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. 2020 Jul 13;9:e58943. doi: 10.7554/eLife.58943

Figure 1. Endogenously-tagged γ-tubulin-GFP localises to a fraction of branchpoints and dendritic bubbles within class I da neurons.

(A) Fluorescent confocal images of the proximal region of a class I da neuron expressing mCD8-RFP (greyscale) within a living 3rd instar larva expressing endogenously-tagged γ-tubulin-GFP (green). Left panel (A) shows an overlay of the GFP and RFP signals, right panel (A’) shows only the GFP signal with the outline of the neuron drawn for clarity; white, blue and orange arrowheads indicate γ-tubulin-GFP puncta/accumulations within dendritic stretches, branchpoints, and dendritic bubbles, respectively. (B,C) Selected images of γ-tubulin-GFP-positive branchpoints (B) or dendritic bubbles (C) from living neurons as in (A). Individual mCD8-RFP channel images (greyscale) have been included for clarity. (D–F) Confocal images show branchpoints (D,E) or dendritic bubbles (F) from 3rd instar larvae expressing endogenous γ-tubulin-GFP fixed and immunostained for GFP (green), GM130 (magenta) and HRP (greyscale). γ-tubulin-GFP signal was rarely observed co-localising with GM130 and HRP signal (D), and frequently observed independent of the Golgi markers at both branchpoints (E) and dendritic bubbles (F).

Figure 1.

Figure 1—figure supplement 1. Class I and class IV da neuron morphology.

Figure 1—figure supplement 1.

(A) A fluorescent confocal image of the anterior region of a living 3rd instar larva where mCD8-RFP and mCD4-tdGFP are expressed in class I (magenta) and class IV (green) neurons, respectively. (B,C) Enlarged images of a class I (B) and a class IV (C) da neuron.
Figure 1—figure supplement 2. Ectopically expressed γ-tubulin-GFP is strongly enriched in branchpoints and dendritic bubbles and ectopically expressed ManII is not a reliable marker of Golgi outposts in class I da neurons.

Figure 1—figure supplement 2.

(A) Fluorescent confocal images of the proximal region of a class I da neuron expressing 221-gal4 > UAS-γ-tubulin-GFP (green) and 221-gal4 > UAS ManII-mCherry (magenta) within a living 3rd instar larva. Left panel shows an overlay of the GFP and mCherry channels, middle and right panels show the GFP and mCherry channels, respectively. Blue and orange arrowheads indicate enrichments of ectopically expressed γ-tubulin-GFP within branchpoints and dendritic bubbles, respectively. (B,C) Fluorescent confocal images of distal dendrites (B) or a soma (C) from different class I da neurons expressing 221-gal4 > UAS ManII-GFP fixed and immunostained with antibodies against HRP (greyscale) and GFP (green). Arrowheads in (B) indicate HRP puncta that represent internal membrane, at least some of which could be Golgi outposts. Note that UAS-ManII-GFP spreads into regions of the class I dendrites, including dendritic bubbles, that do not contain HRP puncta, suggesting that over-expressing ManII in class I neurons leads to ‘leaking’ of the protein outside of Golgi outposts within dendrites. In contrast, the UAS-ManII-GFP signal always colocalises with the HRP signal within soma (C).