Journal: The Journal of Cell Biology

Article Title: Mitoguardin-2–mediated lipid transfer preserves mitochondrial morphology and lipid droplet formation

doi: 10.1083/jcb.202207022

Figure Lengend Snippet: MIGA2 binds and transfers lipids between membranes. (A) Left: Native mass spectra of hMIGA2 long -6xhis purified from bacteria, showing exclusive presence of the dimeric protein. Each charge state is further accompanied by a series of up to four lipid bound peaks. Right: Expansion of the 20 + charge state of native MS for hMIGA2 long -6xhis dimer, showing up to four glycerophospholipids bound, with the average mass of 752 ± 5 Da. (B) Untargeted lipidomics of 3xFLAG-hMIGA2 long , purified from mammalian (Expi293) cells. At left: Distribution of lipid classes bound to hMIGA2 long . At right: Distribution of glycerophospholipids bound to hMIGA2 long as compared to their abundance in cells . (C) In the end-point transfer assay, hMIGA2 constructs are tethered between “heavy” acceptor liposomes (containing 0.75 M sucrose; lipid composition: 65% DOPC, 30% PE, and 5% PI[4,5]P 2 ) and light donor liposomes containing a single species of fluorescent lipid (containing no sucrose; lipid composition: 63% DOPC, 30% PE, 2% NBD- or Rh-lipid, 5% DGS-NTA). After lipid transfer (after 30 min), proteinase K was added to digest proteins, the light donor and heavy acceptor liposomes were separated by centrifugation, and the fluorescence increase of the heavy acceptor liposomes monitored. Transferred lipid was quantitated based on liposome standards incorporating 2, 1, 0.5, 0.25, or 0% of the appropriate fluorescent lipid. Both hMIGA2 long and hMIGA2 C transport NBD-PE acyl , -PS acyl , -PA acyl , and NBD-PC acyl ; NBD-PS head is transferred to a lesser extent, and Rh-PE head is not transferred, indicating that modification of the lipid headgroup can interfere with transport by MIGA2. In a similar experiment, we monitored transfer of unmodified PS using a PS-specific protein probe (C2-domain of lactadherin ) to assess PS transfer. Donor liposomes initially contained 5% PS. Donor and acceptor liposomes were separated by addition of EDTA and imidazole rather than protease, so as not to destroy the PS-probe. Supernatant (containing donor liposomes) and pellet (acceptor liposomes) fractions, in the presence and absence of MIGA2, were analyzed by SDS-PAGE to quantitate the PS-probe associated with each fraction. Transfer efficiency of acyl chain modified NBD-PS, as monitored by fluorescence, is comparable to that of natural PS, indicating that the acyl chain modification does not interfere with transfer by MIGA2. (D) In the FRET-based transfer assay, donor and acceptor liposomes (compositions indicated) were tethered together in the presence or absence MIGA2 linked to the donor liposomes. The donor liposomes initially contain Rh-PE head and NBD-lipids (-PS acyl , -PC acyl , -PA acyl , -PE acyl ), where FRET between the Rh and NBD initially reduces NBD fluorescence. As lipids are transferred from donor to acceptor liposomes, the Rh- and NBD-labeled lipids are diluted, resulting in reduced FRET and an increase in NBD fluorescence. hMIGA2 long can transport NBD-PS acyl , NBD-PA acyl , NBD-PE acyl , and NBD-PC acyl . A donor only control shows that the fluorescence increase was due to transfer of lipids between liposomes rather than solely lipid extraction by hMIGA2. After the transfer reaction was completed, dithionite was added to rule out the possibility of fusion between donor and acceptor liposomes, which would also result in a fluorescence increase . Each experiment was performed in triplicate. SDs are shown. NBD-PS head transfer is less efficient . (E) A similar FRET experiment was carried out to assess transfer from artificial LDs (rather than donor liposomes) to acceptor liposomes and shows that MIGA2 transfers NBD-PE acyl and NBD-PC acyl between the artificial LDs and liposomes. Positive stain transmission electron microscopy was used to assess the quality of the LD preparation as in .

Article Snippet: C. elegans MIGA (Uniprot Q21096 ) fragments were amplified from C. elegans cDNA library ( )and subcloned into pET28-6xhis-sumo plasmid (V012798; NoVoPro) or pET29 (69871; Addgene) or pCMV10 plasmid (E7658; Sigma-Aldrich) with C-terminal 6xhis tag or N-terminal 3xFLAG tag.

Techniques: Purification, Bacteria, Construct, Liposomes, Centrifugation, Fluorescence, Modification, SDS Page, Labeling, Control, Extraction, Staining, Transmission Assay, Electron Microscopy

Journal: The Journal of Cell Biology

Article Title: Mitoguardin-2–mediated lipid transfer preserves mitochondrial morphology and lipid droplet formation

doi: 10.1083/jcb.202207022

Figure Lengend Snippet: Lipid binding and transfer by MIGA2. (A) 3xFLAG-hMIGA2 long was incubated with NBD-labeled lipids and examined by native PAGE. Phospholipids (NBD-PA acyl , NBD-PC acyl NBD-PS head , NBD-PE acyl ) visualized by their fluorescence, comigrated with protein, visualized by Coomassie blue staining. CH, cholesterol; SM, sphingomyelin. Because migration rates on native gels depend on the mass/charge ratio of the sample, and because MIGA2 dimers can bind multiple lipids at once, MIGA2 incubated with charged lipids migrates as multiple species and is smeared. (B) hMIGA2 long -6xhis was incubated with a 1:1 molar ratio of NBD-PC acyl and unlabeled lipid and examined by native PAGE. (C) Dithionite controls for the FRET transfer assay: After the transfer reaction was completed, dithionite was added to rule out the possibility of fusion between donor and acceptor liposomes, which would also result in a fluorescence increase. The fluorescence reduction after dithionite addition is the same for reactions containing hMIGA2 as those without, indicating that fusion has not occurred. Each experiment was performed in triplicate. SDs are shown. (D) FRET-based transfer experiment for NBD-PS head and the dithionite control. (E) Positive staining TEM micrographs of artificial LDs and liposomes: artificial LDs have a monolayer of phospholipids and TAG core, which are dark under positive staining; liposomes have bilayer membrane of phospholipids and buffer in the core, so that only the membrane is stained dark. Source data are available for this figure: .

Article Snippet: C. elegans MIGA (Uniprot Q21096 ) fragments were amplified from C. elegans cDNA library ( )and subcloned into pET28-6xhis-sumo plasmid (V012798; NoVoPro) or pET29 (69871; Addgene) or pCMV10 plasmid (E7658; Sigma-Aldrich) with C-terminal 6xhis tag or N-terminal 3xFLAG tag.

Techniques: Binding Assay, Incubation, Labeling, Clear Native PAGE, Fluorescence, Staining, Migration, Liposomes, Control, Membrane