Journal: Nature Communications

Article Title: Infection-induced peripheral mitochondria fission drives ER encapsulations and inter-mitochondria contacts that rescue bioenergetics

doi: 10.1038/s41467-024-51680-4

Figure Lengend Snippet: A Representative images of mitochondria labeled with Mito-BFP in uninfected (mock) and HCMV-infected cells at 72 h post infection (hpi). Center ROIs show timelapses of midzone (bottom) and peripheral (top) mitochondria fission. Red arrows indicate the fission site. Scale bar is 10 µm. B Number of peripheral or midzone fission events per cell; cells per replicate ≥10, n ≥ 5 biological replicates, total of 303 cells and 581 fission events analyzed. Exact P values - Midzone: mock vs. 72 hpi = 0.041; Mock vs. 96 hpi = 0.003; Peripheral: Mock vs. 72 hpi = 0.039; mock vs. 96 hpi = 0.015; Mock: Peripheral vs. Midzone = 0.006; 24 hpi: Peripheral vs. Midzone = 0.019; 48 hpi: Peripheral vs. Midzone = 0.049; 96 hpi: Peripheral vs. Midzone = 0.006. C Kymograph of peripheral mitochondria fission with each slice (top to bottom) representing a frame. Images from select frames shown in Supplementary Fig. . D Quantification of contact frequency for three-way contacts between ER, mitochondria, and lysosomes. Data points indicate total contact events divided by total fissions for peripheral and midzone mitochondria fission per replicate; cells per replicate ≥10, n ≥ 3 biological replicates, total of 251 fission events across 214 cells. Exact P values; 48 hpi: Midzone vs. Peripheral = 0.058; 72 hpi: Midzone vs. Peripheral = 0.017; Peripheral: Mock vs. 48 hpi = 0.025; Peripheral: Mock vs. 72 hpi = 0.025. E Representative timelapse of peripheral fission at 72 hpi with mitochondria labeled with Mito-BFP (red), ER with GFP-Sec61β (cyan), and lysosomes with mCh-Rab7 (yellow). Top white arrows point to the fission site, middle white arrows point to mitochondria–lysosome contact, and the bottom white arrows point to ER–mitochondria contact (4 × 4 µm ROIs). F Quantification of total fusion events observed per cell. Ten cells per replicate, n = 3 biological replicates, 350 fusion events across 120 cells. Exact P values; Mock vs. 24 hpi = 0.067; Mock vs. 48 hpi = 7.34E-08; Mock vs. 72 hpi = 2.35E-13. G Comparative quantification of all OPA1 peptides (OPA1), peptides found in S-OPA1 (S-OPA1) or peptides that are only found in L-OPA1 (L-OPA1). Quantification by DIA-MS, n = 3. H Quantification of mitochondria fusion as well as midzone and peripheral fission following control (OMP25) or UL37x1 expression. n = 30 cells, 239 fission events and 148 fusion events analyzed. Exact P values; Peripheral: pUL37x1 vs. OMP25 = 0.370; Midzone: pUL37x1 vs. OMP25 = 8.878E-08; Fusion: pUL37x1 vs. OMP25 = 4.429E-13; Total fission events: pUL37x1 vs. OMP25 = 3.34E-05. I Quantification of OPA1 abundance following either control or expression of full-length pUL37x1, or a mutant lacking the transmembrane domain (UL37x1 dTM). Quantification by DIA-MS, n = 2 replicates. hpi hours post infection, mito mitochondria, ER endoplasmic reticulum, mCh mCherry, s seconds, M mock. Data are presented as mean values with 95% confidence intervals. Black asterisks indicate statistical comparison to uninfected cells, while green asterisks indicate comparison between conditions at the same timepoint. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 using a Welch’s t test. Source data are provided as a Source Data file.

Article Snippet: Cells were infected with pUL37x1-GFP AD169 HCMV as described above and plunge-frozen at 72 hpi in ethane/propane mixture cooled to liquid nitrogen temperature, using an automated plunger Leica EM GP2.

Techniques: Labeling, Infection, Control, Expressing, Mutagenesis, Comparison

Journal: Nature Communications

Article Title: Infection-induced peripheral mitochondria fission drives ER encapsulations and inter-mitochondria contacts that rescue bioenergetics

doi: 10.1038/s41467-024-51680-4

Figure Lengend Snippet: A Quantification of peripheral or midzone fission events leading to MENC formation. Each data point represents a biological replicate; Mock = 67, 24 hpi = 67, 48 hpi = 73, 72 hpi = 71 total cells analyzed for each timepoint; Midzone = 3, peripheral = 4 biological replicates. B Cartoon representation of a MENC. Cutout shows PTPIP51 and VAP-B localization to the mitochondria and ER interfaces of MENCs, respectively. C Left: Slice through a tomogram and right: 3D segmentation of an HCMV-infected cell at 72 hpi showing Mito-ER encapsulation (whole volume shown in Supplementary Movie ). The mitochondrial and ER membranes are outlined in blue and brown, respectively. Scale bar is 200 nm. Representative of one biological replicate with 23 cells across two grids being selected for FIB-milling. Out of these, 6 lamella sites were successfully polished and used for cryo-ET data acquisition. In total 34 tilt series were acquired of which 27 contained mitochondria and ER in close proximity. D Live-cell microscopy of pUL37x1-induced MENC formation. White arrows point to MENCs. (ROI = 10 × 10 μm). E Representative images of MFN2 KO-induced MENC formation. Timelapse shown top to bottom. (ROI = 3 × 3 μm). F Quantification of MFN2 KO-induced MENC formation. Ten mitochondria per cell, 10 cells per condition, n = 3 biological replicates; total of 600 mitochondria. G PTPIP51 interaction with pUL37x1 as quantified by targeted MS (PRM). H Left: PTPIP51-ATG interaction abundance as quantified by DDA-MS. (2 biological replicates, except for 24 hpi). Right: Cartoon representation indicating the function of PTPIP51 interactors the ATG5/12/16L complex in autophagosome maturation. I Quantification of LC3 intensity in MENC and non-MENC mitochondria at 72 hpi. Shown as LC3 intensity per cell. 10 MENC and non-MENC mitochondria quantified per cell; 10 cells per replicate; n ≥ 3 biological replicates, 600 mitochondria across 30 cells. J Representative microscopy showing colocalization of acidified mitochondria (as shown through mtKeima ratiometric imaging) with the ER as well as the lack of acidification in MENC mitochondria. Scale bar = 10 µm. hpi hours post infection, mito mitochondria, ER endoplasmic reticulum, mCh mCherry, s seconds, M mock, MENC mitochondria–ER encapsulation, KO knockout. Data are presented as mean values with 95% confidence intervals. Black asterisks indicate statistical comparison to uninfected cells, while green asterisks indicate comparison between conditions at the same timepoint. * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001 using a Welch’s t test. Source data are provided as a Source Data file.

Article Snippet: Cells were infected with pUL37x1-GFP AD169 HCMV as described above and plunge-frozen at 72 hpi in ethane/propane mixture cooled to liquid nitrogen temperature, using an automated plunger Leica EM GP2.

Techniques: Infection, Encapsulation, Tomography, Microscopy, Imaging, Knock-Out, Comparison

Journal: Nature Communications

Article Title: Infection-induced peripheral mitochondria fission drives ER encapsulations and inter-mitochondria contacts that rescue bioenergetics

doi: 10.1038/s41467-024-51680-4

Figure Lengend Snippet: Reagents used in this study

Article Snippet: Cells were infected with pUL37x1-GFP AD169 HCMV as described above and plunge-frozen at 72 hpi in ethane/propane mixture cooled to liquid nitrogen temperature, using an automated plunger Leica EM GP2.

Techniques: Concentration Assay, Plasmid Preparation, Clone Assay, Control