ESCRT-II role in cell biology, development and disease

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Microbiology, School of Life Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

The ESCRT machinery is comprised of four distinct complexes (-0, -I, -II and -III) that have crucial roles in the endosomal and autophagic pathways. In the endocytic pathway, ESCRTs target ubiquitinated cargo to endosomes and aid in the formation of the intraluminal vesicles making them essential for regulation of receptor signaling, cell survival, proliferation and polarity. In the autophagic pathway, functional ESCRTs are vital for efficient fusion of autophagic vesicles with the endocytic pathway and for degradation of both endosomal and autophagic vesicle cargos. In many model systems, mutations in the ESCRT machinery are typically linked to a variety of different pathologies, including cancer and neurodegeneration. In the autophagic pathway, ESCRT-deficient cells cause an accumulation of protein aggregates, which are hallmarks of neurodegenerative disease. Cancer and neurodegenerative diseases are the second and third most common causes of death respectively, and place a large economic and social burden on the community. Development of treatments and therapies is largely dependent on our understanding of the underlying signaling pathways. Studying such pathways in simple eukaryotic models has many benefits, the cellular machinery of lower organisms is similar to mammalian cells but the structure of the genome, tissues and organs is simpler and more amenable to experimental study. In this research we knocked down the expression of the ESCRT-II subunits, Vps22, Vps25 and Vps36 in the simple eukaryotic model Dictyostelium and studied its role in endocytosis and autophagy. Our findings show ESCRT-II subunits localizing on endosomal and autophagosomal membranes and co-localizing with mammalian markers including: Rab7, ubiquitin and LC3B. Our novel findings also show ESCRT-II subunits co-localizing with endolysosomal proteins CLN5 and CLN7 and calmodulin. Decreased expression of ESCRT-II displayed no effect of axenic growth, but showed increased growth on bacterial lawns and increased phagocytic and macropinocytic uptake. These results suggest that ESCRT-II has a negative endocytic role. Decreased expression of ESCRT-II also caused defects in morphogenesis with co-transformants exhibiting thicker shorter stalks and increased DIF-1 induced vacuolization and cell death, which suggests that this complex has a negative autophagic role. Our research also examined the broader functions of ESCRT-II, and we have uncovered unexpected links with the TORC1 pathway, a kinase complex that controls cellular metabolism.