Figure 5. Large Akita puncta accumulate in LNPK KO cells.

(A) Control (Ctrl) and LNPK KO cells were analyzed for the accumulation of large Akita puncta (≥0.5 µm²) by confocal microscopy (top). Arrows point to large puncta. Bar graph shows the % of cells with large Akita-sfGFP puncta (bottom). (B) Maximum intensity projections showing Akita puncta in Ctrl and LNPK KO cells used for the 3D reconstructions shown in Videos 3 and 4. (C) Frames from representative 3D reconstructions of Z stacks of a Ctrl cell (left, 27.5 s in Video 3) and an LNPK KO cell (right, 39 s in Video 4) are shown. Arrowheads mark small Akita puncta, while arrows mark the large puncta. Scale bar for top images in (B) and (C), 10 µm. The insets that are enlarged at the bottom, 1 µm. (D) Ctrl or LNPK KO cells stably expressing mCherry-RTN3 were examined for colocalizing Akita-sfGFP puncta. Arrowheads in the inset indicate Akita puncta colocalizing with RTN3 puncta (top). Arrows show large Akita puncta (≥0.5 µm²) in LNPK KO cells that do not colocalize with RTN3 (bottom). (E) Bar graph for the data shown in (D) of % Akita-sfGFP puncta of different sizes that colocalize with mCherry-RTN3 puncta. (F) Control cells expressing Akita-sfGFP and mCherry-SEC61β were analyzed by confocal microscopy. Arrows mark Akita puncta. (G) Bar graph for the Ctrl data shown in (D) and (F) of % Akita-sfGFP puncta of different sizes that colocalize with either mCherry-RTN3 puncta or mCherry-SEC61β puncta. To identify Akita, RTN3, or SEC61 puncta, the Yen threshold algorithm, which identifies punctate accentuations on the network, was used. We then identified colocalized puncta (Akita-RTN3, Akita-SEC61) using the Boolean image calculator in ImageJ. Colocalizing puncta were calculated as follows: Akita puncta colocalized with RTN3 puncta/total Akita puncta × 100% or Akita puncta colocalized with SEC61 puncta/total Akita puncta × 100%. Scale bars in (A), (D), and (F), 10 µm. Error bars in (A), (E), and (G) represent SEM; n = 3 independent experiments. Approximately 30–40 cells were examined/experiment for (A), and 15–30 cells/experiment for (E) and (G). NS: not significant (p≥0.05); *p<0.05, ***p<0.001, Student’s unpaired t-test.

Figure 5—figure supplement 1. Large Akita puncta accumulate when CLIMP63 is overexpressed.

(A) Maximum intensity projections showing Akita puncta in Ctrl cells expressing mCherry-CLIMP63 (left) or Ctrl cells expressing mCherry-CLIMP63 and mTurquoise2-RTN4 (right). These images were used for the 3D reconstructions in Videos 5 and 6. (B) Frames from representative images of 3D reconstructions of Z stacks of cells showing Akita puncta in Ctrl cells expressing mCherry-CLIMP63 (left, 14 s frame in Video 5), or Ctrl cells expressing mCherry-CLIMP63 and mTurquoise2-RTN4 (right, 3.8 s frame in Video 6). Arrows mark large Akita puncta, while arrowheads mark small puncta. Scale bar for top images in (A) and (B), 10 µm. Insets that are enlarged at the bottom, 1 µm. (C) Endoplasmic reticulum (ER) sheets proliferate when mCherry-CLIMP63 is overexpressed. Bar graph showing the % of cells with mostly sheet-like ER for the data in (A). (D) Bar graph showing the % of cells with large Akita puncta (≥0.5 µm²) for the data in (A). (E) The mean Akita puncta size increases in cells when CLIMP63 is overexpressed. Bar graph showing the mean Akita puncta size per cell for the data in (A) and (B). (F) The mean Akita puncta size increases in LNPK KO cells. Bar graph showing the mean Akita puncta size per cell for the data shown in Figure 6B. Approximately 30–40 cells/experiment were quantitated for (A–D) and 15 cells/experiment were quantitated for (E) and (F). NS: not significant (p≥0.05); *p<0.05; **p<0.01, Student’s unpaired t-test.

Figure 5—figure supplement 2. The delivery of Akita to lysosomes is reduced in LNPK KO cells or when CLIMP63 is overexpressed.

(A) Ctrl and LNPK KO cells expressing Akita-sfGFP and LAMP1-mCherry were treated with DMSO or Baf and examined by confocal microscopy (rows 1–4). Bottom two rows, control cells expressing Akita-sfGFP, mTurquoise2-CLIMP63 and LAMP1-mCherry were treated with DMSO or Baf. (B) Quantitation of Akita-sfGFP in LAMP1-mCherry structures for the data shown in (A). The DMSO control was set to 1.0. Scale bar in (A), 10 µm. Error bars in (B) represent SEM, n = 3 independent experiments. Approximately 20–30 cells/experiment were quantitated. NS: not significant (p≥0.05); *p<0.05, Student’s unpaired t-test.

