Behavioral Responses to Hypoxia in Drosophila Larvae Are Mediated by Atypical Soluble Guanylyl Cyclases

The three Drosophila atypical soluble guanylyl cyclases, Gyc-89Da, Gyc-89Db, and Gyc-88E, have been proposed to act as oxygen detectors mediating behavioral responses to hypoxia. Drosophila larvae mutant in any of these subunits were defective in their hypoxia escape response—a rapid cessation of feeding and withdrawal from their food. This response required cGMP and the cyclic nucleotide-gated ion channel, cng, but did not appear to be dependent on either of the cGMP-dependent protein kinases, dg1 and dg2. Specific activation of the Gyc-89Da neurons using channel rhodopsin showed that activation of these neurons was sufficient to trigger the escape behavior. The hypoxia escape response was restored by reintroducing either Gyc-89Da or Gyc-89Db into either Gyc-89Da or Gyc-89Db neurons in either mutation. This suggests that neurons that co-express both Gyc-89Da and Gyc-89Db subunits are primarily responsible for activating this behavior. These include sensory neurons that innervate the terminal sensory cones. Although the roles of Gyc-89Da and Gyc-89Db in the hypoxia escape behavior appeared to be identical, we also showed that changes in larval crawling behavior in response to either hypoxia or hyperoxia differed in their requirements for these two atypical sGCs, with responses to 15% oxygen requiring Gyc-89Da and responses to 19 and 25% requiring Gyc-89Db. For this behavior, the identity of the neurons appeared to be critical in determining the ability to respond appropriately

Heterogeneous Intracellular Trafficking Dynamics of Brain-Derived Neurotrophic Factor Complexes in the Neuronal Soma Revealed by Single Quantum Dot Tracking

<div><p>Accumulating evidence underscores the importance of ligand-receptor dynamics in shaping cellular signaling. In the nervous system, growth factor-activated Trk receptor trafficking serves to convey biochemical signaling that underlies fundamental neural functions. Focus has been placed on axonal trafficking but little is known about growth factor-activated Trk dynamics in the neuronal soma, particularly at the molecular scale, due in large part to technical hurdles in observing individual growth factor-Trk complexes for long periods of time inside live cells. Quantum dots (QDs) are intensely fluorescent nanoparticles that have been used to study the dynamics of ligand-receptor complexes at the plasma membrane but the value of QDs for investigating ligand-receptor intracellular dynamics has not been well exploited. The current study establishes that QD conjugated brain-derived neurotrophic factor (QD-BDNF) binds to TrkB receptors with high specificity, activates TrkB downstream signaling, and allows single QD tracking capability for long recording durations deep within the soma of live neurons. QD-BDNF complexes undergo internalization, recycling, and intracellular trafficking in the neuronal soma. These trafficking events exhibit little time-synchrony and diverse heterogeneity in underlying dynamics that include phases of sustained rapid motor transport without pause as well as immobility of surprisingly long-lasting duration (several minutes). Moreover, the trajectories formed by dynamic individual BDNF complexes show no apparent end destination; BDNF complexes can be found meandering over long distances of several microns throughout the expanse of the neuronal soma in a circuitous fashion. The complex, heterogeneous nature of neuronal soma trafficking dynamics contrasts the reported linear nature of axonal transport data and calls for models that surpass our generally limited notions of nuclear-directed transport in the soma. QD-ligand probes are poised to provide understanding of how the molecular mechanisms underlying intracellular ligand-receptor trafficking shape cell signaling under conditions of both healthy and dysfunctional neurological disease models.</p></div

Intracellular trafficking dynamics of QD-BDNF complexes in the neuronal soma.

<p>Left panels: QD-BDNF trajectories (colored line) in relation to plasma membrane (left images) and magnified (yellow inset, right images). Middle panels: QD-BDNF colored trajectories in detail. Confined motion is circled in red (with corresponding MSD plots). Right panels: QD-BDNF position plots as a function of time, colored lines correspond to QD-BDNF trajectories in left panels, and gray bars represent average speeds. (A) QD-BDNF complexes undergoing intracellular trafficking that consists of phases of directed transport (blue curve in position plot), followed by confined motion (2 minutes), and a slower directed transport (yellow curve in position plot). MSD shows confined phase has a slow component (red circle). (B) QD-BDNF endosome trafficking that consists of an extended phase of confined motion (∼60 s; MSD plot, red circle), followed by rapid transport. (C) QD-BDNF endosome trafficking at a constant speed (35 s) along a curvilinear path (20 µm) within the neuron.</p

Photophysical properties of QD-BDNF-tagged TrkB receptors in live neurons.

