Morrison, Erin N

Using Drosophila deficiency (Df) and Over Expression (OE) (GAL4/UAS>dCas9-VPR; sgRNA) gene systems, it was demonstrated that Dmel_CG31381 and Dmel_CG11089 are functional tRNA isopentenyltransferase (EC 2.5.1.8) genes (tRNA IPT1 and IPT2) critical to the first committed step in insect cytokinin biosynthesis. IPT Df mutants showed significant decreases in total CK levels and IPT1/IPT2 transcript levels compared to parent lines. IPT OE mutants showed significant increases in total CK levels and IPT1/IPT2 transcript levels compared to parent lines. Further, endogenous CK analyte levels and qPCR relative fold gene expression of Dmel_CG31381 and Dmel_CG11089 (tRNA IPT1 and IPT2) genes demonstrated expression patterns with functional confirmation corresponding to the predicted IPT mutant variants. The functional confirmation of tRNA IPT1 and IPT2 as the first committed step was further supported by the bioinformatic detection of putative gene homologs to corroborate seven remaining enzyme transcripts supporting the novel description of a CK biosynthesis pathway in insects.

Author Keywords: Cytokinin Biosynthesis, Drosophila, gene expression, Insect Gall, mass spectrometry, tRNA IPT

The neuronal ceroid lipofuscinoses (NCLs), commonly known as Batten disease, are a family of inherited neurodegenerative lysosomal storage disorders. CLN7 disease is a subtype of NCL that is caused by mutations in the MFSD8 gene. MFSD8 encodes a lysosomal transmembrane protein that is predicted to play a role in transporting small substrates across membranes. However, little is known about its role and substrate specificity. Previous work identified an ortholog of human MFSD8 in the social amoeba Dictyostelium discoideum and reported its localization to endocytic compartments. In this study, the effects of mfsd8 loss during Dictyostelium growth and multicellular development were further characterized. Dictyostelium mfsd8- cells displayed increased rates of proliferation and pinocytosis in liquid media. During growth, loss of mfsd8 altered lysosomal enzymatic activities and reduced the intracellular and extracellular levels of autocrine proliferation repressor A. mfsd8- cells grown on a lawn of bacteria formed plaques in a shorter period of time compared to WT cells, providing additional support for the enhanced growth of mfsd8- cells. Upon starvation, the aggregation of mfsd8- cells was delayed, and mfsd8- cells formed more mounds that were smaller in size, which may be attributed to the reduced cell-substrate adhesion and altered lysosomal enzymatic activities observed for mfsd8- cells. Following aggregation, tipped mound formation was delayed, however, loss of mfsd8 did not affect the timing of slug/finger and fruiting body formation. Additionally, slug migration was reduced in mfsd8- cells. These aberrant phenotypes, excluding fruiting body formation, were effectively or partially rescued when Mfsd8-GFP was introduced into mfsd8- cells. Overall, these results show that Mfsd8 plays a role in regulating growth and developmental processes in Dictyostelium via lysosomal-associated functions.

Author Keywords: CLN7, Dictyostelium discoideum, Lysosomes, MFSD8, Neuronal Ceroid Lipofuscinoses