PFK-1.1?
To answer this question, we examined the dynamic formation of ALDO-1 and PFK-1.1 condensates by co-expressing the two proteins together and observed that hypoxia triggers their co-clustering. We now include this in Figure 7E and Video 8.
The authors speculate that hypoxia acts via diminished energy (altered ATP AMP ratios). Can this be measured? To support this hypothesis, the authors may wish to test if similar phase separation is triggered by mitochondrial poisons.
We currently lack sensors that can reliably measure, in vivo, the subcellular changes in energy or metabolic flux in C. elegans neurons. However, we previously did test mitochondrial mutants and observed that in those mutants we observe glycolytic condensates (Jang et al., 2016), supporting the idea that defects in energy production promotes the formation of glycolytic condensates.
**Minor comments:** Is 21% O2 not hyperoxic for worms?
While C. elegans are known to prefer lower percentage of oxygen than those in air, in the lab animals are reared in normal air. We therefore used 21% oxygen present in air as our normoxic conditions.
Can the authors speculate more on how do these condensates exhibit "memory" (how they're able to cluster in the same place repeatedly)? Is there any role for the cytoskeleton in mediating nucleation and/or condensation of PFK and glycolytic enzymes?
When we were testing the reversibility of PFK-1.1 condensates, we were not expecting the reappearance of PFK-1.1 condensates in the same place repeatedly. Our current speculation is that, because many glycolytic enzymes, such as PFK-1.1, are allosterically regulated by nucleotides, AMP/ATP ratio may play a role on where glycolytic condensates appear. In other words, the specific synaptic areas, where PFK-1.1 condensate repeatedly reappeared, may have different AMP/ATP ratio that may trigger the condensation of the glycolytic proteins in those locationsupon conformational changes in PFK-1. We can’t exclude, currently, the presence of nucleating factors at synapses that facilitate PFK-1 clustering, but we have not yet identified them. We now include a discussion of this (lines 494-519).
Do the authors think that these clusters are effectively G-bodies from yeast?
G-bodies from yeast also shows glycolytic proteins changing from its diffuse localization to punctate localization in response to hypoxia (Jin et al., 2017). G-bodies, like *C. elegans *glycolytic condensates, are forms of subcellular glycolytic organization within eukaryotic cells. Yet, G-bodies take 24 hours to form, while we observe the glycolytic clusters in *C. elegans *within minutes of hypoxic conditions. We will need to understand the composition and function of both to determine if these forms of glycolytic subcellular organization represent the same structure. We note that glycolytic clusters have also been observed in some human cancer cell lines (Kohnhorst et al., 2017). Observation of glycolytic compartments in multiple different species and cell types suggest that, although the regulation, composition and formation kinetics of the glycolytic condensates may differ, compartmentalization of glycolytic enzymes may be a conserved feature. We now add a sentence discussing this (line 535-537).
Reviewer #3 (Significance (Required)):
It is much appreciated that this study tackles the cell biology of signaling and metabolism, which is a fascinating but difficult to study aspect of molecular biology. This work conclusively documents the dynamic reorganization of metabolic enzymes in vivo in response to physiological stimuli. Such reorganization had been proposed previously but was controversial and difficult to study in a controlled way. This work not only confirms previous observations but further demonstrates that the dynamic reorganization is mediated by a liquid-liquid phase separation. What is lacking is a demonstration that this phase separation is physiologically important. Such observations would generate interest from a much broader audience; the present audience presently targeting people specifically interested in non-membrane organelles per se. The reviewer has expertise in cell signalling and its regulation by phase separation.
As we explain for Reviewer 1, we focus this study on the biophysical characterization of the condensates, and how that results in compartmentalized enrichment of glycolytic proteins. Examination of the functional significance of the phase separation to the enzymatic reactions *in vivo *is not currently possible because we lack probes we can use *in vivo *to measure the metabolites resulting from the reaction. We have now added discussion acknowledging this and framing its significance in the context of what has been published in the field (lines 484-492). For example, a recent manuscript in ChemRxiv demonstrated, in vitro, that the enzymatic activity of glycolytic proteins, hexokinase and glucose-6 phosphate dehydrogenase, promote these enzymes condensing into liquid droplets. The authors further found that the condensation accelerated the glycolytic reactions (Ura et al., 2020). This raises the question whether glycolytic proteins compartmentalize, and form condensates, in vivo, which we address in this manuscript. We capture this point in (lines 444-464) where we explain that, while it has long been hypothesized that glycolytic proteins like PFK-1 could be compartmentalized, this remained controversial due to lack of dynamic *in vivo * imaging. In our study, and through a systematic examination of endogenous PFK-1.1 via the use of a hybrid microfluidic-hydrogel device, we conclusively determine that PFK-1.1 indeed displays distinct patterns of subcellular localization in specific tissues in vivo.
