Date of Award

Spring 5-10-2016

Degree Type

Thesis

Degree Name

Master of Science in Integrative Biology (MSIB)

Department

Biology

Major Professor

Susan M.E. Smith

First Committee Member

Susan M.E. Smith

Second Committee Member

Donald J. McGarey

Third Committee Member

Scott J. Nowak

Abstract

In 1972, J. Woodland Hastings and colleagues predicted the existence of a proton selective channel that opens in response to depolarizing voltage (HV1) across the vacuole membrane of bioluminescent dinoflagellates and conducts protons into specialized luminescence compartments (scintillons), thus causing the pH drop that triggers the light flash. RNA-Seq data from several luminescent dinoflagellate species provided candidate HV1 genes. When expressed in mammalian cells, the predicted HV1 from Lingulodinium polyedrum displays the hallmark properties of bona fide proton channels, including time-dependent opening with depolarization, perfect proton selectivity, and characteristic pH dependent gating. RT-PCR and Western blotting confirm expression of HV1 in L. polyedrum and isolated scintillons. Fluorescence confocal microscopy of L. polyedrum cells stained with antibodies to luminescence proteins luciferase (LCF), luciferin binding protein (LBP) and to HV1 (LpHV1) reveal structures consistent with HV1’s proposed function in bioluminescence. Isolated scintillons immunostained with antibody to LpHV1 displayed LpHV1 expression, showing that LpHV1 is present in this organelle. In addition, proteomics analysis demonstrated that isolated scintillon preparations contain peptides that map to LpHV1, including a portion of the epitope used to raise the antibody. These results indicate that LpHV1 is the voltage gated proton channel that triggers bioluminescence in L. polyedrum.

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