| dc.description.abstract |
Cilia, small antennae, can be found on nearly every cell in the human body and are important for human health and vision. Defects in cilia can lead to a variety of diseases, such as the Alström Syndrome (ALMS, OMIM #203800). This rare autosomal-recessive disorder occurs in 1 to 9 out of 1 million individuals. More than 300 mutations in the ALMS1 gene are known to cause a broad range of clinical defects most notably retinal dystrophy, Type 2 Diabetes, and truncal obesity. The ALMS1 gene codes for a ~0.5 MDa protein, that localizes to the centrosome and the basal body of cilia. So far, the molecular function of ALMS1 and its influence on the function of cilia is still elusive. Based on current literature and own data, I hypothesize that ALMS1 interacts with basal body proteins to enable proper cilia morphology and transport to the cilium. To address this hypothesis phenotypical and protein complex analyses were conducted.
CRISPR/Cas9 mediated ALMS1 KO in hTERT-RPE1 cells was used to identify and decipher ALMS1 function in cilia biology. ALMS1-deficient cells presented shorter cilia and an impaired cellular proliferation compared to control cells. In addition, centrosomal proteins, such as γ-tubulin and CEP250, were investigated. While γ-tubulin remains unchanged, suggesting a function upstream of ALMS1, CEP250 localization was mildly but significantly reduced upon ALMS1 loss. Furthermore, cilia and cilia-related markers were examined, such as RPGR, which exhibits a compressed appearance, while others, such as IFT-88, were unchanged compared to the control. Further investigation is needed to understand the functional relevance of the observed phenotypic changes in the context of cilia biology as well as disease-related mechanisms.
Furthermore, CRISPR/Cas9 mediated endogenously tagged ALMS1 HEK293T cells were used for protein complex analysis to further elucidate ALMS1 function. Investigation of protein-protein interaction revealed, that ALMS1 interacts among others with centrosomal and microtubule-associated proteins. Notably, the centrosomal protein 70 kDa (CEP70) was identified for the first time and appeared as a highly abundant ALMS1 interaction partner. Additional investigations, including deletion analysis, identified ALMS1 associating with the TPR domain and CT end of CEP70. By silencing CEP70 in hTERT-RPE1 cells a reduced ALMS1 localization at the basal body was observed, indicating CEP70 upstream of ALMS1. Nevertheless, further research is necessary to understand ALMS1 and CEP70 function in cilia-related processes, and disease-related mechanisms. |
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