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Molecular mechanisms of the assembly and function of BBSome
Prasai, Avishek ; Huranová, Martina (advisor) ; Varga, Vladimír (referee) ; Bosáková, Michaela (referee)
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...
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Searching for the common function of the BBSome across the evolution and development
Mašková, Kristýna ; Štěpánek, Ondřej (advisor) ; Čajánek, Lukáš (referee)
The BBSome is a protein complex whose function is associated with ciliary trafficking. It has been found that the BBSome is evolutionarily conserved among ciliated organisms. The disruption of the BBSome leads to cilia dysfunctions and affects many signalling pathways. In humans, genetic defects in the BBSome are the cause of Bardet-Biedl Syndrome, which is a pleiotropic disease. The BBSome is studied separately in various model organisms and cell lines to understand the molecular functions of the BBSome. These studies have not been compared to see if there is a common BBSome function, which could be why the BBSome is evolutionarily conserved among ciliated organisms. In this bachelor's thesis, the knowledge about the BBSome was summarized and compared to identify a putative common function of the BBSome among various model organisms or cell types. It seems that there has not been found any BBSome function that could be identified as the common function because the BBSome has many specialized functions in different organisms and tissues. The only distant similarity is BBSome-dependent ciliary retrograde transport, which has been described in most of the studied model organisms and cell types.
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Role of Bardet-Biedl syndrome (BBS) protein complex in T cells
Niederlová, Veronika ; Štěpánek, Ondřej (advisor) ; Černý, Jan (referee)
BBSome is a protein complex crucial for trafficking of specific cargoes to the primary cilium. Although primary cilia are typically not present in cells of haematopoietic origin, such as T cells, recent research has revealed striking parallels between the primary cilium and the immunological synapse. Amongst other similarities, both structures are supposed to use the same transport machinery involving Rab8 and IFT20, the close interaction partners of BBSome. The first goal of this thesis was to investigate the role of BBSome in the biology of T cells. Using RT-qPCR, we have shown that BBSome subunits are expressed in lymphoid tissues and T cells. Studies of localization of BBSome subunits in Jurkat cell line and primary OT-I T cells revealed that the subunits have distinct localization patterns with BBS4 localizing to the centrosome and BBS1, BBS5, and BBip10 having dispersed localization. After the contact with an antigen presenting cell, BBS4 re-localizes to the immunological synapse. Mutations in BBSome encoding genes cause Bardet-Biedl syndrome (BBS), a rare ciliopathy presenting with multiorganic symptoms. The second goal of this thesis was to examine the associations between BBS and the immune system. Examination of medical records of more than 450 BBS patients revealed that autoimmune...
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