EMBO/soda-nel huggingface dataset:
The calcium transporter ANNEXIN1 mediates cold-induced calcium signaling and freezing tolerance in plants (2020)
Figure 2

Figure 2. OST1 interacts with the Ca2+-permeable transporter AtANN1.

A Time-course analysis of cytosolic free calcium concentration ([Ca2+]cyt) dynamics in 10-d-old wild type (Col-0), atann1, and atann1 AtANN1 complementation line (#10) after treatment with ice-cold water or ice-cold water containing 10 mM LaCl3 (arrow shows the time point of treatment). Luminescence was recorded at 1-s intervals.

B Quantification of the cold-induced [Ca2+]cyt changes shown in (A). Peak [Ca2+]cyt indicates the highest [Ca2+]cyt after treatment.

C GST pull-down assay showing that OST1 interacts with AtANN1 in vitro. Purified recombinant GST-OST1, GST-OST1G33R, or GST proteins from E. coli were immunoprecipitated with GST beads and then incubated with MBP-His-AtANN1. Precipitated proteins were detected with anti-GST and anti-His antibodies.

D Interaction of OST1 and AtANN1 detected by bimolecular fluorescence complementation (BiFC) assays. The construct combinations were co-transformed into N. benthamiana leaves and expressed for 2 d. The signal was detected by confocal microscopy. Scale bar, 25 µm. The fluorescence intensity was scanned using the ImageJ plot profile tool. The y-axes indicate relative pixel intensity.

E,F Interaction of OST1 and AtANN1 detected by Co-IP assays in Arabidopsis. Twelve-day-old OST1-Myc overexpressing plants grown on MS medium at 22°C were placed at 4°C for 0, 0.5, 2 h, and total proteins were extracted and immunoprecipitated with anti-Myc agarose beads. The wild type (Col-0) treated with 4°C for 2 h was used as control. The OST1-Myc protein was detected with anti-Myc antibody and the AtANN1 protein was detected with anti-AtANN1 antibody. Representative pictures are shown in (E) and relative protein level in (F).

G Co-IP assay showing the interaction between OST1 and AtANN1 in vivo. The construct combinations were expressed in N. benthamiana leaves. Total proteins were extracted after the N. benthamiana leaves treated with 4oC or 22oC and immunoprecipitated with anti-Myc agarose beads. The proteins were detected with anti-Myc and anti-GFP antibodies.

Data information: In (A and B), the data represent means ± SD of 20 seedlings. Three independent experiments showed a similar result. In (F), the data represent means ± SD of three technical replicates. Different letters represent significant difference at P < 0.05 (one-way ANOVA). In (C-G), representative data were shown, and at least three independent experiments showed a similar result.
The calcium transporter ANNEXIN1 mediates cold-induced calcium signaling and freezing tolerance in plants (2020)
Figure 3

Figure 3. Genetic analysis of OST1 and AtANN1.

A-C Freezing phenotype (A), survival rate (B) and ion leakage (C) of Col-0, ost1-3, atann1, and atann1 ost1-3. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min). Representative pictures are shown in (A).

D,E OST1 gene expression (D) and protein levels of OST1 and AtANN1 (E) in Col-0, OST1-Myc, atann1 and OST1-Myc atann1. OST1 and AtANN1 proteins were detected with anti-Myc and anti-AtANN1 antibodies. HSP90 was used as a control.

F-H Freezing phenotype (F), survival rate (G), and ion leakage (H) of Col-0, OST1-Myc, atann1 and OST1-Myc atann1. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, -5°C, 30 min; CA, -8°C, 40 min). Representative pictures are shown in (F).

Data information: In (B, C, G and H), the data represent means ± SE of three independent experiments, each with three technical repeats (n=19 seedlings for each repeat). Different letters represent significant difference at P < 0.05 (one-way ANOVA). In (D), the data represent means ± SD of three technical replicates.
The calcium transporter ANNEXIN1 mediates cold-induced calcium signaling and freezing tolerance in plants (2020)
Figure 4

Figure 4. AtANN1 is phosphorylated by OST1 at S289.

