De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of Bbs1

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate

Molecular mechanisms of the assembly and function of BBSome

Bardet Biedl syndrome is a genetic disorder caused by the dysfunction of the BBSome, an octameric cargo adaptor protein complex. The BBSome facilitates the transport of signaling receptors into and out of the primary cilium, a microtubule based sensory organelle of the cell. The first part of this thesis focuses on the elucidation of the assembly of the BBSome in living cells. We generated a library of human and mouse cells lines deficient in the individual BBSome subunits and transduced them with the other YFP tagged subunits. We employed biochemical assays, immunofluorescence and quantitative fluorescence microscopy techniques to analyze the individual steps in the BBSome assembly pathway. We revealed that the BBSome assembly occurs sequentially in spatially regulated steps. We showed that BBS4 nucleates the assembly of a pre-BBSome at the pericentriolar satellites. The translocation of the pre-BBSome to the ciliary base is facilitated by BBS1. We also revealed that in a BBS chaperonin deficient cell line, BBS12 KO cells, a small fraction of the BBSome and/or BBSome sub-complexes are still able to form and localize to the cilium. This could suggest that the BBS chaperonins might act later in the BBSome assembly pathway providing a means for quality control for the BBSome. Ciliary ectocytosis..

Molekulární mechanizmy formování a funkce BBSomu

Bardet Biedl syndrome is a genetic disorder caused by the dysfunction of the BBSome, an octameric cargo adaptor protein complex. The BBSome facilitates the transport of signaling receptors into and out of the primary cilium, a microtubule based sensory organelle of the cell. The first part of this thesis focuses on the elucidation of the assembly of the BBSome in living cells. We generated a library of human and mouse cells lines deficient in the individual BBSome subunits and transduced them with the other YFP tagged subunits. We employed biochemical assays, immunofluorescence and quantitative fluorescence microscopy techniques to analyze the individual steps in the BBSome assembly pathway. We revealed that the BBSome assembly occurs sequentially in spatially regulated steps. We showed that BBS4 nucleates the assembly of a pre-BBSome at the pericentriolar satellites. The translocation of the pre-BBSome to the ciliary base is facilitated by BBS1. We also revealed that in a BBS chaperonin deficient cell line, BBS12 KO cells, a small fraction of the BBSome and/or BBSome sub-complexes are still able to form and localize to the cilium. This could suggest that the BBS chaperonins might act later in the BBSome assembly pathway providing a means for quality control for the BBSome. Ciliary ectocytosis...Bardetův Biedlův syndrom (BBS) je genetická porucha způsobená dysfunkcí BBSomu, což je oktamerický adaptorový proteinový komplex. BBSome transportuje signální receptory dovnitř a ven z cílií, což jsou senzorické buněčné organely vystavěné na V první části práce jsme se zaměřili na objasnění formování BBSomu v živých buňkách. Vytvořili jsme knihovnu lidských a myších buněčných linií, kterým chyběly jednotlivé podjednotky BBSomu a vnesli jsme do nich ostatní podjednotky označené YFP proteinem. K analýze jednotlivých kroků formování BBSomu jsme použili biochemické testy, imunofluorescenci a kvantitativní fluorescenční mikroskopii. Odhalili jsme, že sestavování BBSomu probíhá postupně v různých částech buňky. Ukázali jsme, že BBS4 iniciuje rních satelitech. Přemístění pre bázi a finální zkompletování BBSomu umožňuje BBS1. Odhalili jsme také, že v buňkách, kde chybí BBS12 chaperonin, se formuje malá část BBSomu a/nebo subkomplexů . To naznačuje, že BBS chaperoniny zajištují Ciliární ektocytóza odstraňuje přebytečné ciliární signální receptory prostřednictvím odštěpení apikální části ciliární membrány, což je regulované polymerizací aktinu. Faktory které spouštějí polymerizaci aktinu v místě ektocytózy, zůstávají neznámé. Ve druhé části práce jsme odhalili, že CDC42, člen rodiny RHO GTPáz, spouští polymerizaci...Katedra buněčné biologieDepartment of Cell BiologyFaculty of SciencePřírodovědecká fakult

De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of <i>Bbs1</i>

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate