Bridge-like lipid transfer protein family member 2 suppresses ciliogenesis

  1. Jan Parolek
  2. Christopher G. Burd

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Abstract

Bridge-like lipid transfer protein family member 2 (BLTP2) is an evolutionary conserved protein with unknown function(s). The absence of BLTP2 in Drosophila melanogaster results in impaired cellular secretion and larval death, while in mice ( Mus musculus), it causes preweaning lethality. Structural predictions propose that BLTP2 belongs to the repeating β-groove domain-containing (also called the VPS13) protein family, forming a long tube with a hydrophobic core, suggesting that it operates as a lipid transfer protein (LTP). We establish BLTP2 as a negative regulator of ciliogenesis in RPE-1 cells based on a strong genetic interaction with WDR44, a gene that also suppresses ciliogenesis. Like WDR44, BLTP2 localizes to membrane contact sites involving the endoplasmic reticulum and the tubular endosome network in HeLa cells and that BLTP2 depletion enhanced ciliogenesis in RPE-1 cells grown in serum-containing medium, a condition where ciliogenesis is normally suppressed. This study establishes human BLTP2 as a putative LTP acting between tubular endosomes and ER that regulates primary cilium biogenesis.

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  1. Version published to 10.1091/mbc.e24-02-0065
  2. Review Commons

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  3. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    This is a manuscript that focuses on understanding the function of BLTP2 protein in regulation of ciliation. BLTP family of proteins recently emerged as a lipid transporters in several contexts, such as formation and extension of autophagic membrane. The entire study is build on very intriguing observation that BLTP2 depletion leads to enhanced ciliation in RPE1 cells grown in the presence of serum. That makes BLPT2 a negative regulation of cilia formation that appears to mediate serum-dependent inhibition of ciliation in RPEs. Since molecular machinery governing serum-dependent inhibition of cilia formation remains poorly understood, this study does have a potentially high significance and interest. Unfortunately, most of the study (besides Figure 1) is done using Hela cells. It is a puzzling choice since Hela cells do not form cilia. Additionally, mots analyses are fairly descriptive and do not really lead to any specific hypothesis. As the result author's conclusion is very vague, specifically stating that "BLTP2 may suppress ciliogenesis by altering the lipid dynamics and/or densities of unidentified integral membrane proteins that suppress ciliogenesis". Thus, as it stands, this study does not really lead to new insights in BLPT2-dependent regulation of cilia formation.

    Few specific other comments are listed below.

    1. Figure 1. Two different individual siRNAs for each target should be used (unless authors show rescues) to minimize the possibility of off target effects.
    2. Figure 2-4. Not quite sure what was the rationale to study BLTP2 localization in HeLa cells instead of RPE1. Considering that the main focus of this manuscript is ciliation, one would want to see extensive analysis of localization of BLPT2 in RPE1 cells.
    3. The key to this manuscript (considering its focus on ciliation) would be to look at BLPT2 dynamics in the RPE1 cells in the presence and absence of serum, especially during ciliary vesicle and cilia formation. Instead, authors do most of their analysis in HeLa cells that do not even form cilia.

    Significance

    This is a manuscript that focuses on understanding the function of BLTP2 protein in regulation of ciliation. BLTP family of proteins recently emerged as a lipid transporters in several contexts, such as formation and extension of autophagic membrane. The entire study is build on very intriguing observation that BLTP2 depletion leads to enhanced ciliation in RPE1 cells grown in the presence of serum. That makes BLPT2 a negative regulation of cilia formation that appears to mediate serum-dependent inhibition of ciliation in RPEs. Since molecular machinery governing serum-dependent inhibition of cilia formation remains poorly understood, this study does have a potentially high significance and interest. Unfortunately, most of the study (besides Figure 1) is done using Hela cells. It is a puzzling choice since Hela cells do not form cilia. Additionally, mots analyses are fairly descriptive and do not really lead to any specific hypothesis. As the result author's conclusion is very vague, specifically stating that "BLTP2 may suppress ciliogenesis by altering the lipid dynamics and/or densities of unidentified integral membrane proteins that suppress ciliogenesis". Thus, as it stands, this study does not really lead to new insights in BLPT2-dependent regulation of cilia formation.