A,B OST1 phosphorylates AtANN1 (A) and AtANN4 (B) in vitro. Purified recombinant MBP-His-OST1 was incubated with GST-AtANN1, GST-AtANN4 or GST in kinase reaction buffer with 1 µCi [γ-32P] ATP for 30 min at 30°C, followed by separation with SDS-PAGE. Phosphorylated AtANN1 and AtANN4 were detected by autoradiography. Recombinant OST1, AtANN1 and AtANN4 were stained by Coomassie brilliant blue (CBB).

C In-gel kinase assays of OST1 in Col-0 and ost1-3 mutant under cold stress. Twelve-day-old Col-0 and ost1-3 mutant were treated at 4°C for 2 h. Total protein extracts were prepared and separated on a SDS-PAGE gel containing 0.2 mg/mL GST-AtANN1 as a substrate, and incubated with 70 µCi [γ-32P] ATP. Top, autoradiograph; bottom, CBB staining.

D In vitro kinase analysis of the indicated mutant forms of AtANN1 by OST1. After phosphorylation, the proteins were separated by SDS-PAGE and subjected to autoradiography. Top, autoradiograph; bottom, CBB staining.

E OST1 phosphorylates AtANN1 at S289 in vivo in LC/MS analysis. Total proteins were extracted from 12-d-old AtANN1-Myc overexpressing plants treated at 4°C for 0, 10, 30 and 120 min, followed by trypsin digestion. Phosphopeptides were enriched for mass spectrometry analysis. Representative picture for 10 min was shown.

Data information: In (A-C), representative data were shown, and three independent experiments showed a similar result.
The calcium transporter ANNEXIN1 mediates cold-induced calcium signaling and freezing tolerance in plants (2020)
Figure 5

Figure 5. The phosphorylation of AtANN1 by OST1 is required for plant freezing tolerances.

A-C Freezing phenotype (A), survival rate (B), and ion leakage (C) of Col-0, atann1, and atann1 AtANN1S289E (#5) plants. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min). Representative pictures are shown in (A).

D-F Freezing phenotype (D), survival rate (E), and ion leakage (F) of Col-0, Atann1, and atann1 AtANN1S289A (#16) plants. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min). Representative pictures are shown in (D).

Data information: In (B, C, E and F), the data represent means ± SE of three independent experiments, each with three technical repeats (n=25 seedlings for each repeat). **P < 0.01, two-tailed t-test.
The calcium transporter ANNEXIN1 mediates cold-induced calcium signaling and freezing tolerance in plants (2020)
Figure 6

Figure 6. The Ca2+ transport activity of AtANN1 is enhanced by OST1.

A AtANN1 current recordings in Xenopus oocytes. Whole-cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1, OST1, or AtANN1 + OST1 cRNA. Bath solution was described in Materials and Methods. The voltage protocol as well as time and current scale bars for the recordings are shown.

B Current-voltage (I-V) relationship of the steady-state whole-cell currents in Xenopus oocytes described in (A). Negative current is influx of cations into the oocytes.

C Percentage of total Xenophus oocytes in different nA range according to (A).

D AtANN1 current recordings in oocytes. Whole-cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1, AtANN1S289A, OST1, AtANN1 + OST1, and AtANN1S289A + OST1 cRNA.

E Current-voltage (I-V) relationship of the steady-state whole-cell currents in Xenopus oocytes described in (D).

F Percentage of total Xenophus oocytes in different nA range according to (D).

Data information: In (A and D), representative data were shown. In (B and E), the data represent means ± SD of 5 oocytes. Three independent experiments showed a similar result.
The calcium transporter ANNEXIN1 mediates cold-induced calcium signaling and freezing tolerance in plants (2020)
Figure 7

Figure 7. Phosphorylation of AtANN1 by OST1 promotes its Ca2+-binding activity.

