Journal: Traffic (Copenhagen, Denmark)

Article Title: Vps501, a novel vacuolar SNX‐BAR protein cooperates with the SEA complex to regulate TORC1 signaling

doi: 10.1111/tra.12833

Figure Lengend Snippet: YKR078W/Vps501 is a paralog of Vps5 and resides on the vacuolar membrane. (A) Phylogenetic analysis of Vps5‐like SNX‐BAR proteins in selected animals, choanoflagellate and fungi, including other species in the family Saccharomycetaceae indicates the presence of a Vps5‐like protein (YKR078W) in Saccharomyces cerevisiae , referred to here as Vps501. (B) Micrographs of Vps10‐2XGFP in wildtype and indicated mutant cells. Vps501 does not have a role in Vps10 trafficking, despite the phylogenetic similarities with retromer SNX‐BARs. (C) Vps501 localizes to the vacuolar membrane as a N‐terminal GFP fusion protein. GFP‐Vps501 expression is shown as locus integrations using native promoter (top), GPD promoter (bottom) or ectopically expressed as a 2‐micron plasmid (middle). C‐terminal fusions were found to be non‐functional, not shown. Vacuolar membranes are shown using FM4‐64 dye. The scale bar indicates 5 μm. See also Figure . BAR, Bin‐Amphiphysin‐Rvs; GFP, green fluorescent protein; GPD, glyceraldehyde‐3‐phosphate dehydrogenase; SNX, sorting nexin

Article Snippet: His‐tag fusion vector of Vps501 was made by synthesizing the Vps501 ORF as a NdeI and HindIII fragment (Genscript).

Techniques: Membrane, Mutagenesis, Expressing, Plasmid Preparation, Functional Assay

Journal: Traffic (Copenhagen, Denmark)

Article Title: Vps501, a novel vacuolar SNX‐BAR protein cooperates with the SEA complex to regulate TORC1 signaling

doi: 10.1111/tra.12833

Figure Lengend Snippet: Vps501 interacts with subunits of TORC1 and the SEA complex. (A) Mass spectrometry experimental design to identify Vps501 interactors. GFP‐Vps501 and interactors were purified by GFP‐Trap affinity purification and SDS‐PAGE, followed by in‐gel trypsin digestion. Resulting peptides were analyzed and identified by LC–MS/MS according to their relative enrichment. (B) Mass spectrometry analysis using STRING software identified strong interactions with Kog1, a subunit of the TORC1 complex and Sea1 and Seh1, subunits of the SEA complex. Subunits of the SEA complex and TORC1 are represented as nodes in the graphical network. Proteins colored in green (Sea1, Seh1 and Kog1) are those detected by this proteomic study. Lines connecting the nodes represent previously reported interactions. (C) Micrographs show GFP‐Vps501 colocalizes with Sea1‐mCherry, Kog1‐mCherry, Seh1‐mCherry at the vacuolar membrane. The scale bar indicates 5 μm. TOR, target of rapamycin

Article Snippet: His‐tag fusion vector of Vps501 was made by synthesizing the Vps501 ORF as a NdeI and HindIII fragment (Genscript).

Techniques: Mass Spectrometry, Purification, Affinity Purification, SDS Page, Liquid Chromatography with Mass Spectroscopy, Software, Membrane

Journal: Traffic (Copenhagen, Denmark)

Article Title: Vps501, a novel vacuolar SNX‐BAR protein cooperates with the SEA complex to regulate TORC1 signaling

doi: 10.1111/tra.12833

Figure Lengend Snippet: vps501 Δ sea1 Δ cells display a synthetic autophagy defect. (A) Maximum projection micrographs of cells expressing GFP‐Atg8 in wildtype and indicated mutant cells before and after nitrogen starvation. GFP‐Atg8 is found in the vacuole lumen (VL) in wildtype cells after nitrogen starvation, indicating successful autophagic flux has occurred. In atg1 Δ cells, autophagy induction is inhibited and GFP‐Atg8 is absent in the VL. A similar phenotype is also seen only when cells are ablated for both Vps501 and Sea1, indicating a synthetic genetic interaction between Vps501 and Sea1 is critical for autophagy. Cells ablated for Npr2 and Npr3 also show major autophagy defects as single knockouts and likely mask any synthetic phenotypes combined with Vps501 ablation. The scale bar indicates 5 μm. (B) Quantitative immunoblotting was used to detect the amount of GFP‐Atg8 flux before and after autophagy induction. There is a 3‐fold decrease in GFP‐Atg8 flux in vps501 Δ sea1 Δ cells, compared to single deletions, whereas there was no significant difference detected in Npr2 or Npr3 knockout cells. A representative immunoblot is shown. Anti‐Pgk1 was used as a loading control. (C) Graph of quantification of GFP‐Atg8 processing. The results are from three experiments and averaged using the standard error of the mean. Indicated significance is a comparison of wildtype to single deletions or double mutants. * p < 0.05, ** p < 0.01, *** p < 0.001 indicates significance as calculated by Student's t ‐test. See also Figure

Article Snippet: His‐tag fusion vector of Vps501 was made by synthesizing the Vps501 ORF as a NdeI and HindIII fragment (Genscript).

Techniques: Expressing, Mutagenesis, Western Blot, Knock-Out, Control, Comparison

Journal: Traffic (Copenhagen, Denmark)

Article Title: Vps501, a novel vacuolar SNX‐BAR protein cooperates with the SEA complex to regulate TORC1 signaling

doi: 10.1111/tra.12833

Figure Lengend Snippet: SEACIT subunits, Npr2 and Npr3 require Vps501 and Sea1 for vacuolar localization. (A) Npr2‐GFP and Npr3‐GFP localize to the vacuolar membrane in wildtype cells. In vps501 Δ or sea1 Δ cells, Npr2‐GFP and Npr3‐GFP are mislocalized to the vacuole lumen (VL) in 10%–40% of cells. In vps501 Δ sea1 Δ cells, Npr2‐GFP and Npr3‐GFP are mislocalized to the VL in 85% of cells. (B) Graph represents VL localization in wildtype cells or mutants, defined by visually scoring the presence of GFP in the vacuole lumen. The results are from three experiments and averaged using the standard error of the mean. Indicated significance is a comparison of wildtype to single deletions or double mutants. * p < 0.05, ** p < 0.01, *** p < 0.001 indicates significance as calculated by Student's t ‐test

Article Snippet: His‐tag fusion vector of Vps501 was made by synthesizing the Vps501 ORF as a NdeI and HindIII fragment (Genscript).

Techniques: Membrane, Comparison

Journal: Traffic (Copenhagen, Denmark)

Article Title: Vps501, a novel vacuolar SNX‐BAR protein cooperates with the SEA complex to regulate TORC1 signaling

doi: 10.1111/tra.12833

Figure Lengend Snippet: TORC1 and autophagy induction is defective in vps501 Δ sea1 Δ cells. (A) Micrographs of Kog1‐2XGFP localization in wildtype and indicated mutant cells during vegetative growth. Kog1 a subunit of TORC1, has previously been reported to localize to the vacuolar membrane and to dot‐like structures juxtaposed the vacuolar membrane. In vps501 Δ sea1 Δ cells, similar dot‐like structures accumulate suggesting the non‐vacuolar membrane pool of Kog1 are enriched, while the vacuole membrane TORC1 pool appears reduced. Vacuolar membranes are shown using FM4‐64 dye. (B) The number of Kog1 dot‐like structures were quantified in wildtype and indicated mutant cells before and after nitrogen starvation as described in the text. Regardless of starvation conditions, there was a 2‐fold increase in dot‐like structures in vps501 Δ sea1 Δ cells as compared to single mutants or wildtype cells (left graph). Three or more dot‐like structures were found in 80% of vps501 Δ sea1 Δ cells, while single mutants or wildtype cells typically had 0–2 dots (right graph). * p < 0.05, ** p < 0.01, *** p < 0.001 indicates significance as calculated by a one‐way ANOVA from three biological replicates. (C) Quantitative immunoblot analysis of Atg13 in wildtype and indicated mutant cells before and after nitrogen starvation or rapamycin treatment. In wildtype, vps501 Δ or sea1 Δ cells, Atg13 is phosphorylated as indicated by Atg13 band smear during vegetative growth, indicating TORC1 is active. In autophagy induction conditions, TORC1 is inactivated and Atg13 is not phosphorylated indicated by the loss of Atg13 band smear. In vps501 Δ sea1 Δ cells, Atg13 remains phosphorylated indicating autophagy induction is defective in these mutants, regardless of nitrogen starvation or rapamycin (see text for details). (D) Percentage of Atg13 phosphorylation was quantified by determining the proportion of Atg13 to total protein using densitometry. A representative immunoblot is shown. Anti‐Pgk1 was used as a loading control. Indicated significance is a comparison of wildtype to single deletions or double mutants. * p < 0.05, ** p < 0.01, *** p < 0.001 indicates significance as calculated by Student's t ‐test from three biological replicates. TOR, target of rapamycin

Article Snippet: His‐tag fusion vector of Vps501 was made by synthesizing the Vps501 ORF as a NdeI and HindIII fragment (Genscript).

Techniques: Mutagenesis, Membrane, Western Blot, Phospho-proteomics, Control, Comparison

Journal: Traffic (Copenhagen, Denmark)

Article Title: Vps501, a novel vacuolar SNX‐BAR protein cooperates with the SEA complex to regulate TORC1 signaling

doi: 10.1111/tra.12833

Figure Lengend Snippet: Vps501 requires Sea1 for vacuolar localization. (A) Micrographs of GFP‐Vps501 in wildtype and SEA complex mutant cells. GFP‐Vps501 is significantly mislocalized in SEACIT mutant cells but not in SEACAT mutant cells. sec13 Δ cells are nonviable. (B) Vacuolar membrane localization was quantified by comparing GFP‐Vps501 VML/cytosol ratios in wildtype versus SEA complex mutants. A minimum of 100 cells were measured in triplicate; standard error of the mean was calculated. * p < 0.05, ** p < 0.01, *** p < 0.001 indicates significance as calculated by Student's t ‐test

Article Snippet: His‐tag fusion vector of Vps501 was made by synthesizing the Vps501 ORF as a NdeI and HindIII fragment (Genscript).

Techniques: Mutagenesis, Membrane

Journal: Traffic (Copenhagen, Denmark)

Article Title: Vps501, a novel vacuolar SNX‐BAR protein cooperates with the SEA complex to regulate TORC1 signaling

doi: 10.1111/tra.12833

Figure Lengend Snippet: Vps501 non‐canonical Phox (PX) domain is required for localization and function at the vacuolar membrane. (A) Sequence alignments of human SNX1, SNX2 and yeast homologs VPS5 and VPS501 PX domains are shown. Blue indicates residues in the canonical PI3P motif. Magenta indicates key secondary lipid binding site residues. Vps501 canonical PI3P motif is not conserved, however, the non‐canonical His/Tyr motif is conserved. (B) Alanine mutations to His/Tyr residues in the non‐canonical motif resulted in GFP‐Vps501 YKAA mislocalized to the cytosol in vps501∆ , and vps501∆sea1∆ cells, indicating Vps501's recruitment to the vacuolar membrane is dependent on both PI3P recognition and Sea1 binding. The scale bar indicates 5 μm. (C) Ectopic expression of Vps501 YKAA failed to complement vps501∆sea1∆ cells in GFP‐Atg8 processing assays and severely impairs autophagy flux when expressed in sea1∆ cells. (D) Top Graph, quantification of Vps501 localization was defined by vacuolar membrane to cytosol ratios as described in material and methods. GFP‐Vps501 YKAA recruitment to the vacuolar membrane is reduced by ~60% in vps501Δ cells. When combined with ablation of Sea1 vacuolar membrane localization is reduced by ~90%. Bottom Graph, quantification of GFP‐Atg8 processing when Vps501 YKAA is ectopically expressed in wildtype, vps501∆ , or vps501∆sea1∆ cells. The results are from three experiments and averaged (standard deviation). (E and F) PI liposome sedimentation assays and quantification. Query proteins and lipids are indicated on the figure. Vps501 WT bound to liposomes enriched with PI3P or PI(3,5)P. Binding was reduced when PI3P and PI(3,5)P lipids were combined on liposomes. (G and H) PS liposome sedimentation assays and quantification. Yeast SNX‐BARs heterodimer Snx4‐Atg20 has previously been shown to preferentially bind PS‐containing membranes. Vps501 WT also strongly binds to liposomes containing 30% PS, similar to Snx4‐Atg20, whereas Vps501 YKAA PX mutant binding was reduced 2‐fold. Indicated significance is a comparison of wildtype to single deletions or double mutants. * p < 0.05, ** p < 0.01, *** p < 0.001 indicates significance as calculated by Student's t ‐test. SNX, sorting nexin

Article Snippet: His‐tag fusion vector of Vps501 was made by synthesizing the Vps501 ORF as a NdeI and HindIII fragment (Genscript).

Techniques: Membrane, Sequencing, Binding Assay, Expressing, Standard Deviation, Sedimentation, Liposomes, Mutagenesis, Comparison

Journal: Traffic (Copenhagen, Denmark)

Article Title: Vps501, a novel vacuolar SNX‐BAR protein cooperates with the SEA complex to regulate TORC1 signaling

doi: 10.1111/tra.12833

Figure Lengend Snippet: Vps501 cooperates with the SEA complex to inactivate TORC1. Under starvation conditions, Vps501 acts as a stabilizer for the SEA complex to the vacuolar membrane using a non‐canonical PX domain and a direct interaction within the SEA Complex. This interaction promotes SEACIT inactivation of the vacuole and endosomal pools TORC1 via the GTR1/GTR2/EGO complex, resulting in the induction macro and microautophagy. In vps501 Δ sea1 Δ cells, the SEA complex is destabilized and the vacuole pool of TORC1 is mislocalized. TORC1 endosomal pools are enriched and hyperactive, resulting in aberrant Atg13 phosphorylation and defective macro and microautophagy which can lead to the overall destabilization of other vacuolar membrane proteins. TOR, target of rapamycin [Correction added on 22 February 2022, after first online publication: Figure 8 has been corrected.]

Article Snippet: His‐tag fusion vector of Vps501 was made by synthesizing the Vps501 ORF as a NdeI and HindIII fragment (Genscript).

Techniques: Membrane, Phospho-proteomics

Journal: Exploration of Targeted Anti-tumor Therapy

Article Title: Chimeric single-chain variable fragment-human immunoglobulin G crystallizable fragment antibody against GD2 for neuroblastoma targeted immunotherapy

doi: 10.37349/etat.2023.00188

Figure Lengend Snippet: Graphical map of the constructed plasmid vector and PCR analysis of GD2 scFv gene. (A) Graphical map of pFUSE-hIgG1-Fc2 vector created by SnapGene software is shown. Nucleotide sequence of mouse scFv against GD2 was cloned into the vector at EcoRI and NcoI sites before the hinge region of the hIgG1 Fc part. The thin orange arrow indicates an open reading frame, which starts at the codon inside IL2ss and stops at the end of the Fc portion. Zeocin drug resistance gene ( Zeo ) was used for selection; (B) after cloning step, the constructed vector was transformed into DH5-α E. coli and selected by zeocin drug. Five bacterial colonies (as indicated) were picked up and grown. PCR was performed to detect the inserted GD2 scFv gene inside the constructed plasmid vectors. Bacteria clone number 1 was chosen for plasmid extraction. Standard DNA markers (M) in base pairs are shown on the left. Positive and negative controls are also demonstrated. Neg: negative; Pos: positive

Article Snippet: Nucleotide sequence of mouse scFv against GD2 clone 14G2a (a kind gift from Prof. Dr. Suradej Hongeng, Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand) was designed to be inserted into mammalian expression plasmid fusion-hIgG1-Fc2 tag vector (pFUSE-hIgG1-Fc2) using SnapGene software (version 1.1.3, GSL Biotech LLC, San Diego, CA, USA).

Techniques: Construct, Plasmid Preparation, Software, Sequencing, Clone Assay, Selection, Transformation Assay, Bacteria, Extraction

Journal: Exploration of Targeted Anti-tumor Therapy

Article Title: Chimeric single-chain variable fragment-human immunoglobulin G crystallizable fragment antibody against GD2 for neuroblastoma targeted immunotherapy

doi: 10.37349/etat.2023.00188

Figure Lengend Snippet: Determination of scFv-IgG Fc expression in transfected HEK293T cells. (A) Human HEK293T cell line was transfected with pFUSE-scFv-GD2-hIgG1-Fc2 vector and intracellular stained with Alexa Fluor 488-conjugated goat anti-hIgG antibody (GαhIgG-A488), Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (GαmIgG-A488), or without antibodies (no Abs); (B) transfected HEK293T cells were cultured in the presence of zeocin. The transfected cells and un-transfected cells were intracellularly stained with Alexa Fluor 488-conjugated goat anti-hIgG antibody (Blue color) or Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (red color). Overlay histograms are shown

Article Snippet: Nucleotide sequence of mouse scFv against GD2 clone 14G2a (a kind gift from Prof. Dr. Suradej Hongeng, Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand) was designed to be inserted into mammalian expression plasmid fusion-hIgG1-Fc2 tag vector (pFUSE-hIgG1-Fc2) using SnapGene software (version 1.1.3, GSL Biotech LLC, San Diego, CA, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Staining, Cell Culture