Spaced training activates Miro/Milton-dependent mitochondrial dynamics in neuronal axons to sustain long-term memory.

Neurons have differential and fluctuating energy needs across distinct cellular compartments, shaped by brain electrochemical activity associated with cognition. In vitro studies show that mitochondria transport from soma to axons is key to maintaining neuronal energy homeostasis. Nevertheless, whether the spatial distribution of neuronal mitochondria is dynamically adjusted in vivo in an experience-dependent manner remains unknown. In Drosophila, associative long-term memory (LTM) formation is initiated by an early and persistent upregulation of mitochondrial pyruvate flux in the axonal compartment of neurons in the mushroom body (MB). Through behavior experiments, super-resolution analysis of mitochondria morphology in the neuronal soma and in vivo mitochondrial fluorescence recovery after photobleaching (FRAP) measurements in the axons, we show that LTM induction, contrary to shorter-lived memories, is sustained by the departure of some mitochondria from MB neuronal soma and increased mitochondrial dynamics in the axonal compartment. Accordingly, impairing mitochondrial dynamics abolished the increased pyruvate consumption, specifically after spaced training and in the MB axonal compartment, thereby preventing LTM formation. Our results thus promote reorganization of the mitochondrial network in neurons as an integral step in elaborating high-order cognitive processes.

A polarized cell system amenable to subcellular resolution imaging of influenza virus infection.

The life cycle of influenza A viruses (IAV), and notably intracellular trafficking of the viral genome, depends on multiple interactions with the cellular cytoskeleton and endomembrane system. A limitation of the conventional cellular models used for mechanistic study and subcellular imaging of IAV infection is that they are cultured in two dimensions (2D) under non-polarizing conditions, and therefore they do not recapitulate the intracellular organization of the polarized respiratory epithelial cells naturally targeted by IAVs. To overcome this limitation, we developed an IAV-infection assay in a 3D cell culture system which allows imaging along the baso-lateral axis of polarized cells, with subcellular resolution. Here we describe a protocol to grow polarized monolayers of Caco2-TC7 cells on static Cytodex-3 microcarrier beads, infect them with IAV, and subsequently perform immunostaining and confocal imaging, or electron microscopy, on polarized IAV-infected cells. This method can be extended to other pathogens that infect human polarized epithelial cells.