Titel der Forschungsarbeit: Engineering vault proteins and RNA cargos to improve cellular labeling in connectOMICs
Fakultät: TUM School of Natural Sciences
Lehrstuhl: Professur für Neurobiological Engineering
Betreuung: Niklas Armbrust und Prof. Gil Westmeyer
Abstract der Forschungsarbeit
Cells have been found using the first microscope, and also today modern cell biology research heavily relies on microscopy. One of the most complex and important cellular tissue is the brain. Studying neural connections and the formation of neurons in the brain is essential to understand its whole functioning. Neurons connect to each other and form complex networks for signal processing – the study of these networks is called connectOMICs. These neural connections are hard to visualize with microscopy imaging because of the neurons, in particular human ones, can connect to more than 10,000 other neurons.
To study these experimentally, brain tissue is cut into thin slices suitable for microscopy, which results in neurons reaching through thousands of microscopy images due to their length. In order to keep track of a single neuron over many images, it is important to label it. These labelings are then visible for example via fluorescence microscopy or transmission electron microscopy. The quality of these images depends on the labels, and therefore it is important to have access to multiple labeling strategies. Hence, this research aims to develop new tools for labeling cells.
For that, a labeling strategy is chosen where plasmids are transcribed to an RNA (called cargo). In parallel, another transfected plasmid is transcribed and translated as well. The resulting protein monomers assemble to form a protein cage structure, a so-called Vault. The cargo RNA can enter the Vault and bind to it, which helps the cargo to stay stable for a longer time.
Two generations of labeling RNAs were made to enhance Vault-contrast for cell labeling. The first generation failed due to unstable RNA cargos that were degenerated by exonucleases too quickly. To solve this problem, a new plasmid for transfection was built from designed Plasmids in the second generation. The new plasmid was transcribed to a new RNA cargo that was stabilized by circularization. This resulted in improved contrast of multiple vault-labeled cells when looked at via both fluorescent and transmission electron microscopy.
Altogether, the thesis demonstrated how plasmid-based cell labeling can improve contrast in imaging and could thus open exciting new ways for connectOMICs in neuroscience.