Dataset Information

Affinity purification of in vivo assembled whirlin-associated protein complexes from the zebrafish retina - GST

ABSTRACT: Mutations in WHRN lead to Usher syndrome type 2d (USH2d) or to non-syndromic hearing impairment (DFNB31). The WHRN-encoded gene product whirlin directly interacts with the intracellular regions of the other two Usher syndrome type 2 (USH2)-associated proteins, usherin and ADGRV1. Together with PDZD7, these proteins form the transiently present ankle link complex necessary for maturation of the inner ear hair bundle. In photoreceptors, usherin and ADGRV1 are also scaffolded by whirlin and constitute similar fibrous links between the periciliary membrane and the connecting cilium. However, the molecular mechanism of retinal degeneration as a consequence of compromised USH2-associated protein complex formation and function remains elusive. To better understand the function of the USH2-associated proteins in the photoreceptors, we aimed to isolate and characterize whirlin-associated protein complexes from zebrafish photoreceptor cells. We generated transgenic zebrafish that express Strep/FLAG-tagged Whrna, a zebrafish ortholog of human whirlin, under the control of a photoreceptor-specific promoter. Affinity purification of Strep/FLAG-tagged Whrna and associated proteins from adult transgenic zebrafish retinas followed by mass spectrometry analysis identified 19 novel candidate interaction partners. Pull down experiments and dedicated yeast two-hybrid assays confirmed the association of Whrna with 7 of the co-purified proteins. Several of the co-purified proteins are part of the synaptic proteome, which suggest a role for whirlin in the photoreceptor synapse. Future studies will elucidate which of the identified protein-protein interactions contribute to the development of the retinal phenotype observed in USH2d patients.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Danio Rerio (zebrafish) (brachydanio Rerio)

TISSUE(S): Layer Of Retina, Retina

DISEASE(S): Disease Free

SUBMITTER: Karsten Boldt

LAB HEAD: Dr. Karsten Boldt

PROVIDER: PXD034247 | Pride | 2023-12-28

REPOSITORIES: Pride

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Publications

Affinity purification of in vivo assembled whirlin-associated protein complexes from the zebrafish retina.

Schellens R T W RTW Slijkerman R W N RWN Hetterschijt L L Peters T A TA Broekman S S Clemént A A Westerfield M M Phillips J B JB Boldt K K Kremer H H De Vrieze E E Van Wijk E E

Journal of proteomics 20220702

Mutations in WHRN lead to Usher syndrome type 2d or to non-syndromic hearing impairment. The WHRN-encoded gene product whirlin directly interacts with the intracellular regions of the other two Usher syndrome type 2-associated proteins, usherin and ADGRV1. In photoreceptor cells, this protein complex constitutes fibrous links between the periciliary membrane and the connecting cilium. However, the molecular mechanism(s) of retinal degeneration due to compromised formation and function of the USH ...[more]

PMID: 35788411

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Project description:Purpose. XOPS-mCFP transgenic zebrafish experience a continual cycle of rod photoreceptor development and degeneration throughout life, making them a useful model to investigate the molecular determinants of rod photoreceptor regeneration. The purpose of this study was to compare the gene expression profiles of wild type and XOPS-mCFP retinas in order to identify genes that may contribute to the regeneration of the rods. Methods. Wild type and XOPS-mCFP retinal mRNA was subjected to microarray analysis using the Agilent platform. The Ingenuity Pathway Analysis program was used to identify biologically relevant processes that were significantly represented in the dataset. Expression changes were verified by RT-PCR. Selected genes were further examined during retinal development and in adult retinas by in situ hybridization, immunohistochemistry, and using a transgenic fluorescent reporter line. Results. Over 600 genes displayed significant expression changes in XOPS-mCFP retinas compared to wild type controls. Many of the downregulated genes were associated with phototransduction, whereas upregulated genes were associated with several biological functions, including cell cycle, DNA replication and repair, cell development and cell death. RT-PCR analysis of a subset of these genes confirmed the microarray results. Three transcription factors (sox11b, insm1a, and c-myb) displaying increased expression in XOPS-mCFP retinas were also expressed throughout retinal development and in the persistently neurogenic ciliary marginal zone. Conclusions. This study identified numerous gene expression changes in response to rod degeneration in zebrafish, and further suggests a role for the transcriptional regulators sox11b, insm1a, and c-myb in both retinal development and rod photoreceptor regeneration. Two-condition experiment: wild-type vs. XOPS-mCFP retinas. Four biological replicates for each condition. RNA was prepared from one retina for each sample. Each hybridization was accompanied by a dye-swap control, for a total of eight array hybridizations.

Dataset Information

Affinity purification of in vivo assembled whirlin-associated protein complexes from the zebrafish retina - GST

Publications

Affinity purification of in vivo assembled whirlin-associated protein complexes from the zebrafish retina.

Similar Datasets

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