SUMMARY
Eukaryotes have evolved a number of strategies to manage misfolded and aggregated proteins and therein
maintain protein homeostasis (proteostasis) in response to cellular stress. These include proteolytic pathways
such as the ubiquitin proteasome system and restorative pathways in which damaged proteins are rescued by
molecular chaperones and disaggregases. In humans, a breakdown in proteostasis is linked with diverse
pathologies including neurodegenerative diseases and cancer. A cellular hallmark of these diseases is the
presence of cytoplasmic and/or nuclear inclusion bodies which for many years were thought to contribute to
disease establishment and progression. However, a growing body of evidence reveals that inclusion bodies
form as part of a highly-orchestrated cellular protective pathway called spatial protein quality control (SPQC), in
which damaged proteins are sequestered and confined to discrete quality control compartments to be
processed later by the cell. The long-term goals of my research program are to determine in mechanistic detail
how, where, when, and why damaged proteins are recruited to inclusion bodies and to understand the cellular
consequences of dysfunction in this pathway. We are using the filamentous fungus Aspergillus nidulans as an
innovative model system to understand SPQC in highly polarized cells, and we have previously demonstrated
the importance of the microtubule-based transport system in organizing aggregated proteins following heat
shock. The overall Aims in this application are to 1) determine the ultimate cellular fates of endogenous
misfolded proteins following their spatial confinement to inclusion bodies and 2) further elucidate the molecular
mechanisms of SPQC in highly polarized A. nidulans cells. In Aim 1, we will first identify the endogenous
substrates of cytoplasmic and nuclear inclusion bodies in our system using innovative proteomics approaches.
Then, using photoconvertible fluorescent proteins, we will track the spatiotemporal fates of these endogenous
substrates under normal and perturbed conditions to uncover the extent of functional interplay between major
proteostasis pathways and SPQC. In Aim 2, we will test the hypothesis that protein aggregates hitchhike on
moving early endosomes for transportation to protein quality control compartments and examine the
contribution of microtubule dynamics to aggregate reorganization and SPQC. In addition, we will use super-
resolution fluorescence microscopy to resolve the nanoscale organization of cytoplasmic inclusion bodies in
our system, therein gaining new insight into their form and function. Lastly, we will employ an unbiased
genome-wide fluorescence microscopy-based screen to identify novel regulators of SPQC in highly polarized
Aspergillus cells. Consistent with the goals of the AREA program, this research will provide a wealth of
opportunities for hands-on undergraduate research involvement and exposure, while pushing our mechanistic
understanding of this emerging proteostasis pathway forward.