A Time-course analysis of [Ca2+]cyt dynamics between 10-day-old wild type (Col-0) and ost1-3 mutant after treatment with ice-cold water or ice-cold water containing 10 mM LaCl3 (arrow shows the time point of treatment). Luminescence was recorded at 1-s intervals.

B Quantification of the cold-induced [Ca2+]cyt changes shown in (A). Peak [Ca2+]cyt indicates the highest [Ca2+]cyt after treatment.

C MST assays of the calcium-binding affinity of GST-AtANN1, GST-AtANN1S289A, and GST-AtANN1S289E. GST was used as a control.

D A proposed working model. Under normal conditions, OST1 protein kinase interacts with PP2C, which inhibits the kinase activity of OST1. AtANN1 is localized in the cytosol and at the plasma membrane. Under cold stress, OST1 is activated and phosphorylates AtANN1, which, on the one hand, enhances the Ca2+ transport activity of AtANN1, and, on the other, promotes the Ca2+-binding activity of AtANN1. This dual role of phosphorylation results in increased cold stress-induced [Ca2+]cyt. Consequently, AtANN1 indirectly facilitates the expression of CBFs and CORs to positively regulate plant freezing tolerance.

Data information: In (A and B), the data represent means ± SD of 20 seedlings (** P < 0.01, student t-test). Three independent experiments showed a similar result.. In (C), the data represent means ± SD of three biological replicates.

EXPANDED VIEW FIGURES
Two distinct pathways for targeting proteins from the cytoplasm to the vacuole/lysosome. (1997)
Figure 1

Morphological studies of proAPI in the cytosol. Cells were grown in YEPD medium at 30°C to log phase and were prepared for electron microscopy as described in Materials and Methods. Where indicated, immunostaining of API was performed using antibody to the mature protein that detects both the mature and precursor forms of the hydrolase. (A) Immunostaining of API in the vacuole of SEY6210 (wild-type) cell showing a dispersed pattern. (B–D) Immunostaining of TVY1 (pep4) cells showing API in the Cvt complex. The arrow points to a Cvt complex. Higher magnification images are shown in C and D. (E) Freeze-substitution fixation image of Cvt complex in TVY1 cell. (F) Freeze-substitution fixation image of a Cvt complex in strain SEY6210 transformed with a 2μ plasmid encoding API grown in SD medium. (G) Immunostaining of strain SEY6210 containing a 2μ APE1 plasmid grown in SD medium. N, Nucleus; V, vacuole.
Two distinct pathways for targeting proteins from the cytoplasm to the vacuole/lysosome. (1997)
Figure 5

Mutant analysis allows for differentiation between vacuolar delivery by Cvt vesicles and autophagosomes. (A) SEY6210 (wild-type), cvt3, and cvt7 yeast were grown in SD to 1 OD600 U/ml and transferred to SD (-N). Aliquots were collected after 0 h (+) and 4 h (−) incubation and subjected to immunoblotting. The positions of precursor (pro) and mature (m) API are indicated. (B) cvt3 and cvt7 cells were pulse labeled for 10 min. After addition of cold cysteine and methionine, the cells were harvested, washed, and resuspended in either SD or SD(-N) and chased for the indicated times. API was recovered by immunoprecipitation, resolved on SDS polyacrylamide gels, and quantified using a phosphorimager (STORM; Molecular Dynamics, Sunnyvale, CA).
Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy (2022)
Figure 2

Figure 2: INPP4B-generated PI(3)P on late endosomes is retained on endolysosomes

A, B MCF-7 cells expressing NT, INPP4B #1 or INPP4B #2 shRNA were fixed and immunostained with recombinant GST-2xFYVE and LAMP2 antibodies, and co-stained with DAPI (A). Data represent the proportion of 2xFYVE+ lysosomes (n=3 experiments, >40 cells/experiment) (B). Arrows indicate co-localization between 2xFYVE and LAMP2.

