Figures and data
ER-FlucDM-eGFP forms protein aggregates in the nucleus.
(A) Representative images of ER-FlucDM-eGFP expressed in different cell lines. Maximum projected images were shown.
(B) Microscopy images of cells expressing ER-FlucDM-mCherry and mStayGold-Sec61β. Single Z-slice images were shown.
(C) Quantification of the luciferase activity in MCF10A cells expressing different constructs. Unpaired t-test. Data were collected from three independent experiments. Abundance of GFP fusion proteins was determined using protein blotting and was used in normalization.
(D) Representative images to show MCF10A cells expressing ER-FlucDM-mCherry, ERFSHR-FlucDM-eGFP (signal peptide sequence from the N-terminus of FSHR), ER-HaloDM-eGFP or ER-eGFP. Maximum projected images were shown.
(E) Images of ER-FlucDM-eGFP aggregates surrounded by the ER membrane in a MCF10A cell. Single Z-slice images were shown.
(F) TEM images of sectioned MCF10A interphase cells expressing ER-FlucDM-eGFP.
Data represent mean + SD. Scale bar, 10 μm (A to E).
Differential aggregate dynamics in cells at interphase and mitosis.
(A) Representative images of interphase cells co-expressing ER-FlucDM-eGFP and ER-FlucDM-mCherry before and after photobleaching. Yellow rectangles indicate bleached aggregates. Maximum projected images were shown.
(B) Representative images of mitotic cells co-expressing ER-FlucDM-eGFP and ER-FlucDM-mCherry before and after photobleaching. Yellow circles indicate bleached aggregates. Maximum projected images were shown.
(C) Recovery curves of ER-FlucDM-mCherry intensity after photobleaching. 14 ROIs from the ER of interphase cells, 14 aggregates from mitotic cells, 26 aggregates from interphase cells. Data represent mean + SD. A small region of ER-FlucDM-mCherry in the ER network was chosen as a control for photobleaching.
(D) Quantification of the ER-FlucDM-mCherry intensity of bleached aggregates right after bleaching and after 5 min of recovery. 0 min and 5 min refer to the time points in (C). Paired t-test (two-tailed).
Data were collected from four independent experiments. Scale bar, 10 μm.
Aggregates are cleared during cell division.
(A) Representative images of dividing ER-FlucDM-eGFP cells at different cell cycle phases. Grey scale bar: telophase and early G1 cells; black scale bar: interphase and prophase, prometaphase, metaphase, anaphase. Maximum projected images were shown. Scale bar, 10 μm.
(B) Quantification of ER-FlucDM-eGFP aggregates from dividing cells. Count and area are individual cell data. Each grey ring represents aggregates from one cell. Interphase and prophase, 91 cells; prometaphase, 45 cells; metaphase, 60 cells; anaphase, 20 cells; telophase and early G1, 73 cells. From three independent experiments. Unpaired two side Mann-Whitney U-test. Data represent mean ± SD
(C) Representative time-lapse images of cells expressing ER-FlucDM-eGFP released from the G2/M boundary. NEB (nuclear envelop breakdown) is set to be t = 0 min. Maximum projected images were shown. Scale bar, 10 μm
(D) Quantification of aggregate number and area of dividing cells expressing ER-FlucDM-eGFP. Left panel: data of individual cells. Right panel: mean values + SD of single-cell data (left panel). Time point of NEB in each cell was set to 0 min. Aggregate count and total area of each cell at each time point is normalized to its own value at t = 0 min. Aggregate count data are from 61 interphase and 73 dividing cells of four independent experiments. Aggregate area of each cell is calculated from 32 interphase and 34 dividing cells of two independent experiments.
Effects of ER stressors on aggregate clearance.
(A) Relative gene expression levels of chaperones and co-chaperones in cells expressing ER-FlucDM-eGFP. Four independent experiments.
(B) Images of cells treated with DMSO or 1 μM Thaps and fixed at 35 min or 65 min after released from the G2/M boundary. Maximum projected images were shown.
(C) Quantification of cells treated with DMSO or 1 μM Thaps and fixed at 35 min or 65 min after released from the G2/M boundary. 35 min_DMSO, 207 cells; 35 min_Thaps, 173 cells; 65min_DMSO, 244 cells; 65 min_Thaps, 209 cells. Unpaired two side Mann-Whitney U-test. The solid black line in violin plot indicates median of data in the column. Gray dotted lines are quartiles. Each light pink circle indicates measurements of individual cells.
(D) Representative time-lapse images of cells expressing ER-FlucDM-eGFP treated with DMSO or 1 μM Thaps after released from the G2/M boundary. Maximum projected images were shown.