Figure 5—figure supplement 3. hCOL1A1 puncta do not accumulate in LNPK KO cells.

(A) Ctrl and LNPK KO cells expressing EGFP-hCOL1A1 were analyzed for the accumulation of hCOL1A1 puncta by confocal microscopy (top). Bar graph shows the number of EGFP-hCOL1A1 puncta per cell in the control and LNPK KO cells (bottom). Puncta of all sizes were measured. (B) Representative confocal images of WT and LNPK KO cells that contain large hCOL1A1 puncta (≥0.5 µm²) (top). Arrow marks a large punctum. Bar graph (bottom) shows the % of cells with large EGFP-hCOL1A1 puncta for the data shown above. (C, D) G57S Pro-AVP-FLAG accumulated in siSEC24C, but not siSEC24A cells. Cells expressing G57S Pro-AVP-FLAG were depleted of SEC24C, SEC24A, or RTN3 by siRNA (C), or knocked out for LNPK by CRISPR (D). Cells in (C) and (D) were analyzed for the accumulation of large G57S Pro-AVP puncta (≥0.5 µm²) (top). Bar graphs show the % of cells with large G57S Pro-AVP-FLAG puncta in the indicated samples (bottom). Arrows mark the large puncta. (E, F) C28F POMC accumulated in siSEC24C, but not siSEC24A cells. Cells expressing C28F POMC-FLAG were depleted of SEC24C, SEC24A, or RTN3 by siRNA (E), or knocked out for LNPK (F). Cells in (E) and (F) were analyzed for the accumulation of large C28F POMC puncta (≥0.5 µm²) by confocal microscopy (top). Arrows mark the large puncta. Bar graphs (bottom) show the % of cells with large C28F POMC-FLAG puncta (≥0.5 µm²). Scale bars in (A–F), 10 µm. Error bars in (A–F) represent SEM; n = 3 independent experiments. Approximately 20–30 cells were examined in each experiment. NS: not significant (p≥0.05); *p<0.05, **p<0.01,***p<0.001, Student’s unpaired t-test.

Figure 5—figure supplement 4. Proinsulin and Akita colocalize to the same large puncta.

(A) Ctrl and LNPK KO cells were co-transfected with Akita-sfGFP and proinsulin-FLAG and processed for immunofluorescence after 48 hr. A representative LNPK KO cell is shown. Large puncta (≥0.5 µm²) are marked with arrows. (B) Quantitation of control and LNPK KO cells with large puncta that contain Akita-sfGFP or Akita-sfGFP + proinsulin FLAG. (C, D) are the same as (A) and (B) except the cells were co-transfected with G57S Pro-AVP-FLAG and Pro-AVP-HA. (E, F) are the same as (A) and (B) except the cells were co-transfected with C28F POMC-FLAG and POMC-Myc. Scale bars in (A, C, E), 10 µm. Error bars in (B, D, F) represent SEM; n = 3 independent experiments. Approximately 20–25 cells were examined in each experiment. *p<0.05, **p<0.01, Student’s unpaired t-test.

Figure 5—figure supplement 5. Large Akita puncta accumulate in INS1 cells in response to CLIMP63 overexpression.

(A) Insulin-secreting INS1 cells expressing Akita-sfGFP (left) or Akita-sfGFP and mCherry-CLIMP63 (right) were analyzed for the accumulation of large Akita puncta (≥0.5 µm²) by confocal microscopy. The arrow points to a large punctum. The punctum that is marked is shown in (B). Scale bar, 5 µm. (B) A representative image of a 3D reconstruction of a cell expressing Akita-sfGFP and mCherry-CLIMP63 shown in (A). The frames from Video 8 correspond to 9 s and 16 s. Scale bar for top images, 5 µm. Insets that are enlarged at the bottom, 1 µm. (C) Bar graph shows the % of cells with large Akita-sfGFP puncta for the data shown in (A). (D) Proinsulin-sfGFP is processed normally and secreted in a glucose-dependent manner in INS1 cells. To address if the culture conditions used for the experiment in (A) stimulate insulin secretion, INS1 cells were transfected with proinsulin-sfGFP and the culture medium was analyzed. Previous studies have shown that proinsulin-sfGFP is incorporated into secretory granules and processed by prohormone convertases to C-peptide-sfGFP that is co-secreted with insulin into the medium (Liu et al., 2007). The extracellular medium was collected 48 hr after transfection, precipitated with 10% trichloroacetic acid, and secreted C-peptide-sfGFP was detected by western blot analysis with anti-GFP antibody. The extracellular medium from cells cultured in low-glucose medium (2.8 mM glucose) was used as a negative control. Error bars in (C) represent SEM; n = 3 independent experiments. Approximately 15–30 cells/experiment were quantitated. *p<0.05, Student’s unpaired t-test.

