Date of Graduation

12-2014

Document Type

Thesis

Degree Name

Master of Science in Cell & Molecular Biology (MS)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Lehmann, Michael H.

Committee Member

Du, Yuchun

Second Committee Member

Erf, Gisela F.

Keywords

dLipin protein; Drosophila melanogaster; Nuclear and cytoplasmic functions

Abstract

Lipin family proteins are highly conserved proteins present in species ranging from mammals to yeast. Lipin 1, the first Lipin gene identified in fatty liver dystrophy (fld) mutant mice, encodes the bifunctional protein Lipin 1, which can serve as an Mg2+-dependent phosphatidic acid phosphatase (PAP) and transcriptional co-regulator. dLipin, the single Lipin ortholog of Drosophila melanogaster, is required in triglyceride synthesis and fat body development. To study the transcriptional co-regulator activity of dLipin, nuclear receptors were screened to find receptors that interact with dLipin. The genetic interaction data indicated that Drosophila hepatic nuclear receptor 4 (HNF4) was a promising candidate for a protein that cooperates with dLipin in gene regulation. To study the importance of the PAP activity without disturbing the co-regulator activity, mutant flies that only express dLipin protein that lacks the PAP enzymatic activity were generated. The mutant flies showed the normal expression level and pattern of dLipin. The delta PAP mutation is lethal to Drosophila. Ectopically expressed wild type dLipin or GFP-tagged dLipin protein rescued the flies through the early lethal stage until the pupal and, in some instances, the adult stage. The phenotypes of the fat body cells of the rescued animals showed a correlation with the amount of the ectopically expressed dLipin protein. Fat body cells with low PAP enzymatic activity were round, had lost their polygonal shape and were detached from each other. They also contained very small fat droplets. Sufficient PAP enzymatic activity is needed to ensure a normal developmental rate, too. Starvation resistance was greatly impaired in dLipin-GFP-rescued delta PAP mutant heterozygotes that carried a wild-type dLipin allele. In addition, dLipin-GFP proved to be unable to translocate into the cell nucleus and it prevented nuclear translocation of endogenous wild-type dLipin. Together, these data indicate that dLipin-GFP acts in a dominant-negative manner and that nuclear activity of dLipin is required under starvation conditions.

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