(E) Quantification of cells expressing ER-FlucDM-eGFP treated with DMSO or 1 μM Thaps. Time point of NEB in each cell was set to 0 min. Aggregates count and total area of each cell is normalized to its own value at t = 0 min. Black dotted lines are 90 min since NEB. DMSO, 89 cells; Thaps, 65 cells.
Scale bar, 10 μm. Three (C and E) or four (A) independent experiments.
Inhibition of HSP70 and BiP prevents aggregate clearance.
(A) Representative images of cells expressing ER-FlucDM-eGFP treated with DMSO or 50 μM VER and fixed at 65 min after released from the G2/M boundary. Maximum projected images were shown.
(B) Quantification of cells expressing ER-FlucDM-eGFP treated with DMSO or 50 μM VER and fixed at 65 min after released from the G2/M boundary. DMSO, 357 cells; VER, 465 cells.
(C) Time-lapse images of dividing cells expressing ER-FlucDM-eGFP treated with DMSO or 50 μM VER. Maximum projected images were shown.
(D) Representative images of cells expressing ER-FlucDM-eGFP treated with DMSO or different concentrations of YUM70. Maximum projected images were shown.
(E) Quantification of cells expressing ER-FlucDM-eGFP treated with DMSO or YUM70 and fixed at 65 min after released from the G2/M boundary. DMSO, 310 cells; 5 μM YUM70, 258 cells; 20 μM YUM70, 215 cells; 50 μM YUM70, 209 cells; 100 μM YUM70, 287 cells.
Scale bar, 10 μm. Unpaired two side Mann-Whitney U-test. Each solid line in violin plot indicates median of data in the column. Gray dotted lines are quartiles. A, B, D and E are from three independent experiments. C is from two independent experiments
Effects of MG132, APCi and RO-3306 (Cdk1i) on aggregate clearance.
(A) Representative images of cells expressing ER-FlucDM-eGFP arrested at mitosis by MG132 and further treated with Cdk1i to induce mitotic exit. Cells were first treated with 25 μM MG132 for 55 min and mitotic arrested cells were further treated with 25 μM MG132 plus 10 μM RO-3306 or 25 μM MG132 only. Cells were fixed 40 min after the second treatment. Maximum projected images were shown.
(B) Quantification of (A). MG132 only, 442 cells; MG132 + RO-3306, 374 cells.
(C) Time-lapse images of cells expressing ER-FlucDM-eGFP arrested at mitosis by MG132 and further treated with RO-3306 to induce mitotic exit. Cells were treated with 25 μM MG132 for 55 min, and further treated with 25 μM MG132 plus 10 μM RO-3306 or 25 μM MG132 before imaging. Maximum projected images were shown.
(D) Quantification of (C). Remaining aggregates at 40 min were calculated for their aggregates count or area and compared to that of in 0 min. MG132 only, 125 cells; MG132 + RO-3306, 134 cells.
(E) Images of cells expressing ER-FlucDM-eGFP treated with DMSO or APCi (20 μM proTAME and 300 μM Apcin) and fixed at 55 min after released from the G2/M boundary. Maximum projected images were shown.
(F) Quantifications of cells expressing ER-FlucDM-eGFP treated with DMSO or APCi (20 μM proTAME and 300 μM Apcin). DMSO, 450 cells; APCi, 549 cells.
(G) Images of cells at telophase or cells of newly divided upon treatment of DMSO or APCi for 35 to 40 min. Maximum projected images were shown.
(H) Cells at telophase or newly divided cells that have aggregates upon treatment of DMSO or APCi were counted.
(I) Aggregates number and area were counted in cells at telophase or in newly divided cells after treated with DMSO or APCi. Only cells with aggregates were included in the quantification. DMSO, 297 cells; APCi, 319 cells.
Scale bar, 10 μm. Data are from at least three independent experiments and analysed by unpaired two side Mann-Whitney U-test (B, D, F and I) or paired t-test (H).
Perturbation of proteins that regulate ER reorganization in aggregate clearance
(A) Images of cells expressing ER-FlucDM-eGFP after depletion of Atlastin 2 and Atlastin 3 and fixed at 65 min after released from the G2/M boundary. Maximum projected images were shown.
(B) Quantification of cells expressing ER-FlucDM-eGFP after depletion of Atlastin 2 and Atlastin 3 and fixed at 65 min after released from the G2/M boundary. Control, 396 cells; ATL2/ATL3 KD, 362 cells.
