The cellular distribution of mitochondria in response to stress and local energy needs is governed by the relative activities of kinesin and dynein. The mechanism for switching between these two opposite polarity microtubule motors remains unknown. Here, we coupled a novel cellular synthetic cargo transport assay with AlphaFold2-guided mutagenesis to identify a regulatory helix in the mitochondrial adaptor protein (TRAK) that mediates switching between kinesin- and dynein-driven transport. Differences in the helix sequence explain why two near-identical TRAK isoforms transport mitochondria in predominantly opposite directions. Phosphorylation of the regulatory helix by stress-activated kinases causes the activation of dynein and dissociation of kinesin. Our results reveal a molecular mechanism for coordinating the directional transport of mitochondria in response to intracellular signals.

Incentives tend to drive improvements in performance. But when incentives get too high, we can "choke under pressure" and underperform right when it matters most. What neural processes might lead to choking under pressure? We studied rhesus monkeys performing a challenging reaching task in which they underperformed when an unusually large "jackpot" reward was at stake, and we sought a neural mechanism that might result in that underperformance. We found that increases in reward drive neural activity during movement preparation into, and then past, a zone of optimal performance. We conclude that neural signals of reward and motor preparation interact in the motor cortex (MC) in a manner that can explain why we choke under pressure.

Anchoring goals to spatial representations enables flexible navigation but is challenging in novel environments when both representations must be acquired simultaneously. We propose a framework for how Drosophila uses internal representations of head direction (HD) to build goal representations upon selective thermal reinforcement. We show that flies use stochastically generated fixations and directed saccades to express heading preferences in an operant visual learning paradigm and that HD neurons are required to modify these preferences based on reinforcement. We used a symmetric visual setting to expose how flies' HD and goal representations co-evolve and how the reliability of these interacting representations impacts behavior. Finally, we describe how rapid learning of new goal headings may rest on a behavioral policy whose parameters are flexible but whose form is genetically encoded in circuit architecture. Such evolutionarily structured architectures, which enable rapidly adaptive behavior driven by internal representations, may be relevant across species.

Genetically encoded voltage indicators (GEVIs) allow optical recording of membrane potential from targeted cells in vivo. However, red GEVIs that are compatible with two-photon microscopy and that can be multiplexed in vivo with green reporters like GCaMP, are currently lacking. To address this gap, we explored diverse rhodopsin proteins as GEVIs and engineered a novel GEVI, 2Photron, based on a rhodopsin from the green algae Klebsormidium nitens. 2Photron, combined with two photon ultrafast local volume excitation (ULoVE), enabled multiplexed readout of spiking and subthreshold voltage simultaneously with GCaMP calcium signals in visual cortical neurons of awake, behaving mice. These recordings revealed the cell-specific relationship of spiking and subthreshold voltage dynamics with GCaMP responses, highlighting the challenges of extracting underlying spike trains from calcium imaging.

The endoplasmic reticulum (ER) is an important regulator of Ca2+ in cells and dysregulation of ER calcium homeostasis can lead to numerous pathologies. Understanding how various pharmacological and genetic perturbations of ER Ca2+ homeostasis impacts cellular physiology would likely be facilitated by more quantitative measurements of ER Ca2+ levels that allow easier comparisons across conditions. Here, we developed a ratiometric version of our original ER-GCaMP probe that allows for more quantitative comparisons of the concentration of Ca2+ in the ER across cell types and sub-cellular compartments. Using this approach we show that the resting concentration of ER Ca2+ in primary dissociated neurons is substantially lower than that in measured in embryonic fibroblasts.

Spontaneously blinking fluorophores permit the detection and localization of individual molecules without reducing buffers or caging groups, thus simplifying single-molecule localization microscopy (SMLM). The intrinsic blinking properties of such dyes are dictated by molecular structure and modulated by environment, which can limit utility. We report a series of tuned spontaneously blinking dyes with duty cycles that span two orders of magnitude, allowing facile SMLM in cells and dense biomolecular structures.

Techniques that enable precise manipulations of subsets of neurons in the fly central nervous system have greatly facilitated our understanding of the neural basis of behavior. Split-GAL4 driver lines allow specific targeting of cell types in Drosophila melanogaster and other species. We describe here a collection of 3060 lines targeting a range of cell types in the adult Drosophila central nervous system and 1373 lines characterized in third-instar larvae. These tools enable functional, transcriptomic, and proteomic studies based on precise anatomical targeting. NeuronBridge and other search tools relate light microscopy images of these split-GAL4 lines to connectomes reconstructed from electron microscopy images. The collections are the result of screening over 77,000 split hemidriver combinations. In addition to images and fly stocks for these well-characterized lines, we make available 300,000 new 3D images of other split-GAL4 lines.

To survive, animals must be able quickly infer the state of their surroundings. For example, to successfully escape an approaching predator, prey must quickly estimate the direction of approach from incoming sensory stimuli. Such rapid inferences are particularly challenging because the animal has only a brief window of time to gather sensory stimuli, and yet the accuracy of inference is critical for survival. Due to evolutionary pressures, nervous systems have likely evolved effective computational strategies that enable accurate inferences under strong time limitations. Traditionally, the relationship between the speed and accuracy of inference has been described by the "speed-accuracy tradeoff" (SAT), which quantifies how the average performance of an ideal observer improves as the observer has more time to collect incoming stimuli. While this trial-averaged description can reasonably account for individual inferences made over long timescales, it does not capture individual inferences on short timescales, when trial-to-trial variability gives rise to diverse patterns of error dynamics. We show that an ideal observer can exploit this single-trial structure by adaptively tracking the dynamics of its belief about the state of the environment, which enables it make more rapid inferences and more reliably track its own error but also causes it to violate the SAT. We show that these features can be used to improve overall performance during rapid escape. The resulting behavior qualitatively reproduces features of escape behavior in the fruit fly Drosophila melanogaster, whose escapes have presumably been highly optimized by natural selection.

Near-infrared (NIR) fluorescent reporters open interesting perspectives for multiplexed imaging with higher contrast and depth using less toxic light. Here, we propose nirFAST, a small (14 kDa) chemogenetic NIR fluorescent reporter, displaying higher cellular brightness compared to top-performing NIR fluorescent proteins. nirFAST binds and stabilizes the fluorescent state of synthetic cell permeant fluorogenic chromophores (so-called fluorogens), otherwise dark when free. nirFAST displays tunable NIR, far-red or red emission through change of fluorogen. nirFAST allows imaging and spectral multiplexing in live cultured mammalian cells, chicken embryo tissues and zebrafish larvae. Its suitability for stimulated emission depletion nanoscopy enabled protein imaging with subdiffraction resolution in live cells. nirFAST enabled the design of a two-color cell cycle indicator for monitoring the different phases of the cell cycle. Finally, bisection of nirFAST allowed the design of a chemically induced dimerization technology with NIR fluorescence readout, enabling the control and visualization of protein proximity.

bioRxiv preprint: https://doi.org/10.1101/2024.04.05.588310

Motor systems implement diverse motor programs to pattern behavioral sequences, yet how different motor actions are controlled on a moment-by-moment basis remains unclear. Here, we investigated the neural circuit mechanisms underlying the control of distinct courtship songs in Drosophila. Courting males rapidly alternate between two types of song: pulse and sine. By recording calcium signals in the ventral nerve cord in singing flies, we found that one neural population is active during both songs, whereas an expanded neural population, which includes neurons from the first population, is active during pulse song. Brain recordings showed that this nested activation pattern is present in two descending pathways required for singing. Connectomic analysis reveals that these two descending pathways provide structured input to ventral nerve cord neurons in a manner consistent with their activation patterns. These results suggest that nested premotor circuit activity, directed by distinct descending signals, enables rapid switching between motor actions.