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  • The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells.

The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells.

The Journal of biological chemistry (2020-08-08)
Avishek Prasai, Marketa Schmidt Cernohorska, Klara Ruppova, Veronika Niederlova, Monika Andelova, Peter Draber, Ondrej Stepanek, Martina Huranova, Avishek Prasai, Marketa Schmidt Cernohorska, Klara Ruppova, Veronika Niederlova, Monika Andelova, Peter Draber, Ondrej Stepanek, Martina Huranova
ABSTRACT

Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.

MATERIALS
Product Number
Brand
Product Description

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Anti-BBS7 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution
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Anti-β-Actin antibody, Mouse monoclonal, clone AC-15, purified from hybridoma cell culture
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Anti-BBS9 antibody produced in rabbit, affinity isolated antibody, buffered aqueous glycerol solution
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Anti-GFP antibody produced in rabbit, affinity isolated antibody