Holm, M. S. | bioRxiv Science

bioRxiv2024

Depletion of BBSome Subunits Alters Receptor Endocytosis and Promotes EMT via TGF-β Signaling

Background: Ciliopathies are genetic disorders caused by defects in the structure or function of the cilia and their related structures. Bardet-Biedl syndrome (BBS) is a complex ciliopathy with varied symptoms, probably due to altered membrane receptor signalling pathways. Methods: We investigated the role of BBS1 and BBS4 gene deficiencies in retinal epithelial cells using surface membrane proteomics, co-immunoprecipitation, AlphaFold structural modelling, endocytic trafficking colocalization assays, autophagy flux analysis, and epithelial-to-mesenchymal transition (EMT) functional assays including migration and wound healing. Results: Deficiencies in these BBSome components led to shorter cilia and disrupted receptor endocytosis, with accumulation of TGF-{beta}-pathway and EMT-associated proteins on the plasma membrane of BBS1 KO cells. Co-immunoprecipitation and AlphaFold structural modelling indicated that neither BBS1 nor BBS4 forms stable physical complexes with core endocytic components (EEA1, RAB11, RAB7, LAMP2), suggesting an indirect mechanism. BBS1 KO cells showed sustained TGFBR1 recycling, low basal autophagic flux that increases upon TGF-{beta} stimulation, and the most pronounced mesenchymal phenotype. BBS4 KO cells showed reduced receptor recycling, elevated basal autophagic flux that declines upon stimulation, and the strongest canonical TGF-{beta}/SMAD activation with reduced non-canonical ERK1/2 signalling. Conclusions: Increased EMT markers and enhanced migration in BBS1 KO cells highlight the role of BBSome-dependent receptor trafficking as a potential therapeutic target for retinal degeneration in BBS. The divergent autophagic and signalling phenotypes between BBS1 and BBS4 KO reveal subunit-specific contributions to TGF-{beta} pathway dysregulation.

molecular biology↗

bioRxiv2024

TAK1 operates at the primary cilium in non-canonical TGFB/BMP signaling to control heart development

Transforming Growth Factor-Beta-Activated Kinase 1 (TAK1/MAP3K7), along with its upstream regulators TAK1-Binding Protein 2 (TAB2) and the catalytic alpha-subunit of Protein Kinase A (PKA-C/PRKACA), has been identified as a pivotal player in regulation of developmental processes. Haploinsufficiency of TAB2 causes Congenital Heart Disease (CHD) and rare variants in PKA-C and TAK1 cause cardioacrofacial dysplasia (CAFD), and Frontometaphyseal Dysplasia (FMD) and cardiospondylocarpofacial syndrome (CSCFS), respectively, rare multisystem syndromes, where CHD may appear in the clinical spectrum. We hypothesized that TAK1 plays a significant role in heart development and CHD and addressed this by genetic analysis in CHD patient cohorts and experiments in cell and animal models. Exome sequencing data from 1,471 CHD patients with extracardiac anomalies (syndromic CHD, sCHD), 2,405 patients with nonsyndromic CHD (nsCHD) and 45,082 controls showed increased burden of rare TAB2 and TAK1 variants in sCHD, but not in nsCHD. Detailed characterization of tak1-/-and tab2-/- zebrafish mutants revealed cardiac defects (dilated atrium, trabeculation defects, tachycardia and reduced contractility) as well as extracardiac developmental anomalies. RNA sequencing of tak1-/- mutant hearts showed downregulation of genes encoding core cardiac transcription factors, sarcomeric proteins and extracellular matrix proteins. Experiments with cell cultures and analysis of zebrafish larvae and gastruloids indicated that TAK1 via TAB2 and PKA-C is activated at the primary cilium during cardiomyogenesis and that TAK1 activation at this site is enhanced by cardiomyogenic signaling molecules, including ligands of the TGFB/BMP superfamily. Consistent with these findings, CRISPR/Cas9-mediated editing of TAK1 or administration of small molecule inhibitors targeting TAK1 inhibited ciliary signaling and cardiomyocyte differentiation in vitro, while FMD-causing mutations in TAK1 reduced its ciliary localization. In conclusion, our data establishes a central role for TAK1 and its upstream regulators in cardiac development and syndromic CHD, coordinated via the primary cilium.

developmental biology↗