Targeting an anchored phosphatase-deacetylase unit restores renal ciliary homeostasis
- Department of Pharmacology, University of Washington, United States
- Kidney Research Institute, Division of Nephrology, Department of Laboratory Medicine and Pathology, University of Washington, United States
- Institute for Stem Cell and Regenerative Medicine, University of Washington, United States
Figures
Figure 1 with 3 supplements
Loss of AKAP220 enhances ciliogenesis.
(A) Schematic of AKAP220 interaction with selected binding partners. (B) Disruption of this signaling complex upon removal of AKAP220. Protein kinase A (blue), Glycogen synthase kinase-3 (pink), …
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Figure 1—source data 1
Percent ciliated collecting ducts in kidney sections.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig1-data1-v2.xlsx
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Figure 1—source data 2
Length of primary cilia in kidney sections.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig1-data2-v2.xlsx
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Figure 1—source data 3
Rescue of GFP-AKAP220 in AKAP220KO mIMCD3 cells.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig1-data3-v2.xlsx
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Figure 1—source data 4
Percent ciliated cells measured in spheroids.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig1-data4-v2.xlsx
Figure 1—figure supplement 1
Quantification of cilia number in gene-edited mIMCD3 cells.
Immunofluorescent staining of Arl13b (red), acetyl tubulin (green), and DAPI (blue) in serum starved (0.5% FBS, 24 hr) (A) wild-type and (D) AKAP220KO mIMCD3 cells. Gray scale images of Arl13b (B …
Figure 1—figure supplement 2
Deletion of AKAP150 has no effect on primary cilia development.
Crispr-Cas nine gene editing was used to delete the murine anchoring protein AKAP150 in mIMCD3 cells. Double knockout cells were also produced lacking AKAP220 and AKAP150. Immunofluorescent staining …
Figure 1—figure supplement 3
Sequencing data for CRISPR-Cas9 gene edited mIMCD3 cells.
Sequencing analysis data shows (A) intact AKAP150 in wild-type AKAP150, and deletions of AKAP150 in (B) AKAP150KO cells and (C) AKAP220-150 double knockout cells made in AKAP220KO background.
Figure 2 with 2 supplements
AKAP220 influences tubulin deacetylation.
Immunofluorescent detection of acetyl tubulin (green) and DAPI (blue) in (A) wild-type and (B) AKAP220KO mIMCD3 cells. (C) Immunoblot detection of acetylated tubulin (top), alpha tubulin (mid) and …
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Figure 2—source data 1
Densitometry of acetylated tubulin western blots.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig2-data1-v2.xlsx
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Figure 2—source data 2
Densitometry of HDAC6 western blots.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig2-data2-v2.xlsx
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Figure 2—source data 3
Densitometry of HDAC6 in cycloheximide-chase experiments.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig2-data3-v2.xlsx
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Figure 2—source data 4
Percent ciliated AKAP220KO cells transfected with Flag-HDAC6 or HDAC6.DC.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig2-data4-v2.xlsx
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Figure 2—source data 5
Quantification of puncta in proximity ligation assay.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig2-data5-v2.xlsx
Figure 2—figure supplement 1
Further characterization of AKAP220-HDAC6 interface.
HDAC6 accumulates at the base of primary cilia. Immunofluorescent detection of acetyl tubulin (green), HDAC6 (red), and DAPI (blue) in (A) wild-type and (C) AKAP220KO mIMCD3 cells. Gray scale images …
Figure 2—figure supplement 2
Additional Proximity ligation (PLA) controls.
Proximity ligation (PLA) detection of V5-AKAP220/HDAC6 subcomplexes (pink), DAPI (blue) in cells expressing GFP (green) as a transfection marker in mIMCD3 cells (A) transfected with V5-AKAP220 only …
Figure 3 with 4 supplements
Anchored protein phosphatase one is necessary for HDAC6 activity.
(A) Schematic of AKAP220-ΔPP1. Binding partners are indicated. Gene editing deleted the principal phosphatase binding site (KVQF) on AKAP220. (B) Nucleotide sequencing reveals substitution of the …
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Figure 3—source data 1
Percent ciliated DMSO or tubacin-treated mIMCD3 cells.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Characterization of AKAP220-ΔPP1 cell line.
