Analysis of the molecular function of ALMS1

• Applicant: Dr. Tina Beyer
• Subject Area: Cell Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 497060278

The autosomal-recessive disorder Alström syndrome is caused by mutations in one gene, ALMS1, and appears in a high variability in symptoms and disease severity. In general, the symptoms reflect a ciliopathy, a disease caused by malfunction of ciliary proteins. Cilia are microtubule-based cell protrusions, which are involved in multiple cellular and signalling processes. Consequently, defects in ciliary proteins like ALMS1 often result in multiple symptoms, including hearing loss, kidney and heart failure, obesity and retinal degeneration. ALMS1 protein can be found at the basal body, which is required for the ciliary structure itself, and which is involved in polarized cilia docking and selective transport to and into the cilium. ALMS1 protein is thought to be involved in cilia maintenance, transport and signalling processes. However, due to the protein size of 461 kDa which makes ALMS1 analysis difficult, and the variability of symptoms between model organisms and in the disease condition, the molecular function of ALMS1 is not well understood. In the study presented here, we aim at understanding the role of ALMS1 in different cilia-related processes (ciliogenesis, transport and signalling processes) in wildtype as well as in mutant cells. First, we want to gain more information about the ALMS1 protein complex by analysis of endogenously tagged ALMS1 in Hek293T cells generated using the CRISPR/Cas9 technique. Using endogenously tagged cells will enable us to investigate stable (Flag-, GFP-tag) and transient (BioID2-tag) protein complex assembly. In addition, tissue-specific protein-protein interactions will be investigated using the endogenously tagged cells in combination with ciliated mouse tissue lysate. Second, the expression pattern and subcellular localization of ALMS1 and its interacting partners will be analysed in a cell state-dependent manner and in different tissues. Third, the effect of loss of ALMS1 as well as of specific mutations will be investigated. Mutant retinal pigmented epithelial cells will be generated using CRISPR/Cas9 and cytidine deaminases/ conversion base enzymes. To exclude clonal artefacts, the cell cycle progression, viability and migration will be analysed. Hereafter, based on data gained by protein complex, expression and localization analysis, a focused investigation of basal body stability and cilia-related processes will be performed using these mutant cells including cilia assembly and disassembly, basal body protein assembly, protein/ vesicle trafficking and signalling processes.Based on all data gained in this study, we aim to better understand the molecular function of ALMS1 in cilia-related processes which can be used as basis to investigate tissue-specific and disease-related processes in the future.

• DFG Programme: Research Grants