Nanobody-directed targeting of optogenetic tools to study signaling in the primary cilium
- Institute of Innate Immunity, Biophysical Imaging, Medical Faculty, University of Bonn, Germany
- Department of Molecular Sensory Systems, Center of Advanced European Studies and Research (caesar), Germany
- Center for Molecular Signaling (PZMS), Center of Human and Molecular Biology (ZHMB), Saarland University, School of Medicine, Germany
- Lehrstuhl für Biochemie, Universität Bayreuth, Germany
- Research Center for Bio-Macromolecules, Universität Bayreuth, Germany
- Bayreuth Center for Biochemistry & Molecular Biology, Universität Bayreuth, Germany
- Kavli Institute for Systems Neuroscience and Centre for Neural Computation, The Faculty of Medicine, Norwegian University of Science and Technology, Norway
- Department of Neurology and Clinical Neurophysiology, St. Olavs University Hospital, Norway
- Institute of Innate Immunity, Emmy Noether research group, Medical Faculty, University of Bonn, Germany
- Core Facility Nanobodies, University of Bonn, Germany
- Research Group Molecular Physiology, Center of Advanced European Studies and Research (caesar), Germany
Figures
Figure 1
Direct ciliary targeting of optogenetic tools impairs protein function.
(A) Localization of mNphp3(201)-bPAC-mCherry to primary cilia. mIMCD-3 cells expressing mNphp3(201)-bPAC-mCherry were labeled with an anti-acetylated tubulin antibody (cyan, ciliary marker) and with …
Figure 2 with 3 supplements
Targeting optogenetic tools to the primary cilium using nanobodies.
(A) Schematic overview of the targeting approach. Nanobodies were fused to the C terminus of mNphp3(201) for ciliary localization. The protein of interest (POI) is co-expressed with a C-terminal tag …
Figure 2—figure supplement 1
Nanobody-based ciliary targeting.
(A) Localization of the anti-mCherry nanobody to primary cilia. mIMCD-3 cells were transfected with mNphp3(201)-VHHLaM-4-eGFP (green) and stained with an acetylated tubulin antibody (cyan, ciliary …
Figure 2—figure supplement 2
Subcelluar localization of nanobody-targeted optogenetic tools.
(A) HEK-TM cells expressing bPAC. HEK-TM cells stably expressing bPAC-mCherry (red) were fixed and labeled with DAPI (blue). Scale bar: 20 μm. (B) See A. for LAPD-mCherry. (C) HEK-TM cells …
Figure 2—figure supplement 3
Activity measurements in HEK-TM cells.
(A) bPAC activity measurements in HEK-TM cells. HEK293 cells express the CNGA2-TM ion channel, which opens upon cAMP binding and conducts Ca2+ (HEK-TM). bPAC-mCherry was co-expressed with the …
Figure 3
Functional characterization of bPAC in the cell body and cilium.
(A) Schematic overview of the bPAC activity assay in non-ciliated HEK293 cells using R-FlincA (see B-C). (B) HEK293 cells were transfected with bPAC-eGFP and R-FlincA or the non-binding R-FlincA …
Figure 4 with 1 supplement
Functional characterization of nanobody-targeted cAMP biosensor.
(A) Localization of the mNphp3(201)-VHHenhancer-mCherry anti-eGFP nanobody to primary cilia. (B./C) Localization of mlCNBD-FRET in mIMCD-3 cells in the (B) absence or (C) presence of mNPHP3(201)-VHHe…
Figure 4—figure supplement 1
Characterization of the ciliary-targeted cAMP mlCNBD-FRET biosensor.
(A) Localization of mNphp3(201)-mlCNBD-FRET to primary cilia in mIMCD-3 cells. (B) FRET imaging in non-ciliated HEK293 cells expressing mlCNBD-FRET or mNphp3(201)-mlCNBD-FRET under control …
Figure 5 with 1 supplement
Nanobody-based ciliary protein targeting in vivo.
(A) Nanobody localization in the neural tube of a zebrafish embryo. The mRNA of the anti-mCherry mNphp3(201)-VHHLaM-2-eGFP nanobody was injected into nacre zebrafish embryos. Embryos were stained …
Figure 5—figure supplement 1
Nanobody-based ciliary protein targeting in vivo.
(A) Nanobody localization in the neural tube of a zebrafish embryo. The mRNA of the anti-mCherry mNphp3(201)-VHHLaM-2-eGFP (green) nanobody was injected into nacre zebrafish embryos. (B) Ciliary …
Figure 6 with 1 supplement
Controlling cilia length using optogenetics.