<p>(A) Sensitive detection of discrete QD-BDNF complexes in NG neurons (200 pM, left panel) compared to diffuse TrkB labeling with Ax₄₈₈-BDNF (25 nM, right panel).Images are collapsed <i>z</i>-stacks (total cell height, 21–22 µm). QD-BDNFs are also detectable in single <i>z</i>-slice slices (yellow inset). (B) QD-BDNF complexes are detected inside single neuronal somata as single QDs. Representative fluorescent blinking profiles from two QD-BDNFs inside a neuron show square pulses of single ‘on-off’ QD blinking (red asterisks). <i>x</i>-axis  =  intensity, <i>y</i>-axis  =  time. (C) Highly-resolved spatial detection of QD-BDNFs accurately determines membrane vs. cytoplasmic location of QD-BDNF complexes in neurons. 3D models of location of single QD-BDNF complexes (green) with respect to Ax₄₈₈-WGA-labelled membrane (magenta) are computationally processed from raw fluorescence data (inset, 2.7 µm thick <i>z</i>-stack projection taken at neuronal mid-section). (D) QD-BDNF complexes within neuronal somata can be tracked for extended time durations. Single QD-BDNF vs. diffuse Ax₄₈₈-BDNF fuorescence inside a neuron over time (top). Average intensity as a function of excitation duration show quantitative comparison of extended fluorescence stability of QD₆₂₅-BDNF vs. Ax₄₈₈-BDNF (n = 10, middle). Under these extended recording sessions, corresponding DIC images of a representative neuron before and after 10 min of fluorescence excitation shows maintained morphological integrity (bottom). Scale bars: 20 µm (A), 10 µm (C, D).</p

QD-BNDF binds with high molecular specificity to TrkB in live neurons.

<p>(A) Single cell assays show QD-BDNF probes (400 pM) bind preferentially to TrkB-expressing nodose (NG) and cortical (CORT) neurons vs. non-TrkB expressing control N2A neural cell lines. Control streptavidin-QD (400 pM) treatment typically showed non-specific binding levels of 0–2 QD/cell (white arrows; see text for more details). Collapsed <i>z</i>-stack micrographs (total cell height at 22–25 µm for NG, 5–8 µm for CORT and N2A). Scale bars: 10 µm. (B) Population assays by QD fluorescence measurement in neuronal lysates. Neurons treated with either QD-BDNF or streptavidin-QD (400 pM; black bars, red bars/arrows respectively), washed, and lysed. Positive control  =  TrkB receptor, negative control  =  lysis buffer blank.</p

QD-BDNF diffusion dynamics at the plasma membrane, directed transport at neuronal processes, and internalization and recycling in the neuronal soma.

<p>Left panels: QD-BDNF trajectories (colored line) in relation to plasma membrane (left images) and magnified (yellow inset, right images). Middle panels: QD-BDNF colored trajectories in detail. Right panels: MSD or QD-BDNF position plots as a function of time, colored lines correspond to QD-BDNF trajectories in left panels, and gray bars represent speeds. (A) Trajectory and MSD plot for a QD-BDNF complex undergoing Brownian diffusion at the plasma membrane. (B) Trajectory and position plots of a QD-BDNF complex undergoing a mixture of diffusion and rapid transport along a neuronal process. Black arrows point to segments along the colored trajectory where rapid motor transport occurs; corresponding speeds are shown in adjacent position plots. (C)Trajectory and position plots of a QD-BDNF complex undergoing endocytosis that consists of phases of rapid transport as well as a long period of confined motion (red circle, MSD plot). (D) Trajectory and position plot showing a QD-BDNF complex recycling to the plasma membrane by rapid transport along a curvilinear path from deep within a NG neuron.</p