Figures and data
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SY1 aggregates mature into large IBs over time in normal cells, but this process is defective in aged cells.
(A) The maturation process of SY1 aggregates in yeast cells from scattered small aggregates to large single IBs over time. DsRed-SY1 was expressed in yeast with the constitutive promoter TPI1, the phenotype of SY1 aggregates was visualized at 4 h, 16 h and 48 h. Scale bar = 2 μm. (B) Cells with different phenotypes of SY1 IBs are defined as three classes: Class 1 refers to only one IB in a cell, Class 2 refers to two IBs in a cell, and Class 3 indicates three or more IBs in a cell. Scale bar = 2 μm. (C) Time-course study showing that SY1 aggregates mature after 24 h of cultivation, when the cells have traversed the diauxic shift to enter the respiratory and stationary growth phase. IBs were examined every 4 h from 4 to 48 h; green bars indicate percentage of Class 3; white bars indicate percentage of Class 2; red bars indicate percentage of Class 1; the purple curve indicates the optical density of the cell culture, indicated by the vertical axis on the right. Error bars indicate the SD. (D) Maturation of SY1 IBs was defective in old yeast cells. Scale bar = 5 μm. (E) The proportion of cells with SY1 IBs was increased in old cells (61%) compared to young cells (27%). Data were analyzed using the Student’s t-test. Error bars indicate the SD. (F) The number of SY1 IBs in a cell was increased in young cells (2.64±1.29) versus old cells (4.91±3.32). Data were analyzed using the Student’s t-test. Error bars indicate the SD.
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Genome-wide high-content imaging screen identifying the serine C-palmitoyltransferase complex (SPT) as a modulator of SY1 inclusion maturation.
(A) Schematic workflow for identifying mutants that regulate SY1 inclusion maturation. (B) The GO enrichment analysis of genes whose mutants exhibited increased SY1 Class 3 aggregates (ClueGO, cut-off: P < 0.05). (C) The network of the most enriched modules obtained above in 2B. (D-E) The increased SY1 Class 3 aggregates in the SPT mutant (lcb1-4, lcb2-1, tsc3-2) were rescued by backer-introducing the corresponding wild-type gene (a MoBY plasmid carries a WT gene, and p5472 is the empty vector control). Representative images from three independent experiments are shown in D, and quantifications were shown in E. Scale bar = 2 μm. Data were analyzed using the Student’s t-test. Error bars indicate the SD.
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Sphingolipids regulate SY1 IB maturation and cytotoxicity in yeast cells.
(A) A schematic diagram of the sphingolipid pathway in yeast cells. Myriocin blocks sphingolipid synthesis via inhibition of the serine C-palmitoyltransferase (SPT) complex. (B-C) Dihydrosphingosine (DHS) supplementation rescued the deficiency of SY1 IBs maturation in SPT mutants (lcb1-4, lcb2-1, tsc3-2). Representative images from three independent experiments are shown in B, and quantifications are shown in C. Scale bar = 2 μm. Data were analyzed using the Student’s t-test. Error bars indicate the SD. (D-E) Blocking intracellular sphingolipid synthesis with myriocin resulted in defective maturation of SY1 IBs, and exogenous addition of DHS could reverse the effect of myriocin. Representative images from three independent experiments are shown in D, and quantifications are shown in E. Scale bar = 2 μm. Data were analyzed using the Student’s t-test. Error bars indicate the SD. (F) DHS ameliorated the cytotoxicity of SY1 in SPT mutants (lcb1-4, lcb2-1, tsc3-2).
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SY1 IBs associate with mitochondria in yeast cells.
