Genetically-encoded markers for confocal visualization of single dense core vesicles

Abstract

Neuronal dense core vesicles (DCVs) store and release a diverse array of neuromodulators, trophic factors and bioamines. The analysis of single DCVs has largely been possible only using electron microscopy, which makes understanding cargo segregation and DCV heterogeneity difficult. To address these limitations, we developed genetically-encoded markers for DCVs that can be used in combination with standard immunohistochemistry and expansion microscopy, to enable single-vesicle resolution with confocal microscopy.

Keywords: Drosophila; co-transmission; confocal microscopy; expansion microscopy; large dense core vesicle; peptide modulators; vesicle markers.

Extended Data Figure 1:. Endogenous IA2 expression patterns in IA2::mEGFP fusion animals.

a, Schematic diagrams illustrate the CRISPR-engineered fusion of mEGFP to the C-terminus of IA2 in the IA2::mEGFP fly strain. b, GFP staining reveals widespread IA2 expression in the adult brain, third larval instar stage (L3) brain, L3 VNC, L3 body wall and NMJ (left to right). Scale bar: 40 μm, except for 20 μm in the NMJ image.

Extended Data Figure 2:. PDF localizes at the center of IA2-labeled DCVs.

a, Lower magnification images of expanded Drosophila brain show the overall profile of PDF-positive neurons (magenta, PDF staining), in which IA2::mEGFP is expressed (green, GFP staining). Scale bar: 40 μm. b, Super-resolution images from the outlined area in a. Expansion shows that IA2::mEGFP specifically labels the DCVs membrane, with PDF peptide located at the center of the DCVs. Arrowheads indicate examples of single DCVs. Scale bar: 2 μm. c, Enlarged images of the outlined area in b. Scale bar: 0.5 μm. In a-c, magenta indicates PDF and green indicates GFP.

Extended Data Figure 3:. UAS-IA2::mEGFP overexpression increases PDF peptide levels in the projections of sLNv neurons, whereas overexpression of UAS-trIA2::mEGFP does not.

a, Representative images of PDF signal in PDF>IA2::mEGFP, PDF>trIA2::mEGFP and control PDF-GAL4 fly brains. Close-up views of the dotted-line outlined regions are shown in the upper left of each image. Scale bar: 40 μm in each panel and 10 μm in each close-up image. b, Statistical analysis of PDF peptide levels in the outlined regions from a. Data are presented as mean ± SEM, and analyzed by one-way ANOVA with Bonferroni post hoc test as appropriate. Gray dots show individual values. Statistical differences are indicated by letters, and genotypes with the same letter are not significantly different.

Extended Data Figure 4:. Loss of IA2 does not alter SV levels but does reduce DCVs.

a, Cartoon of FRT-IA2::mEGFP-FRT, a line containing recombination sites that allows deletion of the last 8 exons of the IA2 gene with expression of flp recombinase. nos-GAL4 was used to drive recombination and create the null line used in panels b and c; PDF-GAL4 was used to delete IA2 specifically in LNvs in panel d. b, Representative images from WT and IA2-null brains expressing the SV marker Syn::tdTomato in LNvs. Loss of IA2 does not affect SV numbers. c, Representative images of WT and IA2-null brains expressing the DCV cargo ANF::mOrange2 in LNvs. Loss of IA2 significantly reduces DCV number. d, Representative images of brains from animals in which IA2 has been removed only from LNvs. Knock out of IA2 in LNvs reduces PDF staining. Scale bar: 40 μm for each panel in b-d. Data are presented as mean ± SEM, and analyzed by Student’s t test. n.s. indicated no difference; ***P < 0.001. Gray dots show individual values. A.U., arbitrary units.

Extended Data Figure 5:. IA2 does not associate with SVs.