(C) Images of cells expressing ER-FlucDM-eGFP treated with DMSO or 100 nM MLN-8237 and fixed at 65 min after released from the G2/M boundary. Maximum projected images were shown.
(D) Quantification of cells expressing ER-FlucDM-eGFP treated with DMSO or 100 nM MLN-8237 and fixed at 65 min after released from the G2/M boundary. Only round mitotic cells were counted. DMSO, 102 cells; MLN-8237, 288 cells.
(E) Images of cells expressing ER-FlucDM-eGFP treated with DMSO or 331nM nocodazole and fixed at 65 min after released from the G2/M boundary. Maximum projected images were shown.
(F) Quantification of cells expressing ER-FlucDM-eGFP treated with DMSO or 331nM nocodazole and fixed at 65 min after released from the G2/M boundary. Only round mitotic cells were counted. DMSO, 122 cells; nocodazole, 320 cells.
Scale bar, 10 μm. Data are from three independent experiments and analysed by unpaired two side Mann-Whitney U-test.
ER-FlucDM-eGFP forms protein aggregates, related to Figure 1
(A) Microscopy images of cells expressing NLS-FlucDM-eGFP. Two cell lines were used to express NLS-FlucDM-eGFP. Single Z-slice images were shown.
(B) Microscopy images of cells expressing NLS-FlucDM-eGFP and ER-FlucDM-mCherry in MCF10A cells. Single Z-slice images were shown.
(C) Protein blots for ER-FlucDM-eGFP and NLS-FlucDM-eGFP using anti-GFP or anti-β-actin antibodies.
(D) Relative GFP protein intensity in S1(C) was quantitated and plotted. The intensity of β-actin was used in normalization.
(E) Protein blotting to examine solubility of ER-FlucDM-eGFP under different concentrations of detergents, NP-40. Cells expressing ER-FlucDM-eGFP were subjected to 45 °C for 1 hour before harvesting cell lysates in heat shock experiments. Insoluble fractions were quantitated and plotted.
(F) Colocalization of ER-FlucDM-eGFP and Thioflavin T (ThT), a marker for protein aggregates. Single Z-slice images were shown.
(G) Quantification of ER-FlucDM-eGFP aggregate number (left) and total aggregate area (right) of individual cells in different cell lines. MCF10A, 516 cells; A549, 395 cells; MDA-MB-231, 369 cells; U2OS, 528 cells.
(H) Schematic representation of different constructs used in this study.
(I) Quantification of aggregate number in cells expressing ER-eGFP, ER-HaloWT-eGFP, ER-HaloDM-eGFP. ER-eGFP, 612 cells; ER-HaloWT-eGFP, 405 cells; 546 ER-HaloDM-eGFP, 546 cells; ER-FlucDM-eGFP, 516 cells.
Data are from three independent experiments, analysed by unpaired t-test and represented by mean + SD. Scale bar, 10 μm.
FRAP analysis of protein aggregates, related to Figure 2.
(A(A) Recovery curve of ER-HaloDM-eGFP before and after photobleaching. Seven interphase cells from three independent experiments. Data represent mean + SD.
(B) Recovery curve of individual cell data in ER-FlucDM-eGFP before and after photobleaching. 14 ROIs from the ER of interphase cell, 14 aggregates from cells at mitosis, 26 aggregates from cells at interphase. Data were collected from four independent experiments.
(C) Recovery curve of ER-eGFP before and after photobleaching. Seven interphase cells from three independent experiments. Data represent mean + SD.
(D) Quantification of protein fluorescence intensity of different constructs right after photobleaching and 5 min after recovery. 0 min is the protein intensity right after photobleaching and 5 min is the protein intensity 5 min after recovery from photobleaching. At least three independent experiments. Data represent mean ± SD.
Aggregates in dividing cells, related to Figure 3.
(A(A) Images to show ER-FlucDM-eGFP aggregates of dividing cells were surrounded by the membrane stained by the ER-tracker. Scale bar, 10 μm. Single Z-slice images were shown.
(B) TEM images of sectioned MCF10A mitotic cells expressing ER-FlucDM-eGFP.
(C) Time-lapse images of cells expressing ER-HaloDM-eGFP released from the G2/M boundary. NEB (nuclear envelop breakdown) is set to be t = 0 min. Maximum projected images were shown. Scale bar, 10 μm.
(D) Linear regression analysis to show that cell division process is not linearly dependent on aggregates quantity. 74 dividing cells are from four independent experiments.
Effect of Thapsigargin or Tunicamycin on aggregates clearance, related to Figure 4.
(A(A) Proteomic analysis of cells expressing ER-FlucDM-eGFP. Three independent experiments. Proteins involved in ER stress and unfolded protein response (blue) or cell cycle and cell division (pink) were highlighted.