C MCF-7 cells were treated with 0.02% (w/v) saponin for 30 seconds, then fixed and immunostained with INPP4B and either LAMP1 or CD63 antibodies, and co-stained with DAPI. Arrows indicate co-localization between INPP4B and CD63.

D, E MCF-7 cells co-expressing GFP-INPP4B or GFP-vector, and Hrs or NT shRNA, were fixed and immunostained with recombinant GST-2xFYVE and LAMP2 antibodies, and co-stained with DAPI (D). Data represent the proportion of 2xFYVE+ lysosomes (n=3 experiments, >30 cells/experiment) (E). Arrows indicate co-localization between 2xFYVE and LAMP2.

F, G MCF-7 cells co-expressing GFP-INPP4B or GFP-vector, and Hrs or NT shRNA, were fixed and immunostained with recombinant GST-2xFYVE and CD63 antibodies, and co-stained with DAPI (F). Data represent the proportion of 2xFYVE+ late endosomes (n=3 experiments, >30 cells/experiment) (G). Arrows indicate co-localization between 2xFYVE and LAMP2.

H, I MCF-7 cells co-expressing GFP-INPP4B or GFP-vector, and Hrs or NT shRNA, were lysed and immunoblotted with LC3B antibodies, and GAPDH antibodies as a loading control (H). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to GFP-vector;NT shRNA cells which were assigned an arbitrary value of 1 (n=3 experiments) (I).

J INPP4B generates PI(3)P on late endosomes, where it is retained on endolysosomes following late endosome maturation.

Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, C, D, F. p values determined by one-way ANOVA with Tukey post hoc test in B, E, G, or by one-way ANOVA in I.

Source data are available online for this figure.
Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy (2022)
Figure 3

Figure 3: INPP4B promotes PI3Kα-dependent lysosome formation

A, B MCF-7 cells expressing GFP-INPP4B or GFP-vector were fixed and immunostained with LAMP1 or LAMP2 antibodies, and co-stained with DAPI and phalloidin (A). Data represent the number of LAMP1+ or LAMP2+ puncta relative to cell area (µm2) (n=3 experiments, >50 cells/experiment) (B).

C, D Snapshots of Magic Red cathepsin B substrate and Hoechst 33342 captured in live MCF-7 cells expressing GFP-INPP4B or GFP-vector (C). Data represent the number of Magic Red cathepsin B+ puncta per cell (n=3 experiments, >50 cells/experiment) (D).

E, F MCF-7 cells expressing NT or INPP4B #1 shRNA were cultured in growth media or EBSS for the indicated times, then fixed and immunostained with LAMP1 antibodies and co-stained with DAPI and phalloidin (E). Data represent the number of LAMP1+ puncta relative to cell area (µm2) (n=3 experiments, >30 cells per experiment) (F).

G, H MCF-7 cells expressing GFP-INPP4B or GFP-vector were transfected with NT, PIK3CA #1 or PIK3CA #2 siRNA. After 24 hours, cells were fixed and immunostained with LAMP1 antibodies, and co-stained with DAPI and phalloidin (G). Data represent the number of LAMP1+ puncta relative to cell area (µm2) (n=3 experiments, >40 cells per experiment) (H).

Data information: Data is presented as mean ± SD. Scale bar is 10 µm in A, C, E, G. p values determined by two-tailed unpaired t test in B, D, by one-way ANOVA with Tukey post hoc test in F, or by one-way ANOVA in H.

