A distinct Golgi-targeting mechanism of dGM130 in Drosophila neurons

Abstract

GM130 is a matrix protein that is conserved in metazoans and involved in the architecture of the Golgi apparatus. In neurons, Golgi apparatus and dendritic Golgi outposts (GOs) have different compartmental organizations, and GM130 localization is present in both, indicating that GM130 has a unique Golgi-targeting mechanism. Here, we investigated the Golgi-targeting mechanism of the GM130 homologue, dGM130, using in vivo imaging of Drosophila dendritic arborization (da) neurons. The results showed that two independent Golgi-targeting domains (GTDs) with different Golgi localization characteristics in dGM130, together determined the precise localization of dGM130 in both the soma and dendrites. GTD1, covering the first coiled-coil region, preferentially targeted to somal Golgi rather than GOs; whereas GTD2, containing the second coiled-coil region and C-terminus, dynamically targeted to Golgi in both soma and dendrites. These findings suggest that there are two distinct mechanisms by which dGM130 targets to the Golgi apparatus and GOs, underlying the structural differences between them, and further provides new insights into the formation of neuronal polarity.

Keywords: GM130; Golgi apparatus; Golgi-targeting domains; dendrites; neuