Improved volume CLEM revealed that aberrant phagophores and RB1CC1/FIP200-containing clusters appear surround SQSTM1/p62 aggregates in Atg9a-deficient cells.

ATG9A is an important membrane protein in mammalian macroautophagy. The formation of autophagosomes and phagophores is blocked in atg9a KO cells. However, it remains possible that residual membrane formation activity exists in these cells. These precursor structures that precede phagophores are, if they exist, rare and may be difficult to find. Here, we introduce the modified volume correlative light and electron microscopy (CLEM) method to analyze these structures three-dimensionally. In addition to target proteins, mitochondria were labeled as a landmark for precise correlation of slice images by a confocal fluorescence microscope and a focused ion beam scanning electron microscope. We found phagophores and small membrane vesicles near SQSTM1/p62 aggregates in atg9a KO cells, indicating that phagophores could be formed in atg9a-deficient cells, although they were immature and inefficient. Furthermore, we found that RB1CC1/FIP200-positive structures formed clusters around SQSTM1/p62 with ferritin and TAX1BP1. Taken together, our method contributes to the understanding of undiscovered fine structures. Abbreviations: CLEM: correlative light and electron microscopy; EM: electron microscopy; ER: endoplasmic reticulum; FIB-SEM: focused ion beam scanning electron microscopy; FM: fluorescence microscopy; GFP: green fluorescent protein; KO: knock out; MEF: mouse embryonic fibroblast; PBS: phosphate-buffered saline; ROI: region of interest; SEM: scanning electron microscopy.