(B) Changes of aggregate number and total area as a function of time after cells expressing ER-FlucDM-eGFP were treated with DMSO or 1 μM Thaps.
(C) Quantification of aggregate number and total area of cells expressing ER-FlucDM-eGFP treated with DMSO or Tunicamycin (Tuni) during mitosis. DMSO, 542 cells; 200 ng/mL Tuni, 385 cells; 2 μg/mL Tuni, 471 cells; 10 μg/mL Tuni, 439 cells. Four independent experiments. Unpaired two side Mann-Whitney U-test. Each solid thick line in violin plot indicates median of data in the column. Gray lines are quartiles.
Colocalization of BiP and ER-FlucDM-eGFP, related to Figure 5.
(A(A) Immunofluorescence staining of BiP and ER-FlucDM-eGFP in dividing cells. Maximum projected images were shown.
(B) Colocalization analysis of BiP and ER-FlucDM-eGFP. R represents Pearson correlation coefficient (r).
(C) Immunofluorescence staining of BiP and ER-FlucDM-eGFP in interphase cells. Single Z-slice images were shown.
Scale bar, 10 μm. Three independent experiments.
MG132, APCi and RO-3306 treatments on aggregate clearance, related to Figure 6.
(A(A) Quantification of the aggregate number in cells expressing ER-FlucDM-eGFP treated with DMSO or 25 μM MG132 and fixed at 55 min after released from the G2/M boundary. DMSO, 293 cells; MG132, 350 cells. Maximum projected images were shown.
(B) Quantification of the aggregate number in cells expressing ER-FlucDM-eGFP treated with DMSO or 5 μM MG132 and fixed at 55 min since release. DMSO, 295 cells; MG132, 436 cells. Maximum projected images were shown.
(C) Images of cells at telophase or newly divided cells after treated with DMSO or 25 μM MG132 for 35 min. Maximum projected images were shown.
(D) Cells at telophase or newly divided cells that have aggregates upon treatment of 25 μM MG132 were counted. DMSO, 455 cells; MG132, 393 cells.
(E) Aggregates number and area were counted in cells at telophase or in newly divided cells after treated with DMSO or 25 μM MG132. Only cells with aggregates were included in the quantification. DMSO, 294 cells; MG132, 339 cells.
(F) Images of cells at telophase and newly divided cells after treated with DMSO or 5 μM MG132 for 35 min. Maximum projected images were shown.
G) Cells at telophase or newly divided cells that have aggregates upon treatment of DMSO or 5 μM MG132. DMSO, 676 cells; APCi, 646 cells.
(H) Aggregates number and area were counted in cells at telophase or in newly divided cells after treated with DMSO or 5 μM MG132. Only cells with aggregates were included in the quantification. DMSO, 227 cells; MG132, 297 cells.
(I) Quantification of the aggregate number in cells expressing ER-FlucDM-eGFP treated with 5 μM MG132 or 5 μM MG132 + RO-3306.
(J) Images of cells co-expressing ER-FlucDM-mCherry and mGreenLantern-hGeminin (degron).
Left panel: cells were treated with DMSO or APC/Ci after 65 min released from the G2/M. Right panel: after 65 min released from the G2/M, cells were treated with DMSO or APC/Ci for 40 minutes using live-cell imaging. Maximum projected images were shown.
(K) Quantification of mGreenLantern-hGeminin (degron) intensity of cells treated with DMSO or APC/Ci for 40 min.
Scale bar, 10 μm. Data are from at least three independent experiments and analysed by unpaired two side Mann-Whitney U-test (A, B, E, H and I), unpaired t-test (K) and paired t-test (D and G).
Cycloheximide chase experiment and microscopy quantification of cells expressing ER-FlucDM-eGFP, related to Figure 6.
(A(A) Protein blots of ER-FlucDM-eGFP, Cyclin B1, p53 and β-actin under DMSO or 200 μM CHX treatments. Cells were synchronized to the G2/M boundary and released into medium containing DMSO or CHX and lysates were harvested.
(B) Protein levels of p53 and Cyclin B1 in different time points after DMSO or CHX treatments were measured and plotted. Levels of β-actin were used in normalization.
(C) Protein levels of ER-FlucDM-eGFP in different time points after DMSO or CHX treatments were measured and plotted. Levels of β-actin were used in normalization.
(D) Relative intensity of ER-FlucDM-eGFP in early and late dividing cells.
Scale bar, 10 μm. Data are from three independent experiments and analysed by unpaired t-test.