CRISPR-Cas9 gene editing was used to substitute the PP1-targeting KVQF motif in AKAP220 gene to generate AKAP220-ΔPP1 mIMCD3 cells. Sequencing analysis data shows (A) wild-type PP1-binding region in …
Figure 3—figure supplement 2
Investigating if AKAP220-HDAC6 interaction is altered in the AKAP220-ΔPP1 cells.
(A) Western blot of phosphorylated HDAC6 (top panel) in wild-type (lane 1) and AKAP220-ΔPP1 (lane 2) mIMCD3 cells. Ponceau blot (bottom panel) depicts equal loading. (B) Western blot of HDAC6 (top …
Figure 3—figure supplement 3
Serum starvation drives mutant cells to multiciliate.
(A-D) Immunofluorescent staining of Arl13b (red), acetyl tubulin (green), and DAPI (blue) in serum starved (0.5% FBS, 24 hr) (A) AKAP220-ΔPP1 mIMCD3 cells. Gray scale images of (B) Arl13b and (C) …
Figure 3—figure supplement 4
Characterizing tubacin action on cilia number in AKAP220KO cells.
Immunofluorescent staining with ciliary markers acetyl tubulin (green) and Arl13b (red) in (A) DMSO and (B) tubacin-treated AKAP220KO cells. DAPI (blue) serves as a nuclear marker. (C) Expanded gray …
Figure 4 with 1 supplement
Disrupting the AKAP220-PP1 subcomplex impacts actin reorganization.
Gray scale images of acetylated cortactin in (A) wild-type, (C) AKAP220KO, and (E) AKAP220-ΔPP1 mIMCD3 cells. Magnified images of nuclei (top) and three-dimensional surface plots (bottom) of …
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Figure 4—source data 1
FRAP curves for lifeact-GFP in mIMCD3 cells.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig4-data1-v2.xlsx
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Figure 4—source data 2
Half-life measurements for lifeact-GFP in mIMCD3 cells.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig4-data2-v2.xlsx
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Figure 4—source data 3
Mobile fraction measurements for lifeact-GFP in mIMCD3 cells.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig4-data3-v2.xlsx
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Figure 4—source data 4
Intensity of actin in mIMCD3 cells by line plot analysis.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig4-data4-v2.xlsx
Figure 4—figure supplement 1
Distribution of actin in confluent mIMCD3 cell culture.
Immunofluorescent detection of actin (cyan) and DAPI (blue) in (A) wild-type and (C) AKAP220KO mIMCD3 cells. Gray scale images of actin in (B) wild-type and (D) AKAP220KO cells show the distribution …
Figure 5 with 1 supplement
Cilia frequency and development involve f-actin assembly.
(A) Schematic of how actin modulating drugs impact primary cilia. Cytochalasin D depolymerizes actin barriers. Jasplakinolide stabilizes actin filaments. (B–F) Immunofluorescent detection of actin …
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Figure 5—source data 1
Percent ciliated in DMSO or Cytochalasin-D-treated wild-type cells.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig5-data1-v2.xlsx
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Figure 5—source data 2
Cilia length in in DMSO or Cytochalasin-D-treated AKAP220KO cells.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig5-data2-v2.xlsx
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Figure 5—source data 3
Percent ciliated in DMSO or Jasplakinolide-treated AKAP220KO cells.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig5-data3-v2.xlsx
Figure 5—figure supplement 1
Characterizing the effect of actin-modulating drugs on AKAP220-ΔPP1 cilia.
(A-G) Immunofluorescent detection of actin (white), Arl13b (pink), and DAPI (blue) in (A) DMSO and (D) 200 nM Cytochalasi- D-treated AKAP220-ΔPP1 mIMCD3 cells. (Inset) Expanded field of cells …
Figure 6 with 1 supplement
Loss of phosphatase anchoring promotes cilium elongation.
(A) Schematic of cylindrical and retracted cilia morphologies (purple). Super-resolution fixed-cell imaging of Arl13b (gray scale) in (B) wild-type, (C) AKAP220KO, and (D) AKAP220-ΔPP1 cilia. (E) …
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Figure 6—source data 1
Primary cilia length from live cell experiments in mIMCD3 cells.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig6-data1-v2.xlsx
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Figure 6—source data 2
Length of primary cilia in kidney on a chip device.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig6-data2-v2.xlsx
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Figure 6—source data 3
FRAP curved for Arl13b-GFP in mIMCD3 cell lines.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig6-data3-v2.xlsx
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Figure 6—source data 4
Mobile fraction of Arl13b-GFP 3 s after photobleaching.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig6-data4-v2.xlsx
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Figure 6—source data 5
Half-life of Arl13b-GFP in mIMCD3 cell lines.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig6-data5-v2.xlsx
Figure 6—figure supplement 1
Super resolution videos depicting flexibility of AKAP220KO cilia.