  4. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript by Parolek at al demonstrates the role of Bridge-like lipid transfer protein family member 2 (BLTP2) as a negative regulator of ciliogenesis in cultured retinal pigmental epithelial (RPE-1) cells. Based on genetic interaction of BLTP2 with another ciliogenesis negative regulator, WDR44, a RAB11 effector, the authors mechanistically study the regulation of cilia by these proteins. The authors nicely show Hela cell specific colocalization of BLTP2 and WDR44 in the ER-tubular endosome network membrane contact sites (ER-TEN MCS). They demonstrate that the C-terminus of BLTP2 is necessary for localization to the TEN. They also find that the WDR44 network to be present in higher proportion of Hela cells lacking BLTP2. Interestingly, BLTP2 and WDR44 also localize at tips of GFP-Rab8 and GFP Rab10 tubular membrane network in Hela cells. However, RPE-1 cells do not possess WDR44- and BLTP2-associated tubules, so the context of the TEN with the ciliogenesis phenotype in RPE cells, if any, is presently unclear. The rigor and reproducibility of the presented experiments is high and the presented results are novel for cilia, membrane trafficking, memembrane contact sites and lipid transfer fields. Overall, the authors suggest that BLTP2 and WDR44 are common components of a pathway that suppresses ciliogenesis in serum-fed RPE-1 cells.

    Major comments

    How general are the effects of BLTP2 on ciliogenesis? Effects on ciliogenesis in other cells would consolidate the current results in RPE-1 cells.

    Are the effects on ciliogenesis in RPE cells mediated by the role of BLTP2 and WDR44 in TEN network shown in Hela cells? If not, can the authors demonstrate the subcellular localization of these proteins with respect to ciliogenesis, and optionally if possible with the Rab8-Rabin8-Rab11 cascade? For eg., WDR44 knockdown has been shown to increase peri-centrosomal localization of Rabin8 in serum-fed conditions. Does BLTP2 knockdown have similar effects?

    The authors show that the C-term of BLTP2 is necessary for its recruitment into TEN network, irrespective of WDR44 in Hela cells. Does the C-term of BLTP2 also regulate ciliogenesis?

    Optional: Interactions between BLTP2 and WDR44 are currently unclear other than their colocalization. Can the authors further address if there are close physical interactions between these proteins and/or Rab11?

    Minor comments

    Fig 3C. In merge/inset: please check the white bar.

    If possible, show rescue of ciliogenesis phenotype from BLTP2 knockdown or knockout.

    Significance

    The roles of lipid transfer proteins in ciliogenesis are currently unknown. By showing the function of BLTP2 in ciliogenesis, the authors suggest the possibility of lipid transfer between the ER and the endo-lysosomal network in regulating ciliogenesis pathways. The results showing co-localization of BLTP2 and WDR44 in ER-tubular endosome network membrane contact sites (ER-TEN MCS) is nicely done and the effects on TEN upon WDR44 loss is also very intriguing. Recent papers suggest a role of WDR44 in ciliogenesis and in ciliopathies. The current data will be of broad interest to readers in ciliogenesis, intracellular trafficking, and lipid transport fields. From my own expertise in cilia biology, I think the results are exceptionally novel but needs more context to further solidify the role of BLTP2 in ciliogenesis.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    This manuscript by Parolek and Burd addresses the role of the evolutionarily conserved Bridge-like lipid transfer protein 2 (BLTP2) VPS13 family member in negatively regulating ciliogenesis. BLTP proteins are thought to mediate the transfer of lipids between a variety of different organelles, and while BLTP2 loss-of-function in plants and multicellular organism including flies and mice has been connected to various phenotypes, the role of human BLTP2 is poorly understood. In the current study, the authors initially used the Cancer Dependency Map Portal to find a very high mutual co-dependency between the BLTP2 gene and WDR44, a previously well-established negative regulator of primary ciliogenesis. This led the authors to ask whether BLTP2 (similar to WDR44) affects ciliogenesis and indeed they demonstrated that knock-down of either protein in serum-containing RPE1 cells (conditions that normally do not facilitate robust cilia formation) led to an increased percentage of ciliated cells. The authors next addressed the localization of human BLTP2, finding it on ER membranes as well as in linear/tubular structures that correlate with ER subdomains, at least in HeLa cells. Moreover, BLTP2 localization overlapped with that of WDR44. The authors then demonstrated that in BLTP2 knockout cell lines a significantly greater percentage of cells displayed a robust tubular endosome network than in wild-type cells. Finally, the authors show that both BLTP2 and WDR44 tend to localize to the tips of the endocytic tubular network, suggesting that these proteins may localize to contact sites between the ER and (tubular) endosomes, and further, that these contact sites may be required for primary ciliogenesis.

