Molecular structures and conformations of protocadherin-15 and its complexes on stereocilia elucidated by cryo-electron tomography
- Vollum Institute, Oregon Health and Science University, United States
- RNA Therapeutics Institute, UMass Chan Medical School, United States
- Howard Hughes Medical Institute, United States
Figures
Figure 1
Cryo-EM data collection of antibody-stained stereocilia tips supported by cryo-CLEM.
(A) Immunostaining of WT cochlea or PCDH15 KO with polyclonal antibody raised in rabbits against PCDH15 extracellular domain. Scale bars correspond to 10 µm. (B) Immunostaining of WT utricle with …
Figure 2 with 1 supplement
Stoichiometric gold staining of PCDH15 using a monoclonal Fab AuNP conjugate.
(A) FSEC analysis of binding of 39G7 to the PCDH15 extracellular domain. 39G7 addition leads to earlier elution of PCDH15 in full-length constructs and after deletion of EC1 and EC2. Deletion of EC3 …
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Figure 2—source data 1
Original image of polyacrylamide gel shown in Figure 2C.
- https://cdn.elifesciences.org/articles/74512/elife-74512-fig2-data1-v2.zip
Figure 2—video 1
Tomogram of recombinant PCDH15 extracellular domain in complex with 39G7-AuNP conjugate.
Figure 3 with 4 supplements
39G7-AuNP conjugate labels PCDH15 dimer in stereocilia.
(A–D) Representative examples of PCDH15 dimers with two bound AuNPs (A, B) or one bound AuNP (C, D) image in stereocilia. On the left-hand side of each panel, a manually annotate model is shown in a …
Figure 3—video 1
.Tomogram of stereocilium displaying a PCDH15 dimer with two bound 39G7-AuNP conjugates, also depicted in Figure 3A.
Figure 3—video 2
Tomogram of stereocilium displaying a PCDH15 dimer with two bound 39G7-AuNP conjugates, also depicted in Figure 3B.
Figure 3—video 3
Tomogram of stereocilium displaying a PCDH15 dimer with one bound 39G7-AuNP conjugates also depicted in Figure 3C.
Figure 3—video 4
Tomogram of stereocilium displaying a PCDH15 dimer with one bound 39G7-AuNP conjugates, also depicted in Figure 3D.
Figure 4 with 5 supplements
Stereocilia tips frequently harbor more than one copy of PCDH15.
(A) Representative example of stereocilia tip with PCDH15 molecule only found in adjacent shaft. (B, C) Representative examples of stereocilia tips with one copy of PCDH15 at tip. (D, E) …
Figure 4—video 1
Tomogram of stereocilium tip with a single PCDH15 dimer in the shaft region of the tip, also depicted in Figure 4A.
Figure 4—video 2
Tomogram of stereocilium tip with a single PCDH15 dimer at the apex of the tip, also depicted in Figure 4B.
Figure 4—video 3
Tomogram of stereocilium tip with a single PCDH15 dimer at the apex of the tip, also depicted in Figure 4C.
Figure 4—video 4
Tomogram of stereocilium tip with a cluster of multiple PCDH15 dimers at the apex of the tip, also depicted in Figure 4D.
Figure 4—video 5
Tomogram of stereocilium tip with multiple PCDH15 dimers scattered around the tip, also depicted in Figure 4E.
Figure 5 with 4 supplements
PCDH15 complexes are found in clusters.
On the left side of each panel, the tomogram annotation is shown from the side and from top. On the sideview ice thickness is indicated. To the right of the annotation is a projection of the cluster …
Figure 5—video 1
Tomogram of stereocilium tip containing multiple PCDH15 dimers connected to putative CDH23 filaments in a lipid vesicle, also depicted in Figure 5A.
Figure 5—video 2
Tomogram of stereocilium tip containing multiple PCDH15 dimers connected to putative CDH23 filaments in a lipid membrane fragment, also depicted in Figure 5B.
Figure 5—video 3
Tomogram of a stereocilium tip containing multiple putative CDH23 filaments connected to PCDH15 dimers in a lipid vesicle, also depicted in Figure 5C.
Figure 5—video 4
Tomogram of a stereocilium shaft containing multiple putative CDH23 filaments connected to PCDH15 dimers in a lipid vesicle, also depicted in Figure 5D.
Figure 6 with 1 supplement
Tomogram containing intact tip link.
(A) Annotation of tomogram showing a putative intact tip link. The inset at the top left shows a projection of the tomogram. (B) Close-up view of putative tip link density in the tomogram. (C) …
Figure 6—video 1
Tomogram of a stereocilium tip and a stereocilium shaft containing a PCDH15 dimer in the stereocilium tip that is connected to a putative CDH23 filament in the neighboring stereocilium shaft.
The tip also contains PCDH15 dimers not bound to a complex partner. An additional PCDH15-complex is connecting the two stereocilia in the shaft region. Also depicted in Figure 6.
Figure 7 with 1 supplement
Tomogram depicting lateral links containing PCDH15 in small stereocilia.
(A) Annotation of tomogram showing developing stereocilia including PCDH15-containing ‘lateral links.’ The inset at the bottom right shows a projection of the tomogram. (B, C) Close-up of putative …
Figure 7—video 1
Tomogram of stereocilia connected by tens of PCDH15-containing complexes.
Noteworthy complexes that include apparent interaction between putative CDH23 dimers, as well as bends in the putative CDH23 dimer are highlighted. Also depicted in Figure 7.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | Pcdh15 | Uniprot | Q99PJ1 | |
| Cell line (Spodoptera frugiperda) | Sf9 | Thermo Fisher Scientific | 12659017 RRID:CVCL_0549 | |
| Cell line (Homo sapiens) | HEK293 tsa 201 | ATCC | CRL-11268RRID:CVCL_1926 | |
| Recombinant DNA reagent | PCDH15 EC1-EL-YFP expression plasmid | This paper | Created using pEG BacMam (Addgene plasmid # 160451) | |
| Recombinant DNA reagent | 39G7 Fab’ expression plasmid | This paper (created by VectorBuilder) | Backbone described in d oi:10/fhwrn3 | |
| Antibody | Anti-PCDH15 (Rabbit polyclonal) | This paper (created by Genscript) | IF: 10 µg/ml | |
| Antibody | Anti-PCDH15 39G7 (Rabbit monoclonal) | This paper (created by Genscript) | IF: 10 µg/ml | |
| Biological sample (M. musculus) | Inner ear | P6–P9 mice | ||
| Chemical compound, drug | 10 nm gold fiducials | Ted Pella | 90010 | |
| Chemical compound, drug | HAuCL4 | Sigma-Aldrich | 520918 | |
| Chemical compound, drug | 3-MBA | Sigma-Aldrich | 451436 | |
| Chemical compound, drug | mPEG-550-SH | Creative PEGworks | PLS-607 | |
| Software, algorithm | SerialEM | 10.1016 /j.jsb.2005.07.007 | RRID:SCR_017293 | |
| Software, algorithm | IMOD | doi.org.10.1006/jsbi.1996.0013 | RRID:SCR_003297 | |
| Software, algorithm | Topaz | doi.org.10.1038/s41467-020-18952-1 | ||
| Software, algorithm | TomoAlign | doi.org.10.1016/j.jsb.2019.01.005 | ||
| Software, algorithm | Motioncor2 | doi.org.10.1038/nmeth.4193 | RRID:SCR_016499 | |
| Software, algorithm | ChimeraX | doi.org.10.1002/pro.3943 | RRID:SCR_015872 |
