The proper distribution of mitochondria is particularly vital for neurons because of their polarized structure and high energy demand. Mitochondria in axons constantly move in response to physiological needs, but signals that regulate mitochondrial movement are not well understood. Aside from producing ATP, Ca 2+ buffering is another main function of mitochondria. Activities of many enzymes in mitochondria are also Ca 2+ -dependent, suggesting that intramitochondrial Ca 2+ concentration is important for mitochondrial functions. Here, we report that mitochondrial motility in axons is actively regulated by mitochondrial matrix Ca 2+ . Ca 2+ entry through the mitochondrial Ca 2+ uniporter modulates mitochondrial transport, and mitochondrial Ca 2+ content correlates inversely with the speed of mitochondrial movement. Furthermore, the miro1 protein plays a role in Ca 2+ uptake into the mitochondria, which subsequently affects mitochondrial movement.M itochondria are dynamic organelles that constantly move within cells and undergo morphological changes in response to physiological needs (1-3). In neurons, mitochondria are abundantly present throughout different subcellular compartments. The machineries and signals that transport mitochondria from the cell body (where they are synthesized) to the terminal need to be carefully regulated because of the highly polarized structure and lengthy axon of a neuron (4-6). Defects in transport of mitochondria can cause deleterious effects on mitochondrial functions in different parts of neurons, and hence affect neuronal survival and function (2, 3). Recent efforts to investigate mitochondrial transportation have provided significant new information regarding mitochondrial mobility, especially the mechanical components that modulate mitochondrial transport; however, it remains unclear whether intrinsic signals inside of mitochondria also actively regulate mitochondrial movement.Mitochondrial transport is mediated by interactions of the mitochondrial adaptor proteins to the kinesin and dynein motors, as well as binding of the motor proteins to the cytoskeleton track (7,8). It was posited that cytoplasmic Ca 2+ level is a key regulator of mitochondrial trafficking in axons and dendrites, and that intracellular Ca 2+ influx impedes mitochondrial movement by affecting the overall interactions between the mitochondrial adaptor, motor, and cytoskeleton track (9, 10). Two different mechanisms for Ca 2+ -mediated stop in mitochondrial trafficking were proposed. Wang and Schwarz suggested that Ca 2+ binding to the EF hand motif of the mitochondrial adaptor protein miro1 recruits kinesin-1 motor and, hence, derails kinesin-1 from the microtubule track, thereby stopping mitochondrial transportation in axons (10). Macaskill et al. proposed that following Ca 2+ influx induced by glutamate or neuronal activity, Ca 2+ binding to the EF hand motif of the miro1 protein causes miro1 to dissociate from the kinesin-1 motor, and hence halts mitochondrial movement (11). Although the mechanisms propose...

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Mitochondrial Ca uptake is gated by the mitochondrial calcium uniplex, which is comprised of mitochondrial calcium uniporter (MCU), the Ca pore-forming subunit of the complex, and its regulators. Ca influx through MCU affects both mitochondrial function and movement in neurons, but its direct role in mitochondrial movement has not been explored. In this report, we show a link between MCU and Miro1, a membrane protein known to regulate mitochondrial movement. We find that MCU interacts with Miro1 through MCU's N-terminal domain, previously thought to be the mitochondrial targeting sequence. Our results show that the N-terminus of MCU has a transmembrane domain that traverses the outer mitochondrial membrane, which is dispensable for MCU localization into mitochondria. However, this domain is required for Miro1 interaction and is critical for Miro1 directed movement. Together, our findings reveal Miro1 as a new component of the MCU complex, and that MCU is an important regulator of mitochondrial transport. Mitochondrial calcium level is critical for mitochondrial metabolic activity and mitochondrial transport in neurons. While it has been established that calcium influx into mitochondria is modulated by mitochondrial calcium uniporter (MCU) complex, how MCU regulates mitochondrial movement still remains unclear. Here, we discover that the N-terminus of MCU plays a different role than previously thought; it is not required for mitochondrial targeting but is essential for interaction with Miro1, an outer mitochondrial membrane protein important for mitochondrial movement. Furthermore, we show that MCU-Miro1 interaction is required to maintain mitochondrial transport. Our data identify that Miro1 is a novel component of the mitochondrial calcium uniplex and demonstrate that coupling between MCU and Miro1 as a novel mechanism modulating both mitochondrial Ca uptake and mitochondrial transport.

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SummaryUnderstanding the molecular anatomy and neural connectivity of the brain requires imaging technologies that can map the 3D nanoscale distribution of specific proteins in the context of brain ultrastructure. Light and electron microscopy (EM) enable visualization of either specific labels or anatomical ultrastructure, but combining molecular specificity with anatomical context is challenging. Here, we present pan-Expansion Microscopy of tissue (pan-ExM-t), an all-optical mouse brain imaging method that combines ∼24-fold linear expansion of biological samples with fluorescent pan-staining of protein densities (providing EM-like ultrastructural context), and immunolabeling of protein targets (for molecular imaging). We demonstrate the versatility of this approach by imaging the established synaptic markers Homer1, Bassoon, PSD-95, Synaptophysin, the astrocytic protein GFAP, myelin basic protein (MBP), and anti-GFP antibodies in dissociated neuron cultures and mouse brain tissue sections. pan-ExM-t reveals these markers in the context of ultrastructural features such as pre and postsynaptic densities, 3D nanoarchitecture of neuropil, and the fine structures of cellular organelles. pan-ExM-t is adoptable in any neurobiological laboratory with access to a confocal microscope and has therefore broad applicability in the research community.Highlightspan-ExM-t visualizes proteins in the context of synaptic ultrastructureLipid labeling in pan-ExM-t reveals organellar and cellular membranesAll-optical, easily accessible alternative to correlative light/electron microscopyHigh potential for high throughput connectomics studies