LYVAC/PDZD8 is a lysosomal vacuolator

Abstract

Lysosomal vacuolation is commonly found in many pathophysiological conditions, but its molecular mechanisms and functions remain largely unknown. Here, we show that the endoplasmic reticulum (ER)-anchored lipid transfer protein PDZ domain-containing 8 (PDZD8), which we propose to be renamed as lysosomal vacuolator (LYVAC), is a general mediator of lysosomal vacuolation. Using human cell lines, we found that diverse lysosomal vacuolation inducers converged on lysosomal osmotic stress, triggering LYVAC recruitment through multivalent interactions. Stress-induced lysosomal lipid signaling contributed to both the recruitment and activation of LYVAC. By directly sensing lysosomal phosphatidylserine and cholesterol, the lipid transfer domain of LYVAC mediated directional ER-to-lysosome lipid movement, leading to osmotic membrane expansion of lysosomes. These findings uncover an essential mechanism for lysosomal vacuolation with broad implications in pathophysiology.

Figure 1.. Unbiased proteomics reveal LYVAC as an essential mediator of lysosomal vacuolation.

(A) Schematic illustration of the Lyso-TurboID screen. P20-SA↓, streptavidin (SA) pulldown from P20, the pellet fraction after centrifugation at 20,000g. (B) Dot plot of proteins identified by Lyso-TurboID mass spectrometry. The top 959 proteins with ≥10 peptides were analyzed. Orange dots indicate proteins with 1.4-fold peptide enrichment following apilimod treatment. (C) Immunoblot showing the enrichment of LYVAC on lysosomal surface in 293T cells following apilimod treatment. The asterisk indicates the streptavidin band. (D) LYVAC is required for apilimod-induced lysosomal vacuolation. U2OS cells of the indicated genotypes were treated with apilimod, followed by bright-field microscopy to assess vacuole formation. (E) Quantification of average cellular vacuole area in (D). (F) In confocal DIC imaging, apilimod-induced vacuoles exhibited near-complete colocalization with LAMP1-mCherry, a marker of lysosomes. Note, fluorescent lysosomal vacuoles appear slightly smaller than DIC-detected vacuoles. (G) LYVAC is required for lysosomal vacuolation upon PIKfyve deletion. Indicated U2OS cells were infected with PIKfyve CRISPR knockout lentiviruses and imaged seven days post-infection. (H) Quantification of average cellular vacuole area in (G). (I) LYVAC is essential for lysosomal vacuolation induced by FIG4 deletion. Indicated U2OS cells were infected with FIG4 CRISPR knockout lentiviruses and imaged seven days post-infection. (J) Quantification of average cellular vacuole area in (I). Bar, 10 μm. For all vacuole quantifications, cells were randomly and automatically selected using image analysis software; data are presented as mean ± s.e.m.; n = 300 cells per condition, pooled from three independent experiments. Two-way ANOVA with Tukey’s multiple comparison test for (E); ordinary one-way ANOVA with Tukey’s multiple comparison test for (H) and (J).

Figure 2.. LYVAC is a general mediator of lysosomal osmotic vacuolation.

(A) Knockout of LYVAC blocks metoclopramide-induced lysosomal vacuolation. (B) Quantification of average cellular vacuole area in (A). Mean ± sem; n = 300 cells/condition pooled from three independent experiments. (C) Knockout of LYVAC blocks sucrose-induced lysosomal vacuolation. Note that sucrose cannot trigger vacuoles in U2OS cells. (D) Quantification of (C). Mean ± sem; n = 20 random fields pooled from three independent experiments for each condition. (E) Knockout of LYVAC blocks lysosomal vacuolation induced by hypotonic media. (F) Quantification of average cellular vacuole area in (E). Mean ± sem; n = 300 cells/condition pooled from three independent experiments. (G) LYVAC is recruited to LAMP1-positive lysosomes following apilimod treatment. (H) Quantification of LYVAC recruitment in (G). Mean ± sem; n = 62, 52, 55, and 65 random cells, respectively, pooled from three independent experiments. (I) LYVAC is recruited to lysosomes after sucrose treatment. (J) Quantification of sucrose-induced LYVAC recruitment to lysosomes in (I). Mean ± sem. n = 78 and 56 random cells, respectively, pooled from three independent experiments. (K) ATG2A is recruited to lysosomes damaged by LLOME, but not recruited to vacuolating lysosomes following apilimod treatment. (L) Quantification of EGFP-ATG2A puncta in (K). Mean ± sem; n = 35, 43, 42, and 36 random cells, respectively, pooled from three independent experiments. (M) Schematic illustration of ATG2A and LYVAC, two lipid transfer proteins, as specific sensors of different types of lysosomal membrane stress. Bar, 10 μm. Ordinary one-way ANOVA with Tukey’s multiple comparison test for (B), (F), (H) and (L); two-way ANOVA with Tukey’s multiple comparison test for (D); student’s t-test for (J).

Figure 3.. Multivalent weak interactions mediate LYVAC recruitment to osmotically stressed lysosomes.

(A) Structures of different LYVAC domains and dimerization predicted by AlphaFold Multimer. Disordered regions (flexible sequences) were removed. (B) Illustration of LYVAC mutants generated to investigate LYVAC recruitment and lysosomal vacuolation. Key conclusions were summarized on the right. (C) Apilimod-induced lysosomal recruitment of LYVAC is dependent on its TM, C1, and CC domains. U2OS LYVAC knockout cells stably expressing indicated LYVAC mutants with a C-terminal Flag-tag were treated with apilimod and then fixed for immunostaining of Flag and endogenous LAMP1. (D) Quantification of the colocalization between LYVAC and LAMP1 in (C). Mean ± sem; n = 89, 76, 74, 58, 52, 30, 81, 61, 71, 41, 39, 21, 58, 66, 69, 55, 50, 48, 56, and 58 random cells from left to right, pooled from three independent experiments. (E) Lysosomal vacuolation requires most LYVAC domains. U2OS LYVAC-KO cells stably expressing the indicated LYVAC mutants were treated with apilimod for 5 h and subjected to bright-field microscopy. (F) Quantification of average cellular vacuole area in (E). Mean ± sem; n = 300 random cells/condition pooled from three independent experiments. (G) TM-C1-CC serves as a probe for lysosomal osmotic stress. U2OS cells stably expressing TM-C1-CC-Flag were treated with 25 nM apilimod, hypotonic media, 37 μM monesin, or 10 μM nigericin for 1 h to induce lysosomal osmotic stress and then fixed for immunostaining of Flag and endogenous LAMP1. (H) Quantification of the recruitment of potential lysosomal osmotic stress probes. Mean ± sem; n = 48, 53, 52, 29, 58, 34, 73, and 38 random cells from left to right, pooled from three independent experiments. (I) Schematic illustration of lysosomal LYVAC recruitment through multivalent interactions involving TM, C1, and CC domains. Bar, 10 μm. Two-way ANOVA with Tukey’s multiple comparison test.

Figure 4.. Lysosomal osmotic stress activates lipid signaling to promote LYVAC recruitment and vacuole formation.

(A) Mass spectrometry identified the enrichment of ORP family proteins on lysosomes following apilimod treatment. (B) Immunoblot showing apilimod-induced lysosomal enrichment of ORPs. (C) ORP1L is recruited to lysosomes upon apilimod treatment. (D) Quantification of ORP1L recruitment in (C); n = 38, 25, 26, and 24 random cells, respectively. (E) Quantification of lysosomal ORP1L recruitment in U2OS cells from fig. S10A; n = 34, 44, 31, 32, 19, 46, 32, and 29 random cells, respectively. (F) Quantification of apilimod-induced lysosomal recruitment of a PS probe in U2OS cells in fig. S10B; n = 35, 37, 46, 37, 40, 16, 33, 30, and 39 random cells, respectively. (G) Quantification of apilimod-induced lysosomal recruitment of a cholesterol probe in U2OS cells in fig. S10C; n = 32, 20, 14, 15, 13, 19, 23, 26, and 15 random cells respectively. (H) Schematic illustration of lysosomal osmotic stress-induced lipid signaling. (I) Lysosomal recruitment of LYVAC in genetically modified U2OS cells. (J) Quantification of LYVAC/LAMP1 colocalization in (I); n = 25, 25, 53, 59, 53, 38, 52, and 45 random cells, respectively. (K) Knockout of PI4K2A or ORPs suppresses apilimod-induced lysosome vacuolation. (L) Quantification of cellular vacuole area in (K); n = 230 cells/condition. (M-N) Quantification of cellular vacuole area in indicated U2OS cells in fig. S11C–D; n = 300 cells/condition. (O) Schematic illustration of lysosomal lipid signaling upstream of LYVAC. PI4K2A is recruited to osmotically stressed lysosomes where it produces PI(4)P to recruit five ORP family proteins. The ORPs exchange PI(4)P for cholesterol and PS, which promotes LYVAC recruitment and lysosomal vacuolation. Bar, 10 μm. Mean ± s.e.m; total cell numbers were pooled from three independent experiments. Ordinary one-way ANOVA with Tukey’s multiple comparison test for (D); two-way ANOVA with Tukey’s multiple comparison test for (E), (F), (G), (J), (L), (M), and (N).

