Fig 2: Highly mobile small Akita puncta colocalize with SEC24C.A) U2OS cells expressing Akita-sfGFP were treated with MRT68921 for the indicated times and examined for the accumulation of large puncta (≥0.5 µm2). Arrows mark large Akita puncta. (B) Bar graph showing the % of cells with large Akita puncta for the data shown in (A). (C) Cells expressing Akita-sfGFP and mCherry-SEC24C were treated with MRT68921 for 3.5 hr and imaged. Arrowheads show Akita puncta colocalizing with SEC24C, arrows indicate Akita puncta that do not colocalize with SEC24C. (D) Bar graph for the data shown in (C) of % Akita-sfGFP puncta of different sizes that colocalize with mCherry-SEC24C puncta. (E) Cells expressing Akita-sfGFP were treated with MRT68921 for 3.5 hr, and the velocity of differently sized puncta was determined. (F) Cells expressing Akita-sfGFP were treated with MRT68921 for 3.5 hr followed by fluorescence recovery after photobleaching (FRAP) analysis of Akita puncta (average size; ≥ 0.32 µm2) and the endoplasmic reticulum network. Scale bars in (A) and (C), 10 µm. Error bars in (B), (D), and (E) represent SEM; n = 3 independent experiments. Approximately 30–40 cells were examined/experiment (B), 20–30 cells/experiment (D), 35–40 puncta/experiment (E), and 20 puncta/experiment (F). NS: not significant (p≥0.05); *p<0.05, ***p<0.001, Student’s unpaired t-test.

Fig 3: Torin induces the colocalization of SEC24C with LC3B and RTN3, but not FAM134B.(A) U2OS cells expressing EYFP-SEC24C and mCherry-LC3B were treated with Torin 2 for 3.5 hr and examined by confocal microscopy. Arrowheads in the inset indicate SEC24C puncta colocalizing with LC3B. (B) Bar graph showing the % of EYFP-SEC24C puncta colocalizing with mCherry-LC3B puncta for the data shown in (A). (C) Cells expressing mCherry-RTN3 and EYFP-SEC24C were treated with Torin 2 for 3.5 hr. Representative confocal images are shown. Arrowheads in the inset indicate EYFP-SEC24C puncta colocalizing with mCherry-RTN3. (D) Bar graph showing the % of EYFP-SEC24C puncta colocalizing with mCherry-RTN3 or FAM134B-mTurquoise puncta in transiently transfected cells. (E) Cells stably expressing mCherry-RTN3 were transfected with EYFP-SEC24C, mAaus0.5-SEC24A, SEC13-GFP, or EGFP-SEC16A and treated with Torin 2 for 3.5 hr. The % of SEC puncta that colocalized with mCherry-RTN3 puncta was quantified. Scale bars in (A) and (C), 10 µm. Error bars in (B), (D), and (E) represent SEM; n = 3 independent experiments. Approximately 20–30 cells/experiment were analyzed. NS: not significant (p≥0.05); **p<0.01, Student’s unpaired t-test.

Fig 4: Disrupting autophagy leads to the accumulation of Akita puncta that colocalize with SEC24C, and LC3B, but not SEC24A.(A) U2OS cells expressing Akita-sfGFP and mCherry-SEC24C or mCherry-SEC24A were treated with MRT68921 for 3.5 hr. Arrowheads in the inset indicate Akita puncta colocalizing with SEC24C. (B) Bar graph showing the % of cells with multiple Akita-sfGFP puncta colocalizing with mCherry-SEC24C or mCherry-SEC24A puncta 0 or 3.5 hr after treatment with MRT68921. (C) Cells expressing Akita-sfGFP and mCherry-SEC24C were depleted of RTN3 by RNAi and treated with MRT68921 for 0 or 3.5 hr. The % of cells showing multiple Akita-sfGFP puncta colocalizing with mCherry-SEC24C puncta was quantified at the indicated time points. (D) Cells expressing Akita-sfGFP and mCherry-LC3B were depleted of RTN3 and treated with MRT68921 for 0 or 3.5 hr. A representative image for control cells (3.5 hr) is shown. Arrowhead in the inset shows an Akita puncta colocalizing with LC3B. (E) Bar graph showing the % of cells with multiple Akita-sfGFP puncta colocalizing with mCherry-LC3B puncta for the data shown in (D). Scale bars in (A) and (D), 10 µm. Error bars in (B), (C), and (E) represent SEM; n = 3 independent experiments. Approximately 20–30 cells/experiment were examined. NS: not significant (p≥0.05); *p<0.05, **p<0.01, Student’s unpaired t-test.

Fig 5: Akita puncta do not enlarge in the tubules.ER-phagy sites (ERPHS) are formed when Akita puncta associate with RTN3 puncta, LC3B, and SEC24C on the tubules. The association of RTN3 with LC3B occurs in the absence of SEC24C. In LNPK KO cells, the tubular polygonal network collapses and large Akita puncta accumulate in sheet-like endoplasmic reticulum (ER). Large Akita puncta also accumulate in siSEC24C and siRTN3 cells.