Robust model-based analysis of single-particle tracking experiments with Spot-On.

Single-particle tracking (SPT) has become an important method to bridge biochemistry and cell biology since it allows direct observation of protein binding and diffusion dynamics in live cells. However, accurately inferring information from SPT studies is challenging due to biases in both data analysis and experimental design. To address analysis bias, we introduce 'Spot-On', an intuitive web-interface. Spot-On implements a kinetic modeling framework that accounts for known biases, including molecules moving out-of-focus, and robustly infers diffusion constants and subpopulations from pooled single-molecule trajectories. To minimize inherent experimental biases, we implement and validate stroboscopic photo-activation SPT (spaSPT), which minimizes motion-blur bias and tracking errors. We validate Spot-On using experimentally realistic simulations and show that Spot-On outperforms other methods. We then apply Spot-On to spaSPT data from live mammalian cells spanning a wide range of nuclear dynamics and demonstrate that Spot-On consistently and robustly infers subpopulation fractions and diffusion constants

Ketamine Inside Neurons?

Clinically used antidepressants, such as selective serotonin reuptake inhibitors (SSRIs), aid only a fraction of patients. Furthermore, even successful use of SSRIs takes 2 to 6 weeks of maintained medication. Depressed patients need faster help. Since 2000, several clinical studies report that depressed patients given subanesthetic doses of ketamine showed improvement within 2 hours. Trials continue for various dosing regimens, formulations, and populations. It is not understood what causes the therapeutic action of the SSRIs, and it is also not clear how ketamine exerts its effects. The best-known behavioral effect of ketamine is dissociative anesthesia. The drug retains Food and Drug Administration (FDA) approval for anesthesia in special populations as well as for veterinary use. The dissociative effects presumably arise from ketamine’s action to block N-methyl-D-aspartate (NMDA) receptor channels that have been opened by glutamate. The kinetics, equilibrium, and voltage sensitivity of open-channel blockers is a well-studied topic, and recent work shows how ketamine becomes trapped within the channel pore of NMDA receptors at local concentrations of ∼1 µM, which are expected to occur at the clinically effective antidepressant human doses. How might blockade of NMDA receptors lead to the antidepressant effects? Most studies emphasize signal transduction pathways that could be modulated by the locally decreased Ca^(2+) flux through NMDA receptors, especially extrasynaptic GluN2B subunit-containing NMDA receptors. In one series of experiments, the decreased Ca^(2+) flux led to decreased activity of eukaryotic elongation factor 2 kinase, which in turn desuppressed eukaryotic elongation factor 2. This ribosome-binding protein then increased translation of brain-derived neurotrophic factor (BDNF). Many other experiments show that BDNF is released during antidepressant action

A platform for brain-wide imaging and reconstruction of individual neurons

The structure of axonal arbors controls how signals from individual neurons are routed within the mammalian brain. However, the arbors of very few long-range projection neurons have been reconstructed in their entirety, as axons with diameters as small as 100 nm arborize in target regions dispersed over many millimeters of tissue. We introduce a platform for high-resolution, three-dimensional fluorescence imaging of complete tissue volumes that enables the visualization and reconstruction of long-range axonal arbors. This platform relies on a high-speed two-photon microscope integrated with a tissue vibratome and a suite of computational tools for large-scale image data. We demonstrate the power of this approach by reconstructing the axonal arbors of multiple neurons in the motor cortex across a single mouse brain.Howard Hughes Medical InstitutePublished versio

Nuclear microenvironments modulate transcription from low-affinity enhancers

Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. Here, we report that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogaster shavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation

Nuclear microenvironments modulate transcription from low-affinity enhancers

Transcription factors bind low-affinity DNA sequences for only short durations. It is not clear how brief, low-affinity interactions can drive efficient transcription. Here, we report that the transcription factor Ultrabithorax (Ubx) utilizes low-affinity binding sites in the Drosophila melanogaster shavenbaby (svb) locus and related enhancers in nuclear microenvironments of high Ubx concentrations. Related enhancers colocalize to the same microenvironments independently of their chromosomal location, suggesting that microenvironments are highly differentiated transcription domains. Manipulating the affinity of svb enhancers revealed an inverse relationship between enhancer affinity and Ubx concentration required for transcriptional activation. The Ubx cofactor, Homothorax (Hth), was co-enriched with Ubx near enhancers that require Hth, even though Ubx and Hth did not co-localize throughout the nucleus. Thus, microenvironments of high local transcription factor and cofactor concentrations could help low-affinity sites overcome their kinetic inefficiency. Mechanisms that generate these microenvironments could be a general feature of eukaryotic transcriptional regulation

Optimization of fluorophores for chemical tagging and immunohistochemistry of Drosophila neurons.

The use of genetically encoded 'self-labeling tags' with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.This work was supported by Howard Hughes Medical Institute (https://www.hhmi.org), the Medical Research Council (https://mrc.ukri.org; MRC file reference U105188491) and a European Research Council (https://erc.europa.eu) Consolidator grant (649111) to G.S.X.E.J., and a Royal Society (https://royalsociety.org) Dorothy Hodgkin Fellowship to S.C

Economics methods in Cochrane systematic reviews of health promotion and public health related interventions.

Peer reviewedPublisher PD