Figure 5.. Phosphatidylserine and cholesterol on acceptor membranes activate LYVAC-mediated, directional lipid transfer in vitro.

(A) Schematic illustration of the in vitro membrane tethering assay by LYVAC-SMP. (B) When acceptor liposomes contain 20% PS, increased level of cholesterol stimulates higher membrane tethering activity of LYVAC-SMP. (C) Schematic illustration of the in vitro reconstitution of lipid transfer by purified LYVAC-SMP. (D, E) Cholesterol and PS in acceptor liposomes each stimulate LYVAC-SMP–mediated lipid transfer in a dose-dependent manner; Mean ± sem; n = 7 in (D) and n = 6 in (E). (F) The lipid compositions of different acceptor liposomes and related LYVAC activity. PI4P, 5%; PS, 20%, cholesterol: saturating levels. (G, H) Both PS and cholesterol in the acceptor liposome are required for efficient membrane tethering (G) and lipid transfer (H) by LYVAC-SMP; Mean ± sem; n = 4 in (G); n = 3 in (H). (I) Schematic illustration of the in vitro assays to determine the direction of lipid transfer by LYVAC-SMP. (J) Similar membrane tethering by LYVAC-SMP in both the forward and the reverse lipid transfer assays in (I); Mean ± sem; n = 4. (K) LYVAC-SMP preferentially transfers lipids from neutral liposomes, where it is anchored, toward PS/cholesterol-enriched liposomes; Mean ± sem; n = 5 or 6. (L) Illustration of the setup of the molecular dynamics simulation. Note that the forward transfer is more relevant, as the reverse transfer was not favored than forward in vitro, regardless of the lipid substrates. (M) Lipid transfer from the neutral donor to PS/cholesterol-enriched acceptor results in reduced total membrane energy, while the reverse transfer increases it. Each dot represents one simulation. (N) The lipid transfer activity of LYVAC-SMP is increased when the acceptor liposomes have higher luminal osmolarity; Mean ± sem; n = 7. (O) Elevated luminal osmolarity in donor liposomes reduced LYVAC-SMP–mediated lipid transfer; Mean ± sem; n = 6 or 7. See methods for details of all in vitro assays and MD simulations.

Figure 6.. Through PS- and cholesterol-sensing, LYVAC mediates ER-to-lysosome lipid movement and lysosomal vacuolation.

(A) Left: conserved motifs at the SMP tip. Middle: analogy between the FGK/R motifs and established cholesterol-binding CRAC/CARC motifs. Right: Mutations targeting predicted PS-sensing or cholesterol-sensing motifs. (B, C) All mutants of LYVAC-SMP lost activity in membrane tethering (B) and lipid transfer (C) toward PS/Cholesterol-positive acceptor liposomes (acceptor A6); Mean ± sem; n = 4 or 5. (D) Quantification of cellular vacuole area in fig. S16G; Mean ± sem; n = 300 cells/condition. (E) SRS ratiometric imaging of fixed U2OS wild-type (WT) cells reveals increased lysosomal lipid-to-protein ratio following apilimod (25 nM) treatment. Bar, 10 μm. (F) Quantification of the lysosomal lipid-to-protein ratios in WT and LYVAC-KO U2OS cells; Mean ± sem; n = 9 regions of interests (ROIs). (G) Lipid-to-protein ratios in lysosomes and adjacent ER within 1.5 μm of the lysosomal surface. Each dot represents one lysosome or its adjacent ER; lysosome–ER pairs are connected. (H, I) Quantification of the lipid concentration changes on isolated lysosomal (H) and ER (I) membranes in U2OS cells; Mean ± sem; n = 10 random ROIs. (J, K) Quantification of unsaturated lipid concentrations on isolated lysosomal (J) and ER (K) membranes in U2OS cells; Mean ± sem; n = 10 random ROIs. (L) Similarity scores of ER/lysosome lipid spectra before and after apilimod treatment in U2OS cells. Scores were calculated using the average spectra from 8–10 ROIs. (M) The average LAMP1 concentration on individual lysosomes decreases proportionally to the inverse square of lysosome diameter. Each data point represents one lysosome. See also S18C. Each ROI for SRS ratiometric imaging contained 10–12 cells. Approximately 100 cells per condition, from ~9 ROIs pooled across three independent experiments, were used for quantification. Ordinary one-way ANOVA with Tukey’s multiple comparison test for (D); two-way ANOVA with Tukey’s multiple comparison test for (F) and (H) to (L).