Delivery and Removal: Probing cargo sorting and degradation using inducible tools: Probing cargo sorting and degradation using inducible tools

Abstract

Delivery of intracellular cargos to the correct location within the cell is essential to establish and maintain proper cellular functioning. Furthermore, removal of dysfunctional cargo such as misfolded and aggregated proteins is crucial to avoid the toxic effects these proteins might have. In this thesis, I develop and employ new tools to study both delivery and clearance processes. These inducible tools allow control over the timing of specific events and can be used to robustly examine specific cellular pathways. Chapter 2 focusses on the interplay between actin and microtubule motors, in particular with respect to axonal entry. We introduce a new assay to independently recruit 2 different motors to cargo and show that Myosin-V can stall kinesin-driven cargo transport into axons. Furthermore, we describe how Myosin-V recruitment leads to cargo stalling at actin hotspots in the axon initial segment and thereby contributes to proper cargo sorting in the neuron. The rest of this thesis focusses on protein quality control and specifically on a new assay to study how cells can cope with protein aggregates. In Chapter 3, we discuss the establishment of this new assay and demonstrate how it can be used for the study of macroautophagy. In short, we designed a protein that can oligomerize upon addition of a small molecule, resulting in the formation of protein clusters inside living cells. We demonstrate that these clusters behave as aggregates, are recognized by the quality control system and finally targeted for degradation by the autophagy pathway. Moreover, by using a tandem fluorescent tag, these clusters allow us to monitor the delivery in the lysosome for degradation due to the selective loss of EGFP fluorescence. In Chapter 4 we discuss an improvement of the assay, where we replaced the tandem fluorescent tag by a pH sensitive fluorophore. This allows for better compatibility with live cell imaging by freeing up the GFP channel for other markers. Furthermore, we show that the improved assay has better compatibility with FACS analysis and is therefore more suitable for screening approaches. Chapter 5 is dedicated to nuclear quality control. Interestingly, when the same inducible aggregates are formed in the nucleus instead of the cytoplasm, a different cellular response is triggered. We find that proteasomal degradation plays a major role in removal of nuclear aggregates and that nuclear export is also involved. Furthermore we show that aggregate formation in the nucleus leads to nuclear rupture events that could compromise nuclear integrity. Chapter 6 concludes this thesis with a discussion on outstanding research questions.