**REFEREES CROSS-COMMENTING** Globally it seems that all reviewers feel that impact would be increased if the physiological consequence of PFK-1.1 condensates was examined. Other, specific comments seem fair.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #3
Evidence, reproducibility and clarity
Summary:
In this manuscript, the authors focus on the subcellular localization of the key glycolytic enzyme PFK-1.1 in C. elegans, initially in whole animals through GFP tagging of the endogenous locus and subsequently in single cells/tissues using a clever genome editing strategy that permitted tissue-specific expression of GFP-tagged PFK-1.1 from its endogenous locus. They observe that PFK-1.1 localization differs from cell-type to cell-type and can be dynamically reorganized in response to exogenous cues. Focusing on hypoxia, they observe that PFK-1.1 forms foci near synapses in neurons under this stress condition. These foci …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Summary:
In this manuscript, the authors focus on the subcellular localization of the key glycolytic enzyme PFK-1.1 in C. elegans, initially in whole animals through GFP tagging of the endogenous locus and subsequently in single cells/tissues using a clever genome editing strategy that permitted tissue-specific expression of GFP-tagged PFK-1.1 from its endogenous locus. They observe that PFK-1.1 localization differs from cell-type to cell-type and can be dynamically reorganized in response to exogenous cues. Focusing on hypoxia, they observe that PFK-1.1 forms foci near synapses in neurons under this stress condition. These foci are not stress granules and they are dissolved upon re-oxygenation. These condensates have properties of liquid droplets and can mature (harden) over time. PFK-1.1 fused to the CRY domain can trigger condensate formation under normoxic conditions, which can co-recruit WT PFK-1.1 as well as aldolase.
Major comments:
The conclusions are convincing but the impact could be increased if the authors were able to demonstrate the physiological role that the observed phase separation plays in this stress response. Would it be possible to assess glycolytic flux under hypoxia vs normoxia?
The authors should comment on viability during the hypoxia time course.
The co-clustering of ALDO-1 and PFK-1.1::mCh::CRY2 in Figure 7 should be properly quantified/statistically analyzed
A control of mCh::CRY2 + ALDO-1::EGFP is missing from the experiments shown in Figure 7. Is the presence of mCh::CRY2 sufficient to drive ALDO-1::EGFP clustering?
Does hypoxia trigger co-clustering of ALDO-1 and PFK-1.1?
The authors speculate that hypoxia acts via diminished energy (altered ATP AMP ratios). Can this be measured? To support this hypothesis, the authors may wish to test if similar phase separation is triggered by mitochondrial poisons.
Minor comments: Is 21% O2 not hyperoxic for worms? Can the authors speculate more on how do these condensates exhibit "memory" (how they're able to cluster in the same place repeatedly)? Is there any role for the cytoskeleton in mediating nucleation and/or condensation of PFK and glycolytic enzymes? Do the authors think that these clusters are effectively G-bodies from yeast?
Significance
It is much appreciated that this study tackles the cell biology of signalling and metabolism, which is a fascinating but difficult to study aspect of molecular biology. This work conclusively documents the dynamic reorganization of metabolic enzymes in vivo in response to physiological stimuli. Such reorganization had been proposed previously but was controversial and difficult to study in a controlled way. This work not only confirms previous observations but further demonstrates that the dynamic reorganization is mediated by a liquid-liquid phase separation. What is lacking is a demonstration that this phase separation is physiologically important. Such observations would generate interest from a much broader audience; the present audience presently targeting people specifically interested in non-membrane organelles per se. The reviewer has expertise in cell signalling and its regulation by phase separation.
