Cellular Waste Management
Authors:   , Korsten Giel 
Published: January 2025

Cells rely on the proper functioning of a vast amount of proteins. To maintain proper function, proteins must be correctly folded. As a result of disease, stress or ageing, proteins can unfold and cluster together to form aggregates. Such aggregates are found in many diseases, like ALS, Alzheimer’s disease, and Huntington’s disease (HD) and are associated with a large amount of toxic effects. It is therefore crucial to understand the mechanisms employed by cells to combat protein aggregation and remove protein aggregates. In the first part of this thesis, we study aggregate removal via autophagy and disaggregation, two distinct methods for aggregate removal in cells. In chapters 2 and 3 we describe a novel method that enables the induction of protein aggregates in living cells. These Particles Induced by Multimerization (PIMs) consist of multiple homodimerization domains that cluster together to form protein aggregates after exposure to rapalog, a rapamycin analogue. We use PIMs to study aggregate removal via aggrephagy, a form of autophagy aimed at delivering protein aggregates to the lysosome. Our PIMs variants contain various fluorescent molecules that enable visualization of lysosomal delivery. By simultaneously imaging key regulators of the autophagy pathway, we generate a timeline of aggrephagy in live cells. In chapter 4, we target PIM aggregates to the nucleus by appending the PIMs with a nuclear localization signal (NLS). We then use various high throughput imaging approaches and reveal that PIM-NLS aggregates are cleared via disaggregation by the alternative disaggregase VCP and the proteasome. Together, the PIM variants described in this thesis provide a robust tool to study aggregate removal in cells and serves as a valuable platform for high throughput screening approaches. As seen in disease, protein aggregates are not always efficiently cleared, and the localisation of protein aggregates dictates the strategies available for aggregate removal. This likely underlies differential localization of protein aggregate seen in diseases, where aggregation can be localized in specific cellular compartments. For example, HD patients harbour many intranuclear aggregates consisting of the polyglutamine (polyQ) protein huntingtin. In the chapter 5, we aimed to study the cellular toxicity related to these aggregates. We find that polyQ aggregates present in the nucleus cause abundant nuclear envelope (NE) ruptures in cultured cells and cultured rat hippocampal neurons, and show that many of these ruptures are not efficiently repaired. Ruptures coincide with disruption of the nuclear lamina, a key network of proteins that provides mechanical support to the nucleus. We also show accumulation of membrane, repair factors and NE components that indicate failed repair events. Such prolonged ruptures lead to nucleocytoplasmic mixing, which might cause cellular toxicity such as inflammation, DNA damage and disrupted nucleocytoplasmic transport. Combined, this thesis generates new tools to study aggregate clearance in the nucleus and cytosol, and provides novel insight into the toxicity of nuclear aggregates that could be relevant for neurodegenerative disease.