Isoform-Specific Effects of the DOA Kinase on Autophagic Cell Death of the Larval Salivary Glands in Drosophila melanogaster
Date
2017-06-30
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Abstract
Families of protein kinases have been conserved over millions of years,
and they play important roles in many cellular processes such as the cell cycle,
differentiation, and signal transduction (Neiman, 1993). Doa, or Darkener of
apricot, is a locus found in Drosophila melanogaster that encodes a protein
kinase belonging to the LAMMER kinase family. There are seven known protein
isoforms of the DOA kinase, which differ by molecular weight and developmental
specificity (Kpebe and Rabinow, 2008). While the isoforms contain identical Cterminal
catalytic domains, the N-terminal noncatalytic domains are different due
to the use of alternative promoters (Kpebe and Rabinow, 2008). Given the number
of different structures of DOA isoforms and their respective developmental
specificity, it is possible that the isoforms are performing different molecular
functions (Kpebe and Rabinow, 2008).
Macroautophagy, referred to here as autophagy, is a crucial process that
aids in the regulation of cellular homeostasis (Lockshin and Zakeri, 2004).
Autophagy is lysosomally-mediated, and is an important process involved in the
removal and degradation of bulk cytoplasm and long-lived proteins and organelles
(Berry and Baehrecke, 2007). The salivary glands in Drosophila melanogaster
offer an ideal model for studying developmental autophagic cell death in vivo,
since the bulk of the cytoplasm in the larval salivary glands are removed via
autophagic cell death during metamorphosis (Lockshin and Zakeri, 2004).
In this project, interfering RNA (RNAi) constructs encoding short hairpin
RNAs (shRNAs) were used to silence the expression of specific Doa isoforms
through the degradation of homologous mRNA. The constructs each target a
different Doa isoform mRNA, because the N-termini are all different from one
another, and are being expressed from alternative promoters. This approach could
hypothetically reveal different functions of the Doa isoforms. The segregation of
markers (Tb on the balancer chromosome TM6 or TSTL) is used to follow
genotypes of overexpression/RNAi for Doa.
Since RNAi in Drosophila is cell-autonomous, the GAL4-UAS system can
be used to target the expression of the RNAi constructs. By crossing female flies
with a UAS-GFP responder to males with a Salivary Gland (SG) GAL4 driver, it
is possible to express GFP in the salivary glands of the progeny. The salivary
glands can be visualized via fluorescence microscopy, and images taken at 6 hour
intervals should reveal a construct’s effects on autophagic cell death. Differences
in levels of autophagy of the salivary glands between flies with the modifier
construct and the wild-type could be indicative of an isoform’s involvement in the
promotion or inhibition of cell death.
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Keywords
Biology, Drosophila melanogaster, Cell Death, Autophagy, Salivary Glands