Katnip is an uncharacterised yet evolutionary conserved protein which contains 3 repeats of a unique protein domain called DUF4457. Recent work has identified that loss of katnip function in humans, results in Joubert's syndrome, a rare ciliopathy. The loss of katnip in these patients, as well as animal models, caused cilia dysfunction due to the loss of microtubule organisation in the axoneme.
Katnip was also identified in a genetic screen for new autophagy regulators in the social amoeba \textit{Dictyostelium discoideum}. These unicellular organisms have no cilia, and therefore suggests a non-cilia role for katnip which affects autophagy.
This thesis found that the loss of katnip in \textit{Dictyostelium discoideum} results in an autophagy defect due to slower autophagosome formation and degradation. A degradation defect was also identified in phagocytosis and macropinocytosis, both of which rely on lysosomal trafficking for vesicle degradation along with autophagy. Based on these results, katnip mutants were hypothesised to have a general lysosomal trafficking defect.
In order to identify the root cause of this trafficking defect, GFP-katnip was overexpessed in both \Dd cells and multiple mammalian cell lines. GFP-katnip was observed at the centrosome of all these cells with a high cytoplasmic background. Unusually, GFP-katnip was seen to localise to microtubules in highly expressing mammalian cells and upon oxidative damage in \Dd. Therefore, I hypothesis that in addition to binding the centrosome, katnip is able to bind to microtubules, but only under certain conditions. Oxidative damage is used to induce microtubule damage, and therefore, GFP-katnip may be able to bind to points of microtubule damage.
Katnip mutants cells also had a sensitivity to GFP-tubulin expression, which caused an elongation of mitosis, specifically anaphase, and increased tangling of microtubules. I hypothesis that GFP-tubulin may induce more microtubule damage events in these cells and therefore, katnip may function to tolerate microtubule damage.
This thesis confirms a new non-cilia role for katnip, due to the identification of its binding and affecting interphase microtubules. This thesis present katnip as a potential candidate for a microtubule damage protein which can bind to sites of microtubule damage and is necessary for tolerating damage. This loss of microtubule maintenance and accumulation of damage may cause the organisation defect observed in GFP-tubulin expressing cells as well as axoneme microtubules in Joubert's patients. The loss of microtubules organisation results in the general lysosomal trafficking defects, which may also be present in mammalian cells. Therefore, katnip mutant patients may be more susceptible to diseases affected by loss of autophagy including cancers and neurodegenerative diseases.
