SUMMARY Background and key gaps in knowledge: Mitochondria serve as hubs coordinating diverse aspects of cellular signaling, metabolism and inter-organelle dynamics. Our work during the ESI MIRA funding period has revealed multiple roles for MCL-1, an anti-apoptotic BCL-2 protein, in the regulation of mitochondrial morphology and cell state. Detailed mechanistic studies are still needed to understand this most basic aspect of MCL-1 function. The non-apoptotic function of MCL-1 converges and relies on the mitochondrial dynamics machinery. The dynamic nature of the mitochondrial network determines the ability of this organelle to regulate a variety of cellular processes that are tightly linked to cell fate and identity. Mitochondria coordinate these functions by coordinated remodeling, as well as by the physical and functional interaction with other organelles, such as peroxisomes, which also undergo remodeling to coordinate their function. The critical need for controlled mitochondrial and peroxisomal morphology in the maintenance of cellular homeostasis is highlighted by the wide spectrum of neurodevelopmental diseases associated with mutations in the regulators of organelle dynamics. We will combine our expertise in organelle and stem cell biology with state-of-the-art approaches to uncover the mechanisms by which mitochondrial and peroxisomal dynamics maintain neurogenesis. Description of recent progress by PI: In pluripotent stem cells, we reported that MCL-1 is required for maintaining an apoptotic priming state and pluripotency through its regulation of mitochondrial morphology (Rasmussen et al., 2018). In differentiated cells, we found that MCL-1 is also required to maintain cell identity (Rasmussen et al., 2020; Cleveland et al., 2021). This function which is shared with other members of the BCL- 2 family (Joshi et al., 2020) requires the fine-tuned coordination with the mitochondrial dynamics machinery (Rasmussen et al., 2018 and 2020; Joshi et al., 2020). The fundamental role of organelle remodeling in driving cell fate determination, led us to examine the impact of abnormal mitochondrial and peroxisomal fission on neurogenesis (Baum et al., 2020; Robertson et al., 2022, Robertson et al., 2023 in press), using an iPSC-based discovery platform optimized to study organelle biology (Romero-Morales et al., 2019, 2022a, 2022b). Overview of future research program: We propose to build upon our findings from the ESI MIRA by taking a multi-faceted approach aimed at three areas: 1) Continue elucidating how MCL1 controls stem cell identity and differentiation by regulating mitochondrial dynamics and metabolism. 2) Modeling the contribution of mitochondrial and/or peroxisomal fission to cellular transitions during neuronal differentiation. 3) Finally, we will identify the metabolic switches that are engaged by mitochondrial and peroxisomal remodeling during neuronal differentiation. Detailed elucidation of these mechanisms will reveal connections b...