    Major Comments

    This is an interesting manuscript that addresses a poorly understood protein, and potentially provides a novel connection between ER-recycling endosome contact and ciliogenesis. The experimentation is well executed, and the presented data are clear. The strengths of the manuscript lie in partially unveiling new function for BLTP2, functionally associating BLTP2 with WDR44, and the potentially novel relationship between ER-endosome contact sites and ciliogenesis. Moreover, the tubular network of endosomes (which has been primarily implicated in recycling), remains an incompletely understood organelle and this manuscript suggests a new role for it. The weaknesses lie in the preliminary nature of the studies (which in the current form are largely correlative/speculative), and the somewhat descriptive nature of the study. For example, while the proposed connection between ER-endosome contacts, TEN, and cilia is intriguing, there is little attempt to address how such contact sites and TEN might affect ciliogenesis. Developing these ideas would greatly increase the significance of the findings, which as presented are mainly correlative. Other key questions include whether these proteins affect functions previously attributed to tubular endosomes, such as receptor recycling and endocytosis? What is the function of the TEN in maintenance or generation of ER contact sites? There is some literature suggesting that TEN serve in the sorting and/or fission process that might separate Rab5 anterograde transport from Rab4 recycling endosomes, but this is not addressed in the context of the current manuscript. How do ER contact sites and TEN impact ciliogenesis at a molecular level? How might a lipid transfer protein affect primary ciliogenesis? Is the TEN network connected to lipid transfer? Reports hold that PI4,5P2, PI4P, PA and other lipids might be enriched in the TEN; are these (or other lipids in the TEN) affected by BLTP2? In addition, while BLTP2 and WDR44 are linked by localization (and their mutual co-dependency), little attempt has been made to understand how these proteins function together. Do they physically interact? Are Rab8/10 responsible for their recruitment to the tips of endosomes?

    Minor comments:

    Could endogenous Rab proteins be used to address localization of BLTP2 and WDR44 to tubule tips?

    Could the authors please explain how the R=0.61 mutual co-dependency was arrived at? This reviewer used the portal website to find an R value of 0.58. Admittedly these numbers are both very high and the difference between them not significant, but nonetheless they are not identical.

    Significance

    The novelty of this study is that it identifies a new player, BLTP2, involved in the regulation of primary ciliogenesis. Since ciliogenesis impacts so many physiological events and underlies over 100 distinct ciliopathgies, there is clear significance in identifying new proteins that regulate this process (even if hundreds have already been identified). Perhaps the most (potentially) novel finding is the relationship between the ER contact sites, the tubular network of endosomes and ciliogenesis. Few papers have made any connection between tubule generation and ciliogenesis, with the exception of Insinna et al. (Nat Comm., 2019), a paper reporting that the tubular endosome scaffold MICAL-L1 is required for ciliogenesis (Xie et al., J. Cell Sci., 2019), and the recent Biorxiv manuscript by Ott and colleagues suggesting that tubules allow formation of "deconstructed" cilia, none of these connect ciliogenesis to ER contact sites. The limitations of the study are the preliminary nature; as noted, while the individual experiments are clear, substantial data that clearly connect ER contact sites, tubular endosomal networks and ciliogenesis remain largely uncharacterized in this study. In terms of the audience, if a more tangible relationship between ER contact sites/TEN and cilia could be derived, the study would be cross-disciplinary and of interest to a wide audience.

  6. Version published to 10.1101/2023.12.07.570614v1 on bioRxiv