During differentiation, dramatic changes occur in the biochemical processes and the architecture of cells. The subcellular localization of proteins greatly impacts their function and also the transformations during cell differentiation are accompanied or facilitated by protein translocation. In the proposed research project, I want to systematically study how proteins translocate during the differentiation from neuronal stem cells into neurons and astrocytes. To this end, I aim to develop a general and efficient screening method for protein translocation during cell differentiation. I envision to identify protein translocation behavior that is essential for neurodevelopment and direct reprogramming, which I plan to confirm in brain organoids, which include diverse cell types. Additionally, I want to study the underlying mechanisms both initiating and initiated by protein translocation. The method will be based on fluorescence microscopy, which will allow me to observe the dynamics of protein localization in living, differentiating cells. To increase the efficiency of the screening approach, I want to implement a recently published multiplexing strategy, temporally multiplexed imaging (TMI). This method uses the photo-switching kinetics of the utilized fluorescent protein (FP) labels to deconvolute up to 6 signals in a single spectral channel. In this way, I can label several proteins per cell multiplying the efficiency of the method and enabling the screening of a large panel of proteins. I will label these proteins with TMI-compatible FPs through gene editing. Imaging of the edited cells throughout the differentiation process and subsequent automated image analysis will identify translocating proteins. The gained mechanistic knowledge about neuronal cell differentiation will improve our basic understanding of this process eventually facilitating the development of improved disease models as well as medical applications such as reprogramming.