Print

Rajendran, Anbazhagan

Biology

2010

PhD

Bardet-Biedl syndrome (BBS) is a heterogeneous genetic disorder in humans characterized by renal and retinal abnormalities, polydactyly, cystic kidneys, learning disabilities, obesity and male hypogonadism. BBS can be described as a ciliopathy because the clinical symptoms are associated with abnormal cilia. Cilia are tiny hair-like structures present on the surface of almost all tissues in the human body. Cilia are enriched in structural proteins, sensory receptors, ion channels and signaling molecules. They play vital sensory roles such as perception of photons and odorants. In addition, they are involved in the clearance ofmucus lining the pulmonary epithelium and enable cell movement. To date, 14 human BBS genes have been reported. Seven BBS proteins (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8 and BBS9) form a heptameric complex called BBSome complex. The molecular mechanisms ofBBS proteins' tasks in regulating ciliary function are not well understood. Here we report on our use ofParamecium tetraurelia, a eukaryotic unicellular organism covered with cilia, to the study functions of BBS proteins.
All seven genes that are part ofthe BBSome complex as well as BBS3 and 13 are conserved in the Paramecium genome. We are able to immunoprecipitate FLAG tagged BBS9 (FLAGBBS9) or FLAGBBS8 and co-immunoprecipitate other BBS proteins, all of which are known to be BBSome components. These results support the notion that a BBSome complex exists in the Paramecium. We utilized the RNAi feeding method to reveal the significance of the Paramecium BBS proteins. The BBS7 and 9 depleted cells showed short or missing cilia while BBS3, 4 and 8 depleted cells showed slightly short cilia. These findings are consistent with the reports that the BBS proteins are vital for ciliary structure in other model organisms including C. elegans, mice and Chlamydomonas.
When we depleted BBS1, 3,4, 7, 8 or 9 we noticed changes in swimming behavior consistent with defects in the calcium-activated potassium (KCa) conductances but not voltage-gated Ca (Cav) conductances. In addition, the BBS3 and 5 depleted cells showed behavior consistent with disrupted voltage-gated K (Kv) conductances. Moreover, the BBS1 and 7 depleted cells showed disrupted mechanosensitive K (Km) conductance. Hence, we hypothesize that Paramecium BBS proteins are essential for ion channels' function by affecting their localization in cilia.
The exogeneously expressed FLAG tagged small conductance calcium-activated potassium (FLAGSK1a) and FLAG tagged polycystic kidney disease2 (LAGpKD2) channels fail to localize to cilia in cells depleted of BBS7, BBS8 or BBS9. Alternatively, the cellular location of the folate binding protein (FBP), which is involved in the folate chemosensory signal pathways, is not altered in the BBS7, 8 and 9 depleted cells. Thus, we postulate that SK1a and PKD2 channels are trafficked to cilia in a BBSome dependent pathway unlike Cav channels and FBP that are sorted to the cilia in BBSome independent pathways.