Expanding Excellence in Developmental Biology in Oklahoma

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Mutations in centriole duplication (CD) genes are correlated with the incidence of diseases such as primary microcephaly, primordial dwarfism and cancer. However, the effect of many of these disease-associated mutations in CD genes on their respective protein functions is currently unknown. This proposal utilizes one such disease-associated mutation in the conserved CD gene Spindle Assembly Abnormal Protein 6 (HsSAS-6/ SAS-6) as a tool to study SAS-6 function in ciliogenesis and CD. This disease-relevant HsSAS- 6 mutation is associated with the incidence of primary microcephaly and corresponds to sas-6(L69T) in C. elegans. Our long-term goal is to exploit disease-associated alleles of conserved CD genes to better understand their mechanism of action in regulating ciliogenesis and CD. The overall objectives in this application are to determine the effect of the sas-6(L69T) mutation on C. elegans ciliogenesis and CD. The central hypothesis is that the sas-6(L69T) mutation inhibits SAS-6 function leading to impaired ciliogenesis and CD. The rationale for this project is that since this mutation has a known pathological consequence in humans, it will provide important insights into the normal biological function of SAS-6. The central hypothesis will be tested by pursuing two specific aims: 1) Determine the effect of the sas-6(L69T) mutation on C. elegans ciliogenesis; 2) Elucidate the molecular mechanism by which the sas-6(L69T) mutation impairs CD in C. elegans. Under the first aim, the consequences of the sas-6(L69T) mutation on C. elegans ciliogenesis will be determined by performing a thorough microscopic and behavioral analysis of a C. elegans CRISPR strain carrying this mutation. In the second aim, a variety of cell biological and biochemical assays will be performed to clarify the molecular mechanism by which this mutation affects CD. The research proposed in the application is innovative because i) This is the first study investigating the effects of a primary-microcephaly-associated sas-6 mutation in a multicellular eukaryotic animal model. ii) This is the first study that uses a disease-associated sas-6 allele to study SAS-6 function in regulating ciliogenesis. Developing therapeutic or preventative interventions for any disease begins with understanding the fundamental consequences of disease-associated mutations at the level of basic science. The proposed research is significant because there is currently nothing known about the mechanistic effects of this primary microcephaly-associated sas-6 mutation. Understanding the cellular and biochemical consequences of the sas-6(L69T) mutation is the first important step to understanding the mechanism of incidence and progression of primary microcephaly.