Figure 5—figure supplement 6. Akita does not colocalize with SEC24C and LC3B in LNPK KO cells.

(A) Ctrl and LNPK KO cells expressing Akita-sfGFP and mCherry-SEC24C were examined by confocal microscopy. Arrowheads in the inset indicate Akita puncta colocalizing with SEC24C. Arrows indicate large Akita puncta (≥0.5 µm²) that do not colocalize with SEC24C. (B) The loss of LNPK did not lead to a decrease in SEC24C puncta. The average number of SEC24C puncta per cell for the data shown in (A) and graphed in (C). (C) Bar graph showing the % Akita-sfGFP puncta colocalizing with mCherry-SEC24C puncta for the data shown in (A). (D) Akita failed to colocalize with LC3B in the absence of LNPK. Control and LNPK KO cells expressing Akita-sfGFP and mCherry-LC3B were treated with MRT68921 for 3.5 hr. Representative confocal images are shown for treatment with MRT68921 for 3.5 hr. (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, E) represent SEM; n = 3 independent experiments. Approximately 15–30 cells were examined in each experiment. NS: not significant (p≥0.05); *p<0.05, **p<0.01, Student’s unpaired t-test.

Figure 5—figure supplement 7. The velocity of small Akita puncta is unaltered in LNPK KO cells.

(A) Small, but not large, Akita puncta colocalized with RTN3 in MRT68921-treated cells. Ctrl cells expressing Akita-sfGFP and mCherry-RTN3 were treated with MRT68921 for 3.5 hr and the % of Akita puncta of different sizes that colocalized with RTN3 puncta were quantitated. (B) The velocity of Akita-sfGFP puncta of different sizes was analyzed in control and LNPK KO cells by live cell confocal microscopy. (C) LNPK KO cells expressing Akita-sfGFP and mCherry-SEC61β were analyzed by confocal microscopy. Arrowheads mark large Akita puncta that appear to reside in the lumen of the endoplasmic reticulum (ER). (D) Bar graph comparing the colocalization of the % of Akita-sfGFP puncta of different sizes with mCherry-RTN3 puncta (shown in Figure 5D) or mCherry-SEC61β puncta (shown in C) in LNPK KO cells. Scale bar in (C), 10 µm. Error bars in (A), (B), and (D) represent SEM; n = 3 independent experiments. Approximately 15–30 cells/experiment were examined. NS: not significant (p≥0.05); *p<0.05, **p<0.01, ***p<0.001, Student’s unpaired t-test.

Figure 5—figure supplement 8. Large Akita puncta rapidly recover after photobleaching in LNPK KO cells.

(A) Fluorescence recovery after photobleaching (FRAP) analysis of the endoplasmic reticulum (ER) network in Ctrl cells, LNPK KO cells, and large Akita-sfGFP puncta (average size ≥0.35 µm²) in LNPK KO cells. (B) The half time of fluorescence recovery (time 1/2) after photobleaching for the data shown in (A). (C) Left: a control cell showing the region of the ER (see box) that was photobleached in (D). Right: an LNPK KO cell showing the region of the ER (left box) and Akita puncta (right box) that was photobleached in (D). (D) Representative confocal images of the region that was analyzed by FRAP for the data shown in (A). Scale bar in (C), 10 µm. Scale bar in (D), 1 µm. Error bars in (B) represent SD, n = 3 independent experiments. Approximately 20 puncta/experiment were analyzed. NS: not significant (p≥0.05); **p<0.01, Student’s unpaired t-test.

Figure 5—figure supplement 9. Large Akita puncta rapidly recover after photobleaching in siRTN3 cells.

(A) Fluorescence recovery after photobleaching (FRAP) analysis of the endoplasmic reticulum (ER) network in Ctrl cells, siRTN3 cells, and large Akita-sfGFP puncta (average size ≥0.32 µm²) in siRTN3 cells. (B) The half time of fluorescence recovery (time 1/2) after photobleaching for the data shown in (A). (C) Left: a control cell showing the region of the ER (see box) that was photobleached in (D). Right: an RTN3-depleted cell showing the region of the ER (top box) and Akita puncta (bottom box) that was photobleached in (D). (D) Representative confocal images of the region that was analyzed by FRAP for the data shown in (A). Scale bar in (C), 10 µm. Scale bar in (D), 1 µm. Error bars in (B) represents SD, n = 3 independent experiments. Approximately 20 puncta/experiment were analyzed. NS: not significant (p≥0.05); Student’s unpaired t-test.