Project Summary/Abstract ABSTRACT Mitochondria not only provide 90% of the energy required for reading these very lines but they are also responsible for the correct differentiation of the cells lining your gut. The recent recognition that mitochondria play an active role in stem cell fate decisions has moved them from passive power plants to active centers of cell signaling. Our inability to automatically track mitochondria and link them to the fate of a cell is in stark contrast to the increasing relevance of this organelle in stem cell fate decisions and intestinal diseases such as Crohn’s disease and colorectal cancer. Mitochondria have always been too small and too fast for volumetric imaging and tracking. Our preliminary data show that a combination of recently developed lattice light-sheet microscopy and our in-house developed computational image processing pipeline can succeed in the four-dimensional tracking of the entire cellular mitochondrial network. Here we propose to 1) expand our prototype into a general tool that can track mitochondria in multiple cell types and network morphologies, to 2) elucidate the coupling between mitochondrial network morphology and cellular fate, and to 3) create a predictive model that links the mitochondrial network morphology to its underlying signaling drivers and to the fate decisions that are caused by different morphologies. We propose to use intestinal epithelial organoids and the differentiation of intestinal stem cells to paneth cells as a model system for a mitochondria-directed fate determination. This proposal will open a new window into mitochondrial biology that will translate to a large number of mitochondria-related diseases such as cancer and neurological disorders such as epilepsy, Parkinson’s disease, and Alzheimer’s disease.

Project Narrative PUBLIC HEALTH RELEVANCE Abnormalities in renewing the outer cell layers of the intestine lead to disorders of the gastrointestinal tract such as inflammatory bowel diseases and colorectal cancer (respectively ~3 million and ~140 thousand US cases annually) . A change in the morphology of the mitochondrial network is a critical step in this renewal process. The aim of this proposal is to understand these mitochondrial network morphology changes and how they affect the fate of intestinal stem cells during differentiation to develop new therapies for these devastating diseases.