Membrane contact sites regulate vacuolar fission via sphingolipid metabolism

Reviewer #1 (Public Review):

The manuscript investigates the role of membrane contact sites (MCSs) and sphingolipid metabolism in regulating vacuolar morphology in the yeast Saccharomyces cerevisiae. The authors show that tricalbin (1-3) deletion leads to vacuolar fragmentation and the accumulation of the sphingolipid phytosphingosine (PHS). They propose that PHS triggers vacuole division through MCSs and the nuclear-vacuolar junction (NVJ). The study presents some solid data and proposes potential mechanisms underlying vacuolar fragmentation driven by this pathway. However, there are some concerns regarding the strength and interpretation of their lipid data, and the robustness of some conclusions. The manuscript would benefit from addressing these concerns and providing more conclusive evidence to support the proposed conclusions. Overall, the study provides valuable insights into the connection between MCSs, lipid metabolism, and vacuole dynamics, but further clarification will be highly valuable to strengthen the conclusions.

Reviewer #2 (Public Review):

This manuscript investigates the mechanism behind the accumulation of phytosphingosine (PHS) and its role in triggering vacuole fission. The study proposes that membrane contact sites (MCSs) are involved in two steps of this process. First, tricalbin-tethered MCSs between the endoplasmic reticulum (ER) and the plasma membrane (PM) or Golgi modulate the intracellular amount of PHS. Second, the accumulated PHS induces vacuole fission, most likely via the nuclear-vacuolar junction (NVJ). The authors suggest that MCSs regulate vacuole morphology through sphingolipid metabolism.
While some of the results in the manuscript are interesting the overall logic is hard to follow. In my assessment of the manuscript, my primary concern lies in its broad conclusions which, in my opinion, exceed the available data and raise doubts. Here are some instances where this comes into play for this manuscript:

2.) Major points for revision

1.) The rationale to start investigating a vacuolar fission phenotype in the beginning is very weak. It is basically based on a negative genetic interaction with NVJ1. Based on this vacuolar fragmentation is quantified. The binning for the quantifications is already problematic as, in my experience, WT cells often harbor one to three vacuoles. How are quantifications looking when 1-3 vacuoles are counted as "normal" and more than 3 vacuoles as "fragmented"? The observed changes seem to be relatively small and the various combinations of TCB mutants do not yield a clear picture.
2.) The analysis of the structural requirements of the Tcb3 protein is interesting but does not seem to add any additional value to this study. While it was used to quantify the mild vacuolar fragmentation phenotype it does not reoccur in any following analysis. Is the tcb3Δ sufficient to yield the lipid phenotype that is later proposed to cause the vacuolar fragmentation phenotype?
3.) The quantified lipid data also has several problems. i) The quantified effects are very small. The relative change in lipid levels does not allow any conclusion regarding the phenotypes. What is the change in absolute PHS in the cell. This would be important to know for judging the proposed effects. ii) It seems as if the lipid data is contradictory to the previous study from the lab regarding the role of tricalbins in ceramide transfer. Previously it was shown that ceramides remain unchanged and IPC levels were reduced. This was the rationale for proposing the tricalbins as ceramide transfer proteins between the ER and the mid-Golgi. What could be an explanation for this discrepancy? Does the measurement of PHS after labelling the cells with DHS just reflect differences in the activity of the Sur2 hydroxylase or does it reflect different steady state levels.
4.) Determining the vacuole fragmentation phenotype of a lag1Δlac1Δ double mutant does not allow the conclusion that elevated PHS levels are responsible for the observed phenotype. This just shows that lag1Δlac1Δ cells have fragmented vacuoles. Can the observed phenotype be rescued by treating the cells with myriocin? What is the growth rate of a LAG1 LAC1 double deletion as this strain has been previously reported to be very sick. Similarly, what is the growth phenotype of the various LCB3 LCB4 and LCB5 deletions and its combinations.
5.) The model in Figure 3 E proposes that treatment with PHS accumulates PHS in the endoplasmic reticulum. How do the authors know where exogenously added PHS ends up in the cell? It would also be important to determine the steady state levels of sphingolipids after treatment with PHS. Or in other words, how much PHS is taken up by the cells when 40 µM PHS is added?
6.) Previous studies have observed that myriocin treatment itself results in vacuolar fragmentation (e.g. Hepowit et al. biorXivs 2022, Fröhlich et al. eLife 2015). Why does both, depletion and accumulation of PHS lead to vacuolar fragmentation?
7.) The experiments regarding the NVJ genes are not conclusive. While the authors mention that a NVJ1/2/3 MDM1 mutant was shown to result in a complete loss of the NVJ the observed effects cannot be simply correlated. It is also not clear why PHS would be transported towards the vacuole. In the cited study (Girik et al.) the authors show PHS transport from the vacuole towards the ER. Here the authors claim that PHS is transported via the NVJ towards the vacuole. Also, the origin of the rationale of this study is the negative genetic interaction of tcb1/2/3Δ with nvj1. This interaction appears to result in a strong growth defect according to the Developmental Cell paper. What are the phenotypes of the mutants used here? Does the additional deletion of NVJ genes or MDM1 results in stronger growth phenotypes?
8.) As a consequence of the above points, several results are over-interpreted in the discussion. Most important, it is not clear that indeed the accumulation of PHS causes the observed phenotypes.

Reviewer #3 (Public Review):

In this manuscript, the authors investigated the effects of deletion of the ER-plasma membrane/Golgi tethering proteins tricalbins (Tcb1-3) on vacuolar morphology to demonstrate the role of membrane contact sites (MCSs) in regulating vacuolar morphology in Saccharomyces cerevisiae. Their data show that tricalbin deletion causes vacuolar fragmentation possibly in parallel with TORC1 pathway. In addition, their data reveal that levels of various lipids including ceramides, long-chain base (LCB)-1P and phytosphingosine (PHS) are increased in tricalbin-deleted cells. The authors find that exogenously added PHS can induce vacuole fragmentation and by performing analyses of genes involved in sphingolipid metabolism, they conclude that vacuolar fragmentation in tricalbin-deleted cells is due to the accumulated PHS in these cells. Importantly, exogenous PHS- or tricalbin deletion-induced vacuole fragmentation was suppressed by loss of the nucleus vacuole junction (NVJ), suggesting the possibility that PHS transported from the ER to vacuoles via the NVJ triggers vacuole fission.

This work provides valuable insights into the relationship between MCS-mediated sphingolipid metabolism and vacuole morphology. The conclusions of this paper are mostly supported by their results, but there is concern about physiological roles of tricalbins and PHS in regulating vacuole morphology under known vacuole fission-inducing conditions. That is, in this paper it is not addressed whether the functions of tricalbins and PHS levels are controlled in response to osmotic shock, nutrient status, or ER stress.

There is another weakness in their claim that the transmembrane domain of Tcb3 contributes to the formation of the tricalbin complex which is sufficient for tethering ER to the plasma membrane and the Golgi complex. Their claim is based only on the structural simulation, but not on biochemical experiments such as co-immunoprecipitation and pull-down.