C380-GAL4 was used to express UAS-trIA2::mEGFP in larval motor neurons. Green indicates trIA2::mEGFP, magenta indicates CSP, an SV marker. a, Representative image of a motor neuron axon showing no co-localization of trIA2::mEGFP with CSP. b, Representative image of boutons of the CCAP-positive motor neuron 12 showing that trIA2::mEGFP is excluded from regions containing SVs. Scale bar: 2 μm in each panel.

Extended Data Figure 6:. Co-packaging of CCAP and pBurs peptides within individual DCVs in the projections of CCAP-positive motor neurons.

The membrane of DCVs is labeled with trIA2::mEGFP. Green indicates trIA2::mEGFP, red indicates CCAP peptide and magenta indicates pBurs peptide. Scale bar: 2 μm in each panel.

Extended Data Figure 7:. IA2 expression in VAChT-positive (a) and VGluT-positive (b) neurons visualized by conditional tagging of the endogenous gene product.

Left panels show the anterior view, and right panels show the posterior view. Scale bar: 20 μm for each panel.

Extended Data Figure 8:. IA2 expression in DPM (a) and APL (b) neurons visualized by conditional tagging of the endogenous gene product.

To verify that both DPM and APL neurons normally express IA2, we used conditional tagging. Left panels show the anterior view, and right panels show the posterior view. Scale bar: 20 μm for each panel. Dashed white lines indicate the whole brain in a-b. The projection of APL neuron in the mushroom body region (left panel of b) and the calyx region (right panel of b) is outlined by dashed lines.

Fig. 1. Visualizing individual DCVs.

a, Schematic diagrams of Drosophila IA2 transgenes: In the UAS-IA2::mEGFP fly, mEGFP is fused to the C-terminus of IA2 (upper panel). In the UAS-trIA2::mEGFP fly, the C-terminal PTP domain is removed and replaced with mEGFP, followed by IA2’s Leu-motif (lower panel). TM: transmembrane domain, PTP: protein-tyrosine phosphatase domain. b, Cartoon and representative image showing projection (dotted lines) of a trIA2::mEGFP-expressing CCAP neuron. c, Sequential images showing vesicles (arrowheads) moving from head to tail (left panels) or tail to head (right panels). Scale bar: 2 μm in each panel. d, Image depicts vesicle movement along the motor neuron projection over time. Red: individual vesicles moving forward, green: vesicles moving retrogradely, white: stationary vesicles. e, Cartoon illustrating relative levels of vesicle movement. f, Cartoon illustrating the approximately 4.5-fold brain size increase, with 360–900 nm DCVs. g-h, PDF and sNPF peptides are co-packaged into the same DCVs. Lower panels of g show enlarged images of outlined area in upper panels. h shows close-up of the inset in lower panels of g. Green: mEGFP, magenta: PDF, red: sNPF in g-h. Scale bar: 40 μm in upper panels of g, 2 μm in lower panels of g and 0.5 μm in h. i, cartoon of co-packaging.

Fig. 2. Co-localization of DCV IA2 with VMAT and VGAT.

a, Schematic showing CRISPR insertion of Frt-stop-Frt-mEGFP in the 3’ end of the IA2 gene. b-c, IA2 expression in VMAT-positive (b) and VGAT-positive (c) neurons. Left panels show anterior view, right panels show posterior view. Scale bar: 20 μm. d, Co-localization of RFP::VMAT from endogenous VMAT locus with trIA2::EGFP. Left: DPM neuron projections in an expanded fly brain. Right: super-resolution images of the outlined area. Arrowheads indicate DCVs co-labeled by trIA2::mEGFP and RFP::VMAT. Scale bar: 20 μm on left, 2 μm on right. e, Co-localization of RFP::VGAT with trIA2::EGFP. Left: APL neuron projections in an expanded fly brain. Right: super-resolution images of the outlined area. Arrowheads indicate DCVs co-labeled by trIA2::mEGFP and RFP::VGAT. Scale bar: 20 μm on left, 2 μm on right.