Figure 1.
QD-BNDF binds with high molecular specificity to TrkB in live neurons.
(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 z-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.
Figure 2.
QD-BDNF probes activate TrkB signaling and induce neuronal survival.
(A) Embryonic (E) 16.5 NG neurons incubated with QD-BDNF probes showed survival rates equivalent to unmodified BDNF (400 pM), while incubations with streptavidin-QDs did not. Neurons incubated with unmodified BDNF, streptavidin-QD and QD-BDNF (72 hr) are identified by anti-neurofilament (NF68/180) antibodies. (B) Quantitation of results shown in (A). Neuronal survival induced by QD-BDNF was indistinguishable from unmodified BDNF (ANOVA, p<0.0001, Dunnett's post-hoc test, n = 4). Density of neurofilament (NF68/180) immunoreactive (IR) cells increased as a function of BDNF and QD-BDNF concentration. (C) QD-BDNF-dependent survival is TrkB specific as pre-block with anti-TrkB receptors and subsequent QD-BDNF treatment decreased NG neurons survival (ANOVA, p<0.0001, Dunnett's post-hoc test, n = 5). Scale bar: 50 µm.
Figure 3.
Photophysical properties of QD-BDNF-tagged TrkB receptors in live neurons.
(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 z-stacks (total cell height, 21–22 µm). QD-BDNFs are also detectable in single z-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). x-axis = intensity, y-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 z-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).
Figure 4.
Time course of internalization of QD-BDNF-TrkB complexes.
(A) QD-BDNF-TrkB complexes are visualized as fluorescent puncta (green) on the plasma membrane and within NG neurons. Z-stack projections (neuronal mid-section, 2.7 µm thick). WGA-Ax488 labeled plasma membrane (magenta). (B) Quantitative localization of single QD-BDNF complexes (green) in neuronal somata vs. plasma membrane (magenta) following QD-BDNF stimulation (200 pM, 5 min pulse). Each bar is the average of n = 8–10 cells; ∼30–50 QDs/cell for each time point. (C) Endosomal co-localization of QD-BDNF complexes (green) with anti-EEA1, and anti-LAMP1antibodies (Ax488, magenta) following a 5 min pulse of QD-BDNF (200 pM, green) at t = 20 and 60 min. Yellow insets show magnified co-localization (white). Control shown at topmost right is same as BDNF-QD + endosomal antibody labeling conditions except with omission of primary antibody. WGA-Ax488 labeled plasma membrane (cyan). Collapsed z-stack projections of neuronal mid-section (2.7 µm thick). Scale bars = 10 µm.
Figure 5.
QD-BDNF diffusion dynamics at the plasma membrane, directed transport at neuronal processes, and internalization and recycling in the neuronal soma.
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.
Figure 6.
Intracellular trafficking dynamics of QD-BDNF complexes in the neuronal soma.
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.