REFEREES CROSS-COMMENTING Globally it seems that all reviewers feel that impact would be increased if the physiological consequence of PFK-1.1 condensates was examined. Other, specific comments seem fair.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
This paper reports on the condensation of the glycolytic enzyme PFK-1 in response to hypoxic conditions in neurons of C. elegans. The authors employ a microfluidic-hydrogel device to dynamically monitor the relocalisation of PFK-1 from a mostly diffuse state to clusters in response to hypoxia and show that PFK-1 can undergo multiple rounds of PFK-1 clustering and dissolution. The authors work through the key features of a liquid-like compartment (sphericity, fusion, fast internal rearrangements) and give evidence that PFK-1 may have all three. Finally, the authors tag PFK-1 with the light-inducible multimerization domain Cry2 and …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
This paper reports on the condensation of the glycolytic enzyme PFK-1 in response to hypoxic conditions in neurons of C. elegans. The authors employ a microfluidic-hydrogel device to dynamically monitor the relocalisation of PFK-1 from a mostly diffuse state to clusters in response to hypoxia and show that PFK-1 can undergo multiple rounds of PFK-1 clustering and dissolution. The authors work through the key features of a liquid-like compartment (sphericity, fusion, fast internal rearrangements) and give evidence that PFK-1 may have all three. Finally, the authors tag PFK-1 with the light-inducible multimerization domain Cry2 and find that even without light PFK-1 will constitutively form clusters that sequestrate endogenous PFK-1 as well as other glycolytic proteins. The strength of this work is that it is characterizing what appears to likely be phase separation in the context of a whole animal experiencing a stress that it could encounter in the natural world. A limitation of the work is that it is unclear what the functional implications are of condensates of PFK-1 at the molecular or cell scale.
Major comments:
-All experiments were performed using fluorescently tagged PFK-1 expressed from endogenous promoter or from the native genetic locus which is important for excluding overexpression artifacts. However, there is still risk that the GFP tag is driving the assembly process. In order to exclude tag-specific effects that may cause aggregation of the tetrameric PFK-1, ideally a control would be done in which PFK-1 is visualized through immunofluorescence experiments of WT cells. Alternatively, a short tag (e.g HA, His) as epitope for is an alternative .
-For the Cry2-section, the complementation of the pfk-1 mutant supports functionality of the synaptic clustering phenotype. Are there other features of function that can be evaluated or could you look at how Cry-2 vs wt worms recover from different durations of stress or frequencies. Could you see if the Cry-2-fusion will rescue function to a partial-loss-of-function allele or a tetramerization deficient allele? A detailed analysis of the effects of constitutive presence of PFK-1-Cry2 clusters would be necessary to bolster claims that this is fully functional construct. Can enzyme activity be somehow monitored?
-The analysis of the sphericity of clusters (4A) is limited due to the diffraction limit of light which limits an analysis of a compartment of this size. While this is a limitation of the live organism, this should be more clearly acknowledged.
-Fusion experiments (4C) do not fully exclude that clusters overlap instead of merging. It would be beneficial to show the foci for several subsequent frames. One would expect that upon fusion, the condensate size would increase, but video 3 suggests the opposite. It would be useful to quantify condensate size before and after fusion for several separate fusion events.
-an alternative possible experiment would be the tagging of PFK-1 with a photoconvertible fluorophore (e.g. Dendra2) and subsequent analysis of fusion events
-4D). It is unclear if foci are indeed undergoing fission or if two clusters next to each other are moving apart.
-The analysis of side-by-side growth and dissolution kinetics are interesting and a novel view into the non-equilibrium aspects of phase separation in cells.
-Purification of PFK-1 and in vitro reconstitution of condensates would be supportive of liquid-like characteristics although I don't think it is necessary however it would add a lot to the relevance to show enzyme activity is different +/- condensate state but I am not sure if an easy enzymatic assay exists in vitro.
Minor comments:
-Stress granules in other organisms (yeast paper) have different composition depending on stress type. To make the claim that the FPK-1 compartments are independent of SGs one would ideally test multiple different SG markers.
-it should be stated in the main text that the microfluidic-hydrogel device was fabricated following previously published protocols
-Figure 4b: Y-axis should be changed from probability to fraction of occurrence
-The discussion should be less speculative concerning any effects seen in PFK1-Cry2 expressing C. elegans
-it is perplexing that a protein known to tetramerize with no disordered or RNA-binding domains foms condensates like this. Is there anything known from other systems of additional interacting proteins that may have features that promote liquidity and serve to fluidize these assemblies?
Significance
Stimulus-induced phase separation has been observed for dozens of metabolic enzymes from various different pathways (reviewed in Prouteau, 2018). Several studies have published the formation of condensates through PFK-1 in diverse organisms (C. elegans, Yeast, human cancer cells) in response to hypoxia or in some cancer lines also without hypoxia (Jin, 2017, Jang, 2016, Kohnhorst 2017, etc.). A yeast study showed that PFK-1 condensates contain various other glycolytic enzymes and that condensate formation enhances glycolytic rates (Jin, 2017).
This study gives the advance of analyzing the dynamics of PFK-1 condensate formation in vivo in the context of a live animal using a microfluidic-hydrogel device and showing that PFK-1 relocalizes to reversible condensates within minutes of hypoxia. If further appropriate experiments (as mentioned above) are performed, this study would strongly suggest that the underlying process of PFK-1 condensate formation is liquid-liquid phase separation. Ideally, if at all feasible, it would be strengthened if there was some insight into the functional consequences of the condensed assemblies formed in hypoxia. These findings may be interesting to researchers working on glycolysis and metabolism in different cells but particularly in neurons.
Field of expertise
-Phase separation, microscopy, in vitro reconstitution
-no experience with C. elegans biology and do not have a practical handle on ease or difficulties of genetic manipulation of C. elegans or metabolic assays for PFK-1
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Referee #1
Evidence, reproducibility and clarity
Summary
Jang et al., address the important question of spatially localized or compartmentalized metabolic enzymes with a focus on the glycolytic enzyme PFK1. Using a good strategy of inserting a fluorescent tag at the endogenous PFK1 locus with tissue-specific inducible expression in C. elegans, combined with strong quantitative longitudinal imaging and innovative bioengineered microfluidic-hydrogels to control oxygen availability as well as optogenetic approaches, they show PFK1 condensates, which are not stress granules and not seen in normoxia, assemble with hypoxia. PFK1 condensates are dynamic, reversible, localized at the …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary
Jang et al., address the important question of spatially localized or compartmentalized metabolic enzymes with a focus on the glycolytic enzyme PFK1. Using a good strategy of inserting a fluorescent tag at the endogenous PFK1 locus with tissue-specific inducible expression in C. elegans, combined with strong quantitative longitudinal imaging and innovative bioengineered microfluidic-hydrogels to control oxygen availability as well as optogenetic approaches, they show PFK1 condensates, which are not stress granules and not seen in normoxia, assemble with hypoxia. PFK1 condensates are dynamic, reversible, localized at the synapse in neurons, and recruit aldolase, another glycolytic enzyme. Although glycolytic proteins were previously shown to compartmentalize near the plasma membrane, and PFK1 was previously shown to assemble into filaments in vitro and be punctate at the plasma membrane in mammalian cells, evidence for cellular localized PFK1 condensates in animals is highly significant. The work includes strong biophysical characterization of PFK1 phase-separated condensates, but no clear indication of the composition of condensates. More significantly, the findings lack functional significance related to PFK1 activity or glycolytic flux with hypoxia vs normoxia. Despite previous work by this group showing that disrupting subcellular localization of glycolytic enzymes impairs neuronal activity in response with hypoxia, the reader is left with questions on the importance of localized and PFK1 condensates and their make-up .
Major comments:
Key conclusions are convincing, and most experimental approaches, biophysical characterization including thermodynamic principles, and data analysis are exemplary and well described. However, as indicated above, the work is limited to a descriptive analysis of cellular localization of PFK1 condensates and their biophysical properties without insights on functional significance relative to enzyme activity - or at least glycolytic flux or metabolic reprogramming with hypoxia. At best, only correlations can be drawn from hypoxia-induced localized PFK1 condensates and the authors' previous report (Jang et al., 2016) on hypoxia-regulated neuronal activity. Some insight or at least prediction in the discussion on the differences in spatially localized PFK1 in muscle vs neurons with regard to metabolic or energy distinctions should be included.
Despite the strong biophysical analysis of condensates, several important features are not determined. First is at best a rudimentary analysis of the composition of condensates and also how PFK1 is assembled into these structures. For the former, is the core of the condensate predominantly PFK1 with perhaps aldolase only recruited to the periphery or is aldolase an integral component of the structure. Hence, is it a PFK1 condensate or a glycolytic condensate? For the latter question, is there a particular orientation for PFK1 in condensates, i.e a collection of filaments as previously reported, which might provide insight on assembly? Finally, and less critical but also important is the criterion for spherical, which is not well defined, and at least some idea or speculation on determinants for a spherical morphology - compared with filaments that have been reported for other non-glycolytic metabolic enzymes.
Significance
The work is an important advance in our understanding on the self-assembly of metabolic enzymes by showing hypoxia-induced PFK1 condensates in vivo, their spatially-restricted subcellular localization in muscle cells and neurons, and their biophysical properties, the latter being distinct from those of stress granules. Taken together, these findings are more extensive than many previous reports on the assembly of metabolic enzymes into filaments or condensates, but fall short for new insights on functional significance.
Expertise is published on topic
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