Spatial Cell Biology: Dissecting and directing intracellular transport mechanisms

Abstract

Cellular compartmentalization and intracellular transport mechanisms are important to establish and maintain the spatial organisation of proteins and organelles needed to ensure proper cellular functioning. Especially in polarized cells like neurons, the proper distribution of proteins into the right cellular compartment is crucial for correct functioning. In the first part of this thesis, we studied the transport of glutamate receptors into dendrites and synapses. Active long-range transport of recycling endosomes containing these glutamate receptors is mediated by the microtubule cytoskeleton and by motor proteins of the kinesin and dynein families. Myosin-V motor proteins can subsequently transport these endosomes into dendritic spines. Once recycling endosomes fuse with the plasma membrane either in the dendrite or spine, glutamate receptors are released onto the plasma membrane in which they can diffuse laterally. We showed that recycling endosomes in dendritic spines are required to maintain glutamate receptor levels at synapses and synapse architecture. We also found that spine morphology has an impact on how much glutamate receptors can be retained at synapses and thereby contributes to compartmentalization of the receptor. We then focused on the development of a novel optogenetic methods to manipulate organelle transport and positioning in living cells. Using light-sensitive dimerization domains of the LOV and phytochrome classes, we could recruit motor proteins of choice to a specific type of organelle. This led to rapid but highly controllable relocalization of these organelles in the illuminated area of interest allowing us to observe the effect of acute mislocalizations. Moreover, combining these two orthogonal optogenetic systems, we demonstrate simultaneous control of two different organelles. Using these assays we show that recycling endosomes have a local function in axonal outgrowth. A better understanding of the local functions of organelle positioning and the underlying mechanisms that regulate these processes may lead to novel therapeutic targets in diseases such as microvillus-inclusion disease (MVID), neurodegeneration or other developmental defects.