(A) SY1 IBs had no obvious association with the plasma membrane (Pma1-GFP), vacuoles (Vph1-GFP), or ER (Sec63-GFP), but were strongly associated with mitochondria (Tom70-GFP) (See also Figure 4-figure supplement 1A). Scale bar = 2 μm. (B) MitoTracker probe confirmed that all the three classes of IBs were closely associated with mitochondria (arrow heads indicate SY1 aggregates surrounded by mitochondria). Scale bar = 2 μm. n.s., not statistically significant. Data were analyzed using the Student’s t-test. Error bars indicate the SD. (C) Super-resolution microscopy and 3D-SIM construction demonstrated that the SY1 IBs are packed and surrounded by mitochondria. Scale bar = 1 μm. (D) Co-immunoprecipitation assays showing that SY1 physically binds to several mitochondrial membrane proteins, including Tom70, Tom37 and Tim44. The yeast mitochondrial membrane proteins Tom70, Tom37, Sam35 and Tim44 were individually C-terminally tagged with GFP, and the strains were then transformed with a control plasmid (Vector) or a SY1-overproducing plasmid (SY1), respectively. Cells were grown for 24 h and harvested for subsequent IP experiments. (E-F) The association of SY1 IBs with mitochondria decreases when the physical interaction between SY1 and mitochondrial membrane proteins is disrupted (tom70Δ, tom37Δ, tim44). Representative images from three independent experiments are shown in E and quantifications are shown in F. Scale bar = 2 μm. Data were analyzed using the Student’s t-test. Error bars indicate the SD.
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Sphingolipids regulate SY1-induced mitochondrial toxicity in yeast cells.
(A) Overexpression of SY1 decreased mitochondrial membrane potential (indicated by Rho 123 fluor intensity), increased cellular ROS levels (indicated by DCF mean intensity) and reduced cell viability (indicated by ATP level) in yeast cells. Data were analyzed using the Student’s t-test. Error bars indicate the SD. (B) Effects of myriocin treatment on SY1 cytotoxicity in yeast cells as judged by mitochondrial membrane potential / ROS levels / cell viability). Data were analyzed using the Student’s t-test. Error bars indicate the SD. (C) Effects of exogenous addition of DHS on SY1 cytotoxicity in yeast cells based on mitochondrial membrane potential / ROS levels / cell viability. Data were analyzed using the Student’s t-test. Error bars indicate the SD.
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SPTLC2 knockout alters the morphology of SY1 IBs and increases the cytotoxicity in mammalian cells.
(A) The 3D reconstruction showed that SY1 IBs were associated with mitochondria in mammalian HEK293t cells. GFP-SY1 was expressed in HEK293t cells and mitochondria were probed with MitoTracker. The 3D structure was constructed using Imaris software. Scale bar = 10 μm. (B) SY1 expression decreased mitochondrial function in HEK293t cells. Mitochondrial membrane potential was monitored by JC-1 staining, ROS levels were determined by DCF mean intensity values, mitochondrial superoxide was analyzed by MitoSOX mean intensity values. Data were analyzed using the Student’s t-test. Error bars indicate the SD. (C) The 3D structures of spherical and irregular SY1 IBs, as well as their interaction with mitochondria was visualized using Imaris software (See also Figure 6-figure supplement 2). (D) SPTLC2 knockout HEK293t cells had impaired management of SY1 IBs, with increased dispersed irregular IB structures. Scale bar = 20 μm. Data were analyzed using the Student’s t-test. Error bars indicate the SD. (E) SPTLC2 knockout increased SY1 cytotoxicity. Cell viability (indicated by luminescence) / ROS (indicated by DCF mean intensity) / mitochondrial membrane potential (indicated by JC-1 staining) assays were analyzed. Data were analyzed using the Student’s t-test. Error bars indicate the SD.
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Sphingolipids regulate SY1-induced cytotoxicity and SY1 IB morphology in mammalian cells.
(A) Inhibition of the intracellular sphingolipid synthesis pathway by myriocin enhanced SY1 cytotoxicity based on ATP content, intracellular ROS levels and mitochondrial membrane potential assays. Data were analyzed using the Student’s t-test. Error bars indicate the SD. (B) Addition of exogenous DHS rescued the cytotoxicity caused by SY1 expression. Data were analyzed using the Student’s t-test. Error bars indicate the SD. (C) Exogenous addition of DHS to SPTLC2 knockout cells restored the typical spherical SY1 IBs in HEK293t. Statistics of the ratio of cells with dispersed irregular IBs after exogenous addition of DMSO and DHS to HEK293t SPTLC2 knockout cells (See also Figure 7-figure supplement 2). Scale bar = 20 μm. Data were analyzed using the Student’s t-test. Error bars indicate the SD. (D) DHS ameliorated SY1 cytotoxicity by normalizing cell viability, intracellular ROS levels, and mitochondrial membrane potential. Data were analyzed using the Student’s t-test. Error bars indicate the SD.
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