Source data are available online for this figure.
Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy (2022)
Figure 4

Figure 4: INPP4B promotes lysosome reformation

A, B Snapshots of LAMP1-GFP, Magic Red cathepsin B substrate and Hoechst 33342 captured in live HeLa cells transfected with INPP4B or NT siRNA (A). Data represent the number of LAMP1+/Magic Red Cathepsin B+ and LAMP1+/Magic Red Cathepsin B− lysosomes (terminal storage lysosomes) per cell (n=3 experiments, >50 cells/experiment) (B).Yellow arrows indicate LAMP1+/MR Cathepsin B+ lysosomes, and white arrows indicate terminal storage lysosomes.

C Timelapse snapshots of LAMP1-mCherry signals from MCF-7 cells expressing GFP-INPP4B or GFP-vector captured by spinning disk microscopy. Maximum intensity projections of 3 z-planes taken 0.27 µm apart are shown. Arrows indicate lysosome reformation.

D Overview of analysis workflow used to quantify lysosome reformation. Maximum intensity projections of lysosomes were converted to 3-dimensional kymographs using x, y and time dimensions, then segmented and skeletonized to track tubulation and fission events (depicted in red) that branch off the parent lysosome (depicted in black).

E Data represent the number of LAMP1+ reformation events per cell per minute (n=12 GFP-vector cells, n=15 GFP-INPP4B cells).

Data information: Data is presented as mean ± SD. The insets at the lower right of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, C. p values determined by two-way ANOVA with Holm-Sidak post-hoc test in B, or by two-tailed unpaired t test in E.

Source data are available online for this figure.
Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy (2022)
Figure 5

Figure 5: INPP4B promotes PIKfyve-dependent lysosome reformation and autophagic flux

A, B MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 5 µM YM201636 for 2 hours. Cells were fixed and immunostained with recombinant GST-2xFYVE and LAMP2 antibodies, and co-stained with DAPI (A). Data represent the proportion of 2xFYVE+ lysosomes (n=3 experiments, >30 cells/experiment) (B). Arrows show co-localization between 2xFYVE and LAMP2.

C INPP4B generates PI(3)P downstream of PI3Kα signaling, which is predicted to act as a substrate for phosphorylation to PI(3,5)P2 by PIKfyve.

D-F MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 5 µM YM201636 or DMSO as a vehicle control for 2 hours, then YM201636 was washed out for 1, 2 or 4 hours. Cells were fixed and immunostained with LAMP1 antibodies, and co-stained with DAPI and phalloidin (D). Data represent the number of LAMP1+ puncta relative to cell area (µm2) (E), and the lysosome fold change relative to the YM201636-treated condition for each cell line (F) (n=3 experiments, >30 cells per experiment).

G, H MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 1, 2 or 5 µM YM201636 or DMSO as a vehicle control for 4 hours. Cells were lysed and immunoblotted with LC3B antibodies, and GAPDH antibodies as a loading control (G). Data represent the relative LC3B-II levels normalized to GAPDH, and expressed relative to DMSO-treated GFP-vector cells which were assigned an arbitrary value of 1 (n=3 experiments) (H).

Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, D. p values determined by one-way ANOVA in B, E, H, or by two-tailed unpaired t test of the area under the curve in F.

Source data are available online for this figure.
Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy (2022)
Figure 6

Figure 6: SNX2 is a putative PI(3,5)P2 effector that promotes lysosome reformation

A, B MCF-7 cells expressing GFP-INPP4B or GFP-vector were transfected with NT, SNX1 or SNX2 siRNA. After 24 hours, cells were fixed and immunostained with LAMP2 antibodies, and co-stained with DAPI and phalloidin (A). Data represent the number of LAMP2+ puncta relative to cell area (µm2) (n=3 experiments, >40 cells per experiment) (B).

C Timelapse snapshots of LAMP1-mCherry and GFP-SNX2 signals from MCF-7 cells captured by spinning disk microscopy. Maximum intensity projections of 2 z-planes taken 0.27 µm apart are shown.

D, E MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated with 5 µM YM201636 for 2 hours, then YM201636 was washed out for 1 hour. Cells were fixed and immunostained with SNX2 and LAMP1 antibodies, and co-stained with DAPI (D). Data represent the proportion of SNX2+ LAMP2 puncta (n=3 experiments, >40 cells/experiment) (E). Arrows show co-localization between SNX2 and LAMP2.

F HeLa cells were transfected with GFP-SNX2. After 24 hours, cells were treated with 5 µM YM201636 for 2 hours, then YM201636 was washed out for 1 hour. Cells were fixed and subjected to immuno-electron microscopy analysis using GFP (10 nm) antibodies. Arrows show GFP-SNX2 localization to lysosome.

G HeLa cells were transfected with GFP-PIKfyve. After 24 hours, cells were were treated with 5 µM YM201636 for 2 hours, then YM201636 was washed out for 1 hour. Cells were fixed and immunostained with SNX2, GFP and LAMP2 antibodies, and imaged using super resolution microscopy. Arrows show co-localization between GFP-PIKfyve, SNX2 and LAMP2.

H MCF-7 cells expressing LAMP1-mCherry were transfected with NT or SNX2 siRNA. After 24 hours, cells were imaged by spinning disk microscopy for 20 minutes. Representative timelapse snapshots of LAMP1-mCherry from a maximum projection of 3 z-planes taken 0.27 µm apart are shown. Arrow indicates lysosome fission.

I Data represent the number of LAMP1+ reformation events per cell per minute (n=21 NT siRNA cells, n=27 SNX2 siRNA cells).

Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, C, D, G, H, and 100 nm in F. p values determined by one-way ANOVA in with Tukey post hoc test in B, E, or by two-tailed unpaired t test in I.

Source data are available online for this figure.
Endosome maturation links PI3Kα signaling to lysosome repopulation during basal autophagy (2022)
Figure 7

Figure 7: INPP4B and PIKfyve are required for proteotoxic stress response

A, B HeLa cells transfected with INPP4B or NT siRNA were treated for 1 hour with 5 µg/mL puromycin. Cells were fixed and immunostained with ubiquitin antibodies, and co-stained with DAPI and phalloidin (A). Data represent the percentage of cells with ubiquitinated protein aggregates (n=3 experiments, >200 cells/experiment) (B).

C, D HeLa cells were treated with 5 µM YM201636 or DMSO as a vehicle control for 2 hours, then 5 µg/mL puromycin for 1 hour. Cells were fixed and immunostained with ubiquitin antibodies, and co-stained with DAPI and phalloidin (C). Data represent the percentage of cells with ubiquitinated protein aggregates (n=3 experiments, >200 cells/experiment) (D).

E, F MCF-7 cells expressing GFP-INPP4B or GFP-vector were treated for 4 hours with 10 µg/mL puromycin, 5 µm MG132 and either 5 µM YM201636 or DMSO as a vehicle control. Cells were fixed and immunostained with ubiquitin antibodies, and co-stained with DAPI and phalloidin (E). Data represent the percentage of cells with ubiquitinated protein aggregates (n=3 experiments, >200 cells/experiment) (F).

G HeLa cells transfected with INPP4B or NT siRNA were treated with 10 µg/mL puromycin for 2-6 hours, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative cell viability normalized to untreated NT siRNA cells (n=3 experiments).

H HeLa cells were treated 10 µg/mL puromycin ± 5 µM YM201636 or DMSO as a vehicle control for 2-6 hours, then cell viability was assessed using CellTiter-Glo® assays. Data represent the relative cell viability normalized to DMSO-treated cells (n=3 experiments).

Data information: Data is presented as mean ± SD. The insets at the lower right or bottom of each image are higher power regions of the boxed areas. Scale bar is 10 µm in A, C, E. p values determined by one-way ANOVA in with Tukey post hoc test in B, D, F, by two-tailed unpaired t test of the area under the curve in G, or by one-way ANOVA with Tukey post hoc test of the area under the curve in H.

Source data are available online for this figure.