Journal: bioRxiv

Article Title: Selective MOSPD2-STARD3 interaction at ER contact sites governs late endosome/lysosome dynamics and cholesterol homeostasis

doi: 10.64898/2026.03.30.714413

Figure Lengend Snippet: A-B: Immunoprecipitation (anti-Flag) experiments between Flag-STARD3 and GFP-VAP-A, GFP-VAP-B, GFP-MOSPD2, GFP-MOSPD2 RD/LD, and GFP-VAP-A KD/MD (B) in HeLa cells. Approximatively 5 µg of total protein extract was analyzed by Western blot using anti-GFP, anti-STARD3, and anti-GAPDH antibodies. Immunoprecipitated proteins were analyzed using anti-GFP and anti-STARD3 antibodies. C: Immunoprecipitation between endogenous STARD3 and VAP-A, VAP-B and MOSPD2 in HCC1954 cells. Immunoprecipitation was performed using control IgG or anti-STARD3 antibodies in triplicate. Total protein extracts and immunoprecipitated proteins were analyzed by Western blot using anti-MOSPD2, anti-VAP-A, anti-VAP-B, anti-STARD3, and anti-GAPDH antibodies. *: aspecific. D: Principle of the native Holdup assay. Total protein extracts (a) are incubated with streptavidin resin saturated with a biotinylated MSP domain or control resin (b). After reaching equilibrium, unbound proteins are filtered out and quantified by Western blot. Binding intensity = 1 – (C Unbound / C total ). E: Coomassie blue staining of recombinant proteins used for native Holdup experiments: MBP alone or fused to the MSP domains of VAP-A, VAP-B or MOSPD2 (WT and RD/LD mutant), tagged with a 6 His for purification and biotinylated thanks to an AviTag. A total of 25 pmol of each protein was loaded. F: Native Holdup experiments quantifying the interaction between the recombinant MSP domains of VAP-A, VAP-B, MOSPD2, and MOSPD2 RD/LD and endogenous STARD3. Left: western blot analysis of the unbound prey protein (STARD3) in HCC1954 protein extracts after incubation with increasing amounts of the recombinant MSP domains. Right: Binding intensity between the MSP domains and the prey protein (STARD3). Binding curves were fitted using a Hill equation (mean ± SEM from 2 technical replicates), and apparent affinities ( K app ) and maximal binding intensities ( B max ) were calculated (± SD). G: FRAP experiment in HeLa cells co-expressing mCherry-STARD3 and either GFP-VAP-A, GFP-VAP-B, or GFP-MOSPD2. a: images showing STARD3-positive LE/Lys in close apposition to ER-localized GFP-MOSPD2 (top), GFP-VAP-A (middle), or GFP-VAP-B (bottom). Left: colocalization of mCherry-STARD3 (magenta) and GFP (green) pre-bleach. GFP signal (gray) displayed sequentially from left to right: pre-bleach, immediately post-bleach, 3 seconds post-bleach, and 20 s post-bleach. b: Quantification of relative GFP-signal intensity in the bleached ROI during the 2 seconds before bleaching, and the 21 seconds following bleaching in cells expressing GFP-VAP-A (blue curve), GFP-VAP-B (green curve), and GFP-MOSPD2 (red curve). The gray curve represents the GFP signal in the absence of bleaching. Mean values and standard deviations (black bars) are shown. The calculated half-times of recovery (mean t½ ± SD) are indicated.

Article Snippet: The pQCXIP MOSPD2 (WT; RD/LD; ΔCRAL-TRIO; ΔMSP), pQCXIP GFP-MOSPD2 (WT: Addgene #186467; RD/LD #186468), pQCXIP mScarlet-MOSPD2 (WT: Addgene #186472; RD/LD Addgene #186476; W201E: Addgene plasmid #186477; ΔMSP; ΔCRAL-TRIO mutants), GFP-VAP-A (WT: Addgene #104447; KD/MD Addgene #104449), GFP-VAP-B (Addgene plasmid #104448), EGFP-ER[TM(SAC1)] (Addgene plasmid #186475), pQCXIP mScarlet-ER [TM(SAC1)] (Addgene plasmid # 186572), Flag-STARD3, GFP-VAP-A (WT and KD/MD mutant), and GFP-VAP-B, expression vectors were previously described ( , ; ; ; ).

Techniques: Immunoprecipitation, Western Blot, Control, Incubation, Binding Assay, Staining, Recombinant, Mutagenesis, Purification, Expressing

Journal: Scientific Reports

Article Title: Extracellular calcium alters calcium-sensing receptor network integrating intracellular calcium-signaling and related key pathway

doi: 10.1038/s41598-021-00067-2

Figure Lengend Snippet: The schematic model of CaSR mediated signaling, biosynthesis and ER quality control of CaSR invoked through Ca 2+ ext perturbation. Identified proteins from the experiment are colored respectively with fold-enrichment (log 2 (Ca 2+ /EGTA)-positive controls). ( A ) (I) CaSR is activated with 4 mM Ca 2+ ex . (II) Ca 2+ ex /CaSR mediated G-protein signaling through phospho-lipase C(PLC) activation and subsequently, IP 3 invoked release of Ca 2+ from the ER. Ca 2+ dependent Gα i invoked inhibition of acetyl cyclase is induced. ( B ) Alteration in Ca 2+ ER induce amplification of CaSR interactomes. Biosynthesis and processing in the ER. (III) Co-translational translocation: The newly synthesized polypeptide bound to the SRP is directed to the ER membrane to the Sec complex by the SRP receptor, then processed by oligosaccharyl transferases (OST: DDOST and RPN1/2). GRP78 chaperones CaSR polypeptide to ER lumen where glucosidases GANAB allows for the removal of the outermost glucose residues. (IV) Calnexin cycle CANX and/or CRT along with GRP78 promotes folding and PDIA3 catalyzes disulfide formation. (V) ER-exit Properly folded CaSR traffics from ER to the Golgi assisted by VAPA and VAPB as well as p24A. Rab6 assists in retrograde trafficking from Golgi to the ER. 14-3-3 allows the CaSR to remain in the ER in the absence of Ca 2+ . UGGT acts as a check- point for improperly folded CaSR. [VI] Degradation pathway: Terminally misfolded CaSR undergoes ERAD and is ubiquitinated through STUB1. (VII) Endosomal degradation: CaSR from the plasma membrane can be endocytosed assisted by Rab5, Rab18 and CLTC. ( C ) Ca 2+ ER release via CaSR monitored using G-CatchER + (green line). Activated CaSR mediated Ca 2+ i mobilized from ER measured using Fura-red (red line).

Article Snippet: Empty pcDNA3.1, human CaSR with FLAG-tag (FLAG-hCaSR) (between Asp 371 and Thr 372 ) in pcDNA3.1 and EGFP-CaSR (provided by Dr. Chen Zhang, La Jolla Institute of Allergy and Immunology, CA) were used for transfection for negative and positive controls, respectively. pEGFPC1-hVAPA and pcDNA3.1 GRP78 were obtained from Addgene. mCherry-VAPA was constructed from pEGFPC1-hVAPA (Addgen) and mCherry in pcDNA3.1.

Techniques: Control, Activation Assay, Inhibition, Amplification, Translocation Assay, Synthesized, Membrane, Clinical Proteomics

Journal: Scientific Reports

Article Title: Extracellular calcium alters calcium-sensing receptor network integrating intracellular calcium-signaling and related key pathway

doi: 10.1038/s41598-021-00067-2

Figure Lengend Snippet: Interactions of CaSR with assistant proteins, VAPA and GRP78, and Ca 2+ ext modulated CaSR-VAPA interdependent cell membrane expression. Co-immunoprecipitation experiments with either Ca 2+ or EGTA treatments using anti-FLAG antibody on total cell extracts from HEK293 transfected with FLAG-CaSR and VAPA or GRP78 pcDNA3.1 followed by western blot on 50 µg total extract. Analysis with anti-CaSR antibody (( A,B ) insert; second and fifth panel), anti-VAPA antibody (( A ) insert, first and fourth panel), anti-GRP78 (( B ) insert, first and fourth panel) or anti-GAPDH (as control, ( A,B ), third panel) was carried out in triplicate (one representative blot is shown) and is represented in bar graph for output VAPA ( A ) and output GRP78 ( B ). Representative confocal imaging ( C ) showing colocalization of VAPA (red) (in physiological condition (first panel) and 4 mM Ca 2+ (second panel)), or GRP78 (red) (in physiological condition (third panel) and 4 mM Ca 2+ (fourth panel)) with CaSR (green) and ER marker (calreticulin, magenta) along the colocalized region (yellow). Scale bar, 2.5 µm. Comparison of pixel intensity correlation of CaSR and ER with VAPA ( D ) or GRP78 ( E ) in confocal images conducted at the regions between cell nuclei and cell membrane boundaries in single cell image sections of Cos7 cells obtained after treatments with physiological Ca 2+ (DMEM with 2.2 mM) vs 4 mM Ca 2+ . Statistical calculations (Mean ± SD) were determined from five independent cells from different slides obtained with separate transfections (scatter dot). Representative dual-color TIRF images of cells expressing both VAPA and CaSR (( F ), upper panel), expressing only VAPA (( F ), middle panel), and expressing only CaSR (( F ), lower panel). Scale bar, 10 µm. Relative fluorescence intensity changes of membrane expressed CaSR signal in cells with (black line) and without (red line) co-expressing VAPA, represented by a single cell ( G ) and an average representation with n > 5 ( H ). The density of VAPA spots near the cell membrane in cells with (black line) and without (red line) co-expressing CaSR, represented by a single cell ( I ) and an average representation with n > 7 ( J ).

Article Snippet: Empty pcDNA3.1, human CaSR with FLAG-tag (FLAG-hCaSR) (between Asp 371 and Thr 372 ) in pcDNA3.1 and EGFP-CaSR (provided by Dr. Chen Zhang, La Jolla Institute of Allergy and Immunology, CA) were used for transfection for negative and positive controls, respectively. pEGFPC1-hVAPA and pcDNA3.1 GRP78 were obtained from Addgene. mCherry-VAPA was constructed from pEGFPC1-hVAPA (Addgen) and mCherry in pcDNA3.1.

Techniques: Membrane, Expressing, Immunoprecipitation, Transfection, Western Blot, Control, Imaging, Marker, Comparison, Fluorescence