(A) Cilia length of mIMCD-3 cells stimulated for 1 hr with 10 μM Forskolin (solvent: DMSO), normalized to the DMSO control. Data are shown as mean ± S.D., n = 3 with at least 40 cells per …
Figure 6—figure supplement 1
cAMP levels and ciliary length in mIMCD-3 cells expressing LAPD.
(A) Determination of total cAMP levels in WT mIMCD-3 cells or mIMCD-3 LAPD-mCherry cells in the dark or after light stimulation (2 min, 630 nm, 42.3 µW/cm2). cAMP levels have been determined using …
Videos
Video 1
FRET imaging in primary cilia.
mIMCD-3 cells expressing mlCNBD-FRET and mNphp3(201)-VHHenhancer-mCherry have been stimulated with 250 μm IBMX or buffer only.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (mouse) | Nphp3 | NCBI | NM_028721 | |
| Cell line (human) | HEK293 | ATCC | CRL-1573 | |
| Cell line (human) | HEK-TM | Wachten et al., 2006 | ||
| Cell line (mouse) | mIMCD-3 | ATCC | CRL-2123 | |
| Transfected construct (mouse) | See Sup. Table 1 | See Sup. Table 1 | See Sup. Table 1 | |
| Biological sample (Danio rerio) | nacre | Lister et al., 1999 | ||
| Biological sample (Danio rerio) | b-actin:arl13b-gfp | Borovina et al., 2010 | ||
| Biological sample (Danio rerio) | Ubi:zebrabow | Pan et al., 2013 | ||
| Antibody (mouse) | anti-acetylated-Tubulin (mouse, monoclonal) | Sigma Aldrich | T6793 | 1:600 |
| Antibody (rabbit) | anti-GFP (rabbit, polyclonal) | Abcam | ab6556 | 1:500 |
| Antibody (rabbit) | anti-Arl13B (rabbit, polyclonal) | Proteintech | 17711–1-AP | 1:500 |
| Antibody (mouse) | anti-Arl13B (mouse, monoclonal) | Abcam | ab136648 | 1:500 |
| Antibody (donkey) | anti-mouse-Cy5 (donkey) | Dianova | 715-175-151 | 1:500 |
| Antibody (goat) | anti-rabbit-Alexa488 (goat) | Life Technologies | A11034 | 1:500 |
| Recombinant DNA reagent | cADDis cAMP assay kit | Montana Molecular | #D0201/11G | |
| Sequence-based reagent (IDT) | VHHLaM-2, VHHLaM-4 | Gene blocks (IDT) Fridy et al., 2014 | ||
| Sequence-based reagent (Ploegh lab) | VHHEnhancer | Hidde Ploegh, Boston, USA Kirchhofer et al., 2010 | ||
| Commercial assay or kit (Thermo) | Lipofectamine 2000 | Thermo Fisher Scientific | #11668030 | |
| Commercial assay or kit (Molecular Devices) | CatchPoint assay | Molecular Devices | ||
| Commercial assay or kit (Thermo) | Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | 23227 | |
| Commercial assay or kit (Qiagen) | QIAquick PCR purification kit | Qiagen | #28104 | |
| Commercial assay or kit (Thermo) | mMessage mMachine T7 kit | Thermo Fisher Scientific | #AM1344 | |
| Chemical compound, drug (Sigma) | PEI | Sigma Aldrich | #64604–1G | |
| Chemical compound, drug (Merck) | ChemiBLOCKER | Merck Millipore | #2170 | |
| Chemical compound, drug (Polysciences) | Aqua-Poly/Mount | Polysciences | #18606–20 | |
| Chemical compound, drug (Thermo) | DAPI | Thermo Fisher Scientific | D1306 | 1:10.000 |
| Chemical compound, drug (Enzo) | FluoForte | Enzo Life Sciences | ENZ-52015 | |
| Chemical compound, drug (Sigma) | Fluo4-AM | Sigma Aldrich | 93596 | |
| Chemical compound, drug (Thermo) | probenecid | Thermo Fisher Scientific | P36400 | |
| Chemical compound, drug (Sigma) | NKH477 | Sigma Aldrich | N3290 | |
| Chemical compound, drug (Sigma) | Forskolin | Sigma Aldrich | F3917 | |
| Chemical compound, drug (Sigma) | IBMX | Sigma Aldrich | I5879 | |
| Software, algorithm (this paper) | CiliaQ | This publication | https://github.com/hansenjn/CiliaQ; Hansen, 2020; copy archived at https://github.com/elifesciences-publications/CiliaQ |
Additional files
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Supplementary file 1
Plasmids and cloning information.
Plasmids are listed and the ID and sequence of the primers that have been used for cloning are indicated.
- https://cdn.elifesciences.org/articles/57907/elife-57907-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/57907/elife-57907-transrepform-v2.pdf
