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Graduation Term

Summer 2025

Degree Name

Master of Science (MS)

Department

School of Biological Sciences

Committee Chair

Kevin A. Edwards

Committee Member

Thomas M. Hammond

Committee Member

Viktor Kirik

Committee Member

Martin Engelke

Abstract

Many proteins in eukaryotic cells resist diffusion, assembling into complexes or localizing in distinct patterns. This "self-organization" is essential for the complexity of eukaryotes, but it is difficult to predict localization and assembly from protein sequence, or to discern general principles from individual patterns. To survey protein localization signals in an unbiased manner, I tested the effectiveness of screening fluorescent protein traps in live Drosophila embryos with laser scanning confocal microscopy. The traps were generated with the Hostile takeover (Hto) transposon system, in which tissue-specific GAL4 constructs drive expression of a FLAG-mCherry RFP tag fused to a random downstream coding region. In a pilot screen using the embryo epidermis, all embryos expressing a fusion protein with an appearance distinct from that of the Starter Hto element were recovered. The screen yielded a final collection of 41 fly stocks with new protein trap insertions. RFP localization was assayed for all lines using the egg chamber follicle cell epithelium. Most of the fusions concentrated in the nucleus, and the rest were mostly non-nuclear, including several with distinct patterns (junctions, vesicles, and plasma membrane). A second, more selective screen was performed using embryo muscle, recovering five more lines including two with punctate cytosolic patterns. Hto insertion sites were recovered and sequenced for over 30 lines, and examination of the downstream exons provided a model for the likely fusion partner for each insert. There was a strong correlation between the RFP pattern and the fusion candidate. Fusions with a "diffuse nuclear-enriched" distribution tend be out-of-frame fusions to short peptides. But the well-patterned fusions tended to be in-frame and include most or all of the target protein, including the developmentally important proteins PXo, RhoGAP100F, and Qin/Kumo. I conclude that the embryo screen procedure efficiently recovers Hto protein traps useful to the Drosophila community, and that by re-screening these lines for novel patterns we can further enrich for members of important cellular pathways. In a second type of screen, I searched for Hto inserts that produce developmental abnormalities when expressed in the wing. These "phenotypic" protein traps take advantage of the power of Drosophila to place genes in pathways and networks based on their phenotypes. This screen provided novel insights into several understudied genes.

Access Type

Thesis-ISU Access Only

DOI

https://doi.org/10.30707/ETD.1763755358.845535

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