Julia Schessner (Planegg / DE), Adam O'Neill (Planegg / DE), Fatma Uzbas (Planegg / DE), Daniel Itzhak (Planegg / DE), Magdalena Götz (Planegg / DE), Georg Borner (Planegg / DE)
Brain development encompasses a series of cellular differentiation steps ultimately leading to the striking morphology and function of cortical neurons. During this process the organelles within the cell undergo drastic changes in shape and function, driven by altered protein expression and subcellular localization of the proteome. To gain systematic insights into organellar rearrangements during neuronal differentiation, we applied the Dynamic Organellar Maps approach to generate spatial proteomes from three cell types of the developing telencephalon: dorsal and ventral neural stem cells and early maturing neurons. The approach combines subcellular fractionation by differential centrifugation with mass spectrometry-based proteomics and now also phosphorylation analysis, enabling subcellular localization prediction and analysis of relocalizing proteins. We determined the subcellular localization of 8,400 proteins, 3,000 of them at organelle level, along with 42,000 phosphorylation sites, including known and novel drivers for subcellular localization regulation. We identified several thousand full proteome and phosphorylation changes, revealing the main biological processes involved in differentiation, including known signaling pathways, cytoskeletal organization and cell-cell and cell-ECM interactions. At the subcellular level we identified several hundred localization changes ranging across all organelles. These included increased ER reticulation during neuron maturation and altered localization of ubiquitin and RNA interacting proteins between the two neural stem cell states. Since the three cell types have different mitotic turnover, many abundance changes and subcellular shifts were attributed to cell cycle regulation and shed light on how the transition to postmitotic neurons is regulated. Our data will be available online in an interactive database to facilitate data access and reusability for the community.