Mutations in centriole duplication (CD) genes are associated with diseases such as primary microcephaly (MCPH), and cancer. To aid in the development of therapy against these diseases, it is critical to understand how CD proteins normally function within cells. Studying the effects of disease-associated mutations in CD genes can provide important clues into their biological roles. One such mutation in the sas-6 gene (HsSAS6(I62T)) was associated with the incidence of MCPH in a Pakistani family. SAS-6 function is essential for both primary ciliogenesis and CD: defects in each of these processes have been implicated to cause MCPH. Although the MCPH phenotype caused by the HsSAS6(I62T) allele indicates a potential defect in primary cilium function and/or CD, the mechanistic failures caused by this mutation are unknown. Importantly, the effects of this MCPH-associated mutation on ciliogenesis have never been investigated. Over-expression of the HsSAS6(I62T) mutation in U2OS cells yielded monopolar spindles. This suggests that when over-expressed, the mutant protein can have a dominant negative effect on CD through an as yet unknown mechanism. The molecular and developmental consequences of this mutation have never been investigated in the context of a whole multicellular eukaryotic model organism. The function of the SAS-6 protein is conserved among several different species, including humans and C. elegans. In fact, SAS-6 was first identified in C. elegans as being essential for proper CD and was later shown to have a similar role in humans. SAS-6 function is also essential for ciliogenesis in both C. elegans and humans. Although HsSAS-6/SAS-6 is widely recognized as a critical structural component of the centrioles, many gaps still exist in our understanding of how SAS-6 mediates primary ciliogenesis and CD in vivo. One reason for this is that since sas-6 is an essential gene, severely impairing sas-6 activity causes embryonic lethality. Our lab has generated a C. elegans strain carrying the human MCPH-equivalent sas-6(L69T) mutation using CRISPR/Cas9 genome editing. Our preliminary data indicate that this mutation mildly perturbs CD in C. elegans as appears to be the case in humans. By generating a sas-6 allele that has a pathological relevance, we have created an excellent tool to probe the function of SAS-6 in the processes of ciliogenesis and CD. Therefore, in this proposal, we will utilize this new, health-relevant sas-6 allele to study the role of SAS-6 in ciliogenesis and CD.
The long-term goal of my laboratory is to study the effect of disease-associated mutations in conserved ciliogenesis and CD genes. The objectives in this endeavor are to determine the effects of the sas-6(L69T) mutation on C. elegans ciliogenesis and CD. Our preliminary data demonstrate that, the presence of the sas-6(L69T) mutation increases CD failure of a strain that is partially compromised for CD. Based upon these and other preliminary data, our central hypothesis is that the sas-6(L69T) mutation inhibits SAS-6 function leading to impaired ciliogenesis and CD. The rationale for this proposal is that since the human equivalent of the sas-6(L69T) mutation has a pathological consequence, this mutation will provide important insights into the normal biological function of SAS-6. To test our central hypothesis, we will pursue the following 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.
Upon the successful completion of these aims, the expected outcomes are an enhanced knowledge of how SAS-6 regulates ciliogenesis and CD. This application is innovative because this is the first study investigating the consequences of the MCPH-associated sas-6(L69T) mutation in an in vivo model system. Hence, we expect that the successful completion of the aims of this proposal will provide knowledge that will vertically advance the field. Moreover, this proposal is significant because there is currently no cure or treatment options for MCPH. A clearer understanding of the consequences of this mutation will enable a better understanding of the mechanism of incidence and progression of MCPH.
Jyoti Iyer, PhD
Assistant Professor of Chemistry and Biochemistry
University of Tulsa