(A) Wild-type and (B) AKAP220KO primary cilia from mIMCD3 cells transduced with Arl13b-GFP were monitored across a time course of 4 s. The AKAP220 cilium is longer and more flexible compared to the …
Figure 7
Inactivating HDAC6 reduces renal cystogenesis.
Schematic of polycystic kidneys (A) before and (B) after tubacin treatment. (Insets) Tubacin action on the AKAP220-signaling complex in precystic PKD2-/- organoids derived from WA-09 cells. (C-J) …
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Figure 7—source data 1
Quantification of cyst size in DMSO and tubacin-treated iPSCs.
- https://cdn.elifesciences.org/articles/67828/elife-67828-fig7-data1-v2.xlsx
Author response image 1
Inhibition of HDAC6 enhances acetylated cortactin in mIMCD3 cells.
Immunofluorescence images of acetylated cortactin in DMSO or Tubacin-treated (A and B) wildtype, C and (D) AKAP220KO and (E and F) AKAP220-ΔPP1 mIMCD3 cells. Insets depict marked nucleus (yellow …
Videos
Video 1
Recovery of lifeact-GFP upon photobleaching in wild-type mIMCD3 cells.
Video 2
Recovery of lifeact-GFP upon photobleaching in AKAP220KO mIMCD3 cells.
Video 3
Recovery of lifeact-GFP upon photobleaching in AKAP220-ΔPP1 mIMCD3 cells.
Video 4
Recovery of Arl13b-GFP upon photobleaching in wild-type mIMCD3 cells.
Video 5
Recovery of Arl13b-GFP upon photobleaching in AKAP220KO mIMCD3 cells.
Video 6
Recovery of Arl13b-GFP upon photobleaching in AKAP220-ΔPP1 mIMCD3 cells.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Acetylated tubulin | Thermo Fisher | Mouse monoclonal 32–2700 RRID:AB_2533073 | IF(1:5000), WB(1:1000) |
| Antibody | ActinRed-555 | Molecular probes | R37112 | Manufacturer instructions |
| Antibody | Arl13b | Proteintech | Rabbit Polyclonal 17711–1-AP RRID:AB_2060867 | IF(1:5000) |
| Antibody | AKAP220 | Santa Cruz Biotech | Goat polyclonal No longer available | IF (1:500) |
| Antibody | AKAP220 | Rockland | Rabbit polyclonal Custom generated | WB (1:500) |
| Antibody | Amersham ECL Mouse IgG, HRP-linked F(ab')₂ fragment (from sheep) | GE Life Sciences | NA9310 RRID:AB_772193 | WB (1:10000) |
| Antibody | Amersham ECL Rabbit IgG, HRP-linked F(ab')₂ fragment (from donkey) | GE Life Sciences | NA9340 RRID:AB_772191 | WB (1:10000) |
| Antibody | Donkey anti-goat IgG-HRP | Santa Cruz | sc-2020 RRID:AB_631728 | WB (1:10000) |
| Antibody | Donkey anti-Mouse IgG, Alexa Fluor 488 | Invitrogen | A-21202 RRID:AB_141607 | IF (1:500) |
| Antibody | Donkey anti-Mouse IgG, Alexa Fluor 555 | Invitrogen | A-31570 RRID:AB_2536180 | IF (1:500) |
| Antibody | Donkey anti-Mouse IgG, Alexa Fluor 647 | Invitrogen | A-11126 RRID:AB_221538 | IF (1:500) |
| Antibody | Donkey anti-Rabbit IgG, Alexa Fluor 488 | Invitrogen | A-21206 RRID:AB_2535792 | IF (1:500) |
| Antibody | Donkey anti-Rabbit IgG, Alexa Fluor 555 | Invitrogen | A-31572 RRID:AB_162543 | IF (1:500) |
| Antibody | Donkey anti-Rabbit IgG, Alexa Fluor 647 | Invitrogen | A-31573 RRID:AB_2536183 | IF (1:500) |
| Antibody | GFP | Rockland | 600-101-215 goat polyclonal RRID:AB_218182 | WB (1:1000), IF (1:500) |
| Antibody | HDAC6 | Proteintech | 12834–1-AP Rabbit Polyclonal RRID:AB_10597094 | IF (1:200) |
| Antibody | V5 | Invitrogen | R960-25 Mouse monoclonal RRID:AB_2556564 | IF (1:500) |
| Cell line (H. sapein) | HEK293 | RRID:CVCL_0045 | Maintained in Scott lab in DMEM supplemented with 10% FBS | |
| Cell line (M. musculus) | mIMCD3 | RRID:CVCL_0429 | Maintained in Scott lab in DMEM/F12 supplemented with 10% FBS | |
| Chemical compound, drug | DAPI | Thermo Fisher | 62248 RRID:AB_2307445 | IF (1:1000) |
| Chemical compound, drug | Dimethylsulfoxide (DMSO) | Pierce | TS-20688 | Manufacturer’s instructions |
| Chemical compound, drug | DMEM FluoroBrite | Life Technologies | A1896701 | |
| Chemical compound, drug | DMEM/F-12 Hepes | Life Technologies | 11330057 | |
| Chemical compound, drug | DMEM, high glucose | Life Technologies | 11965118 | |
| Chemical compound, drug | Fetal Bovine Serum | Thermo Fisher | A3382001 | |
| Chemical compound, drug | Lipofectamine 2000 Transfection Reagent | Invitrogen | 11668027 | |
| Chemical compound, drug | NuPAGE LDS Sample Buffer 4X | Thermo Fisher | NP0008 | |
| Chemical compound, drug | Opti-MEM, Reduced Serum Medium, no phenol red | Life Technologies | 11058021 | |
| Chemical compound, drug | ProLong Diamond Antifade Mountant | Life Technologies | P36961 RRID:SCR_015961 | Manufacturer’s instructions |
| Chemical compound, drug | Polybrene | Santa Cruz | 134220 | Manufacturer’s instructions |
| Chemical compound, drug | Super Signal West Dura Extended Duration Substrate | Thermo Fisher | 34075 | |
| Chemical compound, drug | TransIT-LT1 Transfection Reagent | Mirus | MIR2300 | |
| Chemical compound, drug | Trypsin-EDTA (0.25%), phenol red | Gibco | 25200056 | |
| Commercial assay or kit | BCA Protein Assay Kit | Thermo Fisher | 23227 | |
| Recombinant DNA reagent | pMD2.G | RRID:Addgene_12259 | gift from Didier Trono; Addgene plasmid #12259 | |
| Recombinant DNA reagent | psPAX2 | RRID:Addgene_12260 | gift from Didier Trono; Addgene plasmid #12259 | |
| Recombinant DNA reagent | Arl13b-GFP | RRID:Addgene_40879 | gift from Tamara Caspary; Addgene plasmid # 40879 | |
| Recombinant DNA reagent | lifeact-GFP | RRID:Addgene_58470 | gift from Dyche Mullins; Addgene plasmid # 58470 | |
| Recombinant DNA reagent | pcDNA-HDAC6-flag | RRID:Addgene_30482 | gift from Tso-Pang Yao; Addgene plasmid # 30482 | |
| Recombinant DNA reagent | pEGFP-HDAC6-DC | RRID:Addgene_36189 | gift from Tso-Pang Yao; Addgene plasmid # 36189 | |
| Software, algorithm | Fiji/ImageJ | ImageJ (http://imagej.nih.gov/ij/) | ||
| Software, algorithm | GraphPad Prism | GraphPad Prism (https://graphpad.com) | ||
| Software, algorithm | SoftWoRx | GE Healthcare | ||
| Other | Bolt 4–12% Bis-Tris Plus Gels | Invitrogen | NW04120BOX |
Additional files
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Source data 1
Original blots included in the manuscript.
- https://cdn.elifesciences.org/articles/67828/elife-67828-data1-v2.zip
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Source data 2
Blots with legends added.
- https://cdn.elifesciences.org/articles/67828/elife-67828-data2-v2.zip
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Transparent reporting form
- https://cdn.elifesciences.org/articles/67828/elife-67828-transrepform-v2.pdf
