Imaging protein and organelle compartmentalization in metabolism and differentiation

Aktiviteetti: TutkimustyypitVäitöskirjan ohjaaja tai sivuohjaaja

Kuvaus

Compartmentalization supports and diversifies biological processes on multiple levels. Cellular localization has become a part of the main feature set defining the protein and tissue characteristics. For this purpose, a variety of techniques has been developed. However, spatiotemporal regulation of compartmentalization and its effects on protein and cellular functions remain undefined for many of the proteins and tissues especially during development.

The overall aim of the thesis was to characterize compartmentalization on organelle, cellular and tissue level. In particular, we have defined: a) compartmentalization and biochemical properties of serine protease LACTB in mitochondria, b) PPT1 enzyme cellular distribution and uncovered new interaction partners and its effects on differentiation in neuronal cells models of CLN1 metabolic disease, and c) metanephric kidney mesenchyme self-organization and differentiation using 3D culture.

By employing fluorescence and electron microscopy, and mass spectroscopy along with other biochemical methods, intermembrane space compartmentalization of LACTB forming stable filaments was pinpointed. High-resolution colocalization and image analysis was utilized to quantify the cellular distribution of overexpressed PPT1-CTAP showing partial localization to lysosomes. Overexpression of wild type and mutant PPT1 in human neuronal cell-lines facilitated the discovery of novel interaction partners and uncovered effects on neuronal differentiation in vitro. Finally, simple 3D mouse kidney mesenchyme culture was developed. Using a set of time laps, fluorescence microscopy, and automated image analysis algorithms, the nephron progenitors’ compartmentalization was characterized in vitro, which resembles pretubular aggregates in vivo. Importantly, in this 3D culture model, nephron progenitors spontaneously differentiate without the exogenous inductive stimulation.

Taken together, these results extended our knowledge of compartmentalization, and metabolism and differentiation on organelle, cellular and tissue level. The findings, when extrapolated in an appropriate context, along with the developed culture method, could be utilized to deepen our understanding on neuronal and kidney development and in other metabolic diseases.
Aikajakso1 tammik. 20192022
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