PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase

  1. Avishek Ghosh
  2. Aishwarya Venugopal
  3. Dhananjay Shinde
  4. Sanjeev Sharma
  5. Meera Krishnan
  6. Swarna Mathre
  7. Harini Krishnan
  8. Sankhanil Saha
  9. Padinjat Raghu

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Abstract

Phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 5-phosphate (PI5P) are low-abundance phosphoinositides crucial for key cellular events such as endosomal trafficking and autophagy. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) is an enzyme that regulates PI5P in vivo but can act on both PI5P and PI3P in vitro. In this study, we report a role for PIP4K in regulating PI3P levels in Drosophila . Loss-of-function mutants of the only Drosophila PIP4K gene show reduced cell size in salivary glands. PI3P levels are elevated in dPIP4K 29 and reverting PI3P levels back towards WT, without changes in PI5P levels, can rescue the reduced cell size. dPIP4K 29 mutants also show up-regulation in autophagy and the reduced cell size can be reverted by depleting Atg8a that is required for autophagy. Lastly, increasing PI3P levels in WT can phenocopy the reduction in cell size and associated autophagy up-regulation seen in dPIP4K 29 . Thus, our study reports a role for a PIP4K-regulated PI3P pool in the control of autophagy and cell size.

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  1. Version published to 10.26508/lsa.202301920
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    Reply to the reviewers

    The authors do not wish to provide a response at this time

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    Referee #3

    Evidence, reproducibility and clarity

    This study by Ghosh et al. proposes a role for phosphatidylinositol 5-phosphate 4-kinase (PIP4K) in regulating PI3P levels in vivo. They use loss-of-function Drosophila model of the only PIP4K gene (dPIP4K29) to investigate the PI3P and PI(3,5)P2 metabolizing enzymes. First, they showed that loss of function of PIP4K leads to reduced cell size in larval salivary glands and this was attributed to the elevated level of PI3P. Then, they modulated enzymes involved in PI3P metabolism (kinases and phosphatases) and propose the implication of the PI3P phosphatase myotubularin (Mtm) and the Pi3k Class III (PI3K59F) in PIP4K-dependent cell seize control. Finally, as PI3P has an established role in autophagy, they modulate the autophagy related gene (atg1) and connect the observed increase of PI3P level to the upregulation of autophagy in dPIP4K29 model. The authors used genetic manipulations of dPIP4K29 models as well as specialized lipidomic expertise (phosphoinositide measurement using mass spectrometry and PI-kinase/phosphatase assays) to address their conclusions. The experimental strategies were well designed and major conclusions were in line with the obtained results.

    Major comments:

    • Are the key conclusions convincing?

    Almost yes, however there is two major concerns for me: Concern 1 is about the level of PIP2/PI4,5P2, the product of PIP4K, in the dPIP4K29 model. This was not measured in the study. The authors claim page 5 that: "This observation suggests that the ability of dPIP4K to regulate cell size does not depend on the pool of PI(4,5)P2 that it generates... based on the fact that re-expression a mutation that hPIP4Kβ[A381E] in the salivary glands of dPIP4K29 (AB1> hPIP4Kβ[A381E]; dPIP4K29) (Figure S1A) did not rescue the reduced cell size. This mutation hPIP4Kβ[A381E] was generated in a study by Kunz et al. (2002) where they demonstrated by in vitro kinase assay that it cannot utilize PI5P as a substrate but can produce PI(4,5)P2 using PI4P as a substrate. In the same study, using MG-63 cells, Kunz et al. propose that the A381E mutation did not metabolize PI5P as it lost its plasma membrane localization. In my opinion the author should strength their claim about the role of dPIP4K independently of PI(4,5)P2 by addressing the level of PI(4,5)P2 in their model biochemically by mass spectrometry as they have this powerful tool and support this by using PH-PLCd probe to detect PI(4,5)P2. Also, as they use completely different model as Kunz et al. they should verify, if possible, the localization of hPIP4Kβ[A381E] vs WT PIP4Kβ in salivary glands.

    Concern 2: Page 7: The author used Mtm tagged constructs (mCherry and GFP) and measure its phosphatase activity toward PI(3,5)P2 and they did not show any obvious activity. I would like to suggest the use of untagged (or small tag construct, Flag or HA) for the expression experiment in S2R+ cell as it is known that active myotubularins in other cell model as well as in vitro have a strong 3-phosphatase activity toward PI(3,5)P2. By looking at the graph FigS2 Bii, we could clearly see a big disparity within mCherry-Mtm data points. This experiment should be more strengthen by additional experimental points but also by using a positive CTRL where PI(3,5)P2 level drops (inhibition of PIKfyve by Apilimod).

    Concern 3: Page 10: "we tagged dPIP4K with the tandem FYVE domain at the C-terminus end of the protein (dPIP4K2XFYVE) to target it to the PI3P enriched endosomal compartment and reconstituted this in the background of dPIP4K29. We did not observe a significant change in the cell size of dPIP4K29" I really don't understand the relevance of this experiment. FYVE tandem will bind to PI3P whenever it was in the cell (Lysosomes, autophagosome). Why the authors claim that the expression of restricted dPIP4K2XFYVE will be restricted to the endosomes. I think that this experiment is confusing and should be removed.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    See concern 1 to 3.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    Yes, the proposed experiments in concern 1-3 are not difficult to address as the authors have all the appropriate tools to manage this.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    Yes. It is not time consuming and not costly according to their expertise, available tools and materials that they used through the study.

    • Are the data and the methods presented in such a way that they can be reproduced?

    Yes

    • Are the experiments adequately replicated and statistical analysis adequate?

    Yes

    Minor comments:

    • Specific experimental issues that are easily addressable.

      1. Address the level of PI(4,5)P2 in dPIP4K29 model by mass spectrometry.
      2. Address the localization of hPIP4Kβ[A381E] vs WT PIP4Kβ in salivary glands.
      3. Test the Mtm phosphatase activity toward PI(3,5)P2 using untagged or small tagged (HA or Flag) Mtm and repeat/homogenize the PI(3,5)P2-phosphatase assay (FigS2ii).

    • Are prior studies referenced appropriately?

    Yes

    • Are the text and figures clear and accurate?

    The figures needsmore organization.

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    NO

    Referees cross-commenting

    Overall, Reviewer #1 and #2 found the study by Ghosh et al interesting well designed and written providing insights into the role of PIP4K in regulating cell seize. However, they comment few points that would be very helpful to improve the study. I am agreeing with both reviewers for the raised comments.

    Significance

    The author addressed how elevated PI3P in dPIP4K29 model impacted cell seize. Indeed, they connected this cell phenotype to the autophagy where PI3P plays a crucial role. However, I am still questioning how deletion of PIP4K enhances PI3P level.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    The role of PIP4K in cellular homeostasis and organismal physiology is still unclear. This study brings additional insights into how PIP4K could be involved in important cellular process such as autophagy by regulating additional phsophoinositides.

    • State what audience might be interested in and influenced by the reported findings.

    Phosphoinositide metabolism

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Phosphoinositides, Myotubularin, endolysosomal trafficking, skeletal muscle.

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    Referee #2

    Evidence, reproducibility and clarity

    The authors utilise a drosophila model to investigate the molecular mechanisms underlying the role of dPIP4K in regulating cell size. They suggest that PIP4K directly regulates PI3P levels in cells, which through upregulating autophagy, can reduce cell size. Overall, this is an interesting and well-designed study. The main downfall of this study is whether the regulation of PI3P levels by dPIP4K is occurs via direct or indirect mechanisms, which is unclear in the data provided in this study.

    More specific comments are as follows:

    Major Comments:

    It's not clear why there are no differences in PI3P/PIP2 levels in Figure 4B, but this is overcome by normalising to organic phosphate levels (4C)? Can differences in PI3P/PIP2 levels be seen in Figure 4B without normalisation if additional controls such as PI3K59F/VPS34 KD were used (as done in figure 5B)? A discussion of this could be useful.

    Figure 4D: Does the A381E mutant of PIP4K affect PI3P levels in cells as it cannot reverse the cell size phenotype in Figure S1B?

    Figure 4G: The conclusion on line 255 that all phosphatase transcripts are unchanged in this figure when two of them appear to have significant reduction appears inaccurate. In addition, changes of transcript levels of these enzymes may not necessarily reflect their overall activity in cells. A localised reduction in MTM levels or activity may well play a role in dPIP4K29 cells even though an overall phosphatase activity is seen increased in the in vitro assay in Figure 4F. Similarly, not clear that the authors can completely rule out a potential activation of PIP3K59/vps34 and subsequent increase in PI3P levels in cells by simply looking at RNA levels. Is there a reason why the authors could not measure the enzyme levels in cells as mentioned in the text? VPS34 activity can be measured in mammalian systems. This is important as PI3PK59 KD does seem to reverse change in cell size (Figure 5A).

    Another method to test the involvement of PI3K59/Vps34 is to target its adaptor proteins. Can the authors distinguish the endosomal and autophagosomal PIP3K59/vps34 complex and PI3P production by looking at drosophila homologues of Atg14 and UVRAG? The majority of PI3P in mammalian cells is found in the endosomal compartment rather than autophagosomal vesicles. If the authors predict that only autophagosomal PI3P levels are changed, then an overall change in enzymatic activity required for PI3P accumulation may not be easy to detect in total cell extracts.

    Figure 5C&D: how specific is the FYVE domain fused probes to endosomal PI3P? Such probes are used in mammalian cells to measure overall PI3P, whether endosomal or autophagosomal. In addition, such probes when expressed in live cells can alter PI3P generation. In line with this comment, FYVE-domain probes can be used to quantify PI3P levels in fixed cells, this method could be used to verify changes in PI3P levels seen in PIP4K mutant flies.

    Minor Comments:

    Fig 1A: this is a slightly confusing diagram and could perhaps be made a little clearer. For an example, the arrows are not clearly differentiating phosphorylation from dephosphorylation events. Also, the choice of colour for the phosphatase arrows (brown-red) and kinases (also appearing brown-red) makes it harder to follow this figure.

    Similar comment applies to S4B: PI could be depicted as an unphosphorylated version of PI3P/PI5P and drown in the centre.

    Line 301: "lipidated Atg8a fuses with the formed omegasome" Atg8a fusion with omegasome is not an accurate description of the early autophagosome biogenesis events.

    A new image (similar to Fig 1A) depicting how PIP4K affect PI3P levels to summarise the findings of this manuscript would be helpful.

    The material and methods is an important section in this paper: a more thorough description of the methods, especially those referred to previous publications would be very helpful. The authors can at least add a brief outline of the methods they followed and include contents of buffers used.

    Significance

    Overall, this is a well designed and written study providing insights into the role of PIP4K in regulating PI3P levels and cell size in Drosophila. The authors develop interesting methods to measure endogenous levels of PI species, which can be useful for the wide research community. As I am not an expert in these Mass Spec analyses, it would be important for these assays to be thoroughly reviewed by a specialist to ensure that the methods used to quantify these phospholipids have been carefully controlled.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Here Ghosh et al describe a potentially novel role for PIP4K in controlling PI3P in Drosophila tissues. Using PIP4K loss of function mutants and innovative lipid measurement techniques the authors try to address how cell size and autophagy are affected and report it may be through a PIP4K-regulated PI3P pool in flies.

    Comments:

    Overall, the paper is interesting, pretty well written and it has a lot of details in it that have addressed most of the questions, however they do refrain from stating that actually the PIP4Ks phosphorylate PI3P. Is it possible to measure the product PI3,4P2 if this were true? The authors claim that the regulation is direct but never show if by expressing dPIP4K it can phosphorylate PI3P to PI34P2. Using their optimized label-free LC-MS/MS methods this should not be trivial or by performing an invitro kinase assay.

    Further, the authors claim there is an increase in autophagy in the dPIP4K animals however they only measured autophagosome numbers. Autophagy flux and lysosome functional assays need to be performed to accurately show this, as it has been demonstrated that the inhibition of PIP4kinases in mammalian cells does indeed cause an increase in the autophagosome pools but because of an autophagosome-lysosome fusion defect which ultimately impairs autophagy not increasing autophagy. This needs to be addressed in the fly system.

    Also, localization studies with PIP4K in Drosophila should be performed to explain the role in autophagy or see if they localize at the same compartments as the enzymes that have been shown to regulate PI3P levels in flies.

    Also of note, PI3P in mammalian epithelial cells has been shown to control cell size through regulation of autophagy (https://pubmed.ncbi.nlm.nih.gov/31941925/), but I guess it's novel in flies.

    Significance

    Overall, as it has already been shown that the PIP4K can regulate PI3P levels in vitro as well as PI3P has been shown to control cell size in mammalian cells so the novelty is diminished as well as how their results really impact autophagy are not complete as the authors only quantified Atg8a puncta. If the authors can show the activity in flies is real by measuring the product PI34P2 this would be compelling evidence. Also, they need to complete localization, autophagy flux assays, westerns of LC3 or p62, etc to accurately state that autophagy is enhanced.

  6. Version published to 10.1101/2022.06.18.496676 on bioRxiv