Fig. 3: Mitochondria–ER encapsulations (MENC) follow peripheral fission and promote bioenergetics.

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 S4). 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.