Journal: iScience
Article Title: The microcephaly-associated protein YIPF5 differentially regulates ER export
doi: 10.1016/j.isci.2026.114791
Figure Lengend Snippet: YIPF5 depletion induces the formation of dynamic SURF4-positive tubules and accelerates the secretion of the SURF4 cargo CAB45 MCF10A WT or YIPF5-KO cells transiently transfected with EGFP-SURF4 (green) and the ER-marker mRFP-KDEL (red) were imaged by Live-cell Airyscan super-resolution microscopy. (A) Maximal projection of typical 90 s time series (t projection) with white indicating EGFP-SURF4 and ER-marker mRFP-KDEL colocalization and arrowheads indicating mRFP-KDEL-negative EGFP-SURF4 tubules. Scale bars, 5 μm. For corresponding and see supplemental information. (B) Quantification of mRFP-KDEL-negative EGFP-SURF4 tubules from time series as in (A). The graph represents EGPF-SURF4-only tubules counted per cell/90 s time series from 28 WT to 23 YIPF5-KO cells from three independent experiments. Welch’s t test, ∗∗∗∗ indicates p < 0.0001. (C) Live-cell Airyscan super-resolution microscopy of MCF10A YIPF5-KO cells stably expressing EGFP-SURF4 (green) and transfected with SEC24C-mCherry (red), marking ERES. Right, magnifications of the boxed area at three different timepoints (T1–3, 0.5 s interval). Arrowheads depict a long tubular structure emanating from an ERES (arrow) and indicate the dynamic extension of GFP-SURF4-positive tubules, while the full arrow points to a Sec24C-mCherry-labeled ERES. Scale bars, 2 μm. (D) Airyscan imaging of fixed YIPF5-KO cells expressing GFP-SURF4 (green) and immunostained for ERGIC-53 (red). Enlarged regions show ERGIC-53-positive labeling (arrows) along SURF4-positive tubules (arrowheads). Scale bars, 10 μm. (E) Intensity profile plots of two regions of interest (ROI1 and 2) from (D). (F and G) HeLa cells transfected with RUSH-CAB45-GFP and stably expressing either shScramble or shRNA3-YIPF were treated with biotin (40 μg/mL) for indicated timepoints. (F) Supernatants were subjected to GFP-trap pull-down, separated by SDS-PAGE, blotted and probed with anti-GFP antibody. (G) Quantification of (F). Displayed is the mean secreted GFP-CAB45 as arbitrary units normalized to 15 min, ±SEM of n = 4 experiments. Statistical test, multiple unpaired t test with Welch correction. For full size blots see F. See also , , and .
Article Snippet: Following seeding, live-cell imaging was initiated (t = 0 h) using the IncuCyte® SX5 Live-Cell Analysis System (Essen BioScience).
Techniques: Transfection, Marker, Super-Resolution Microscopy, Stable Transfection, Expressing, Labeling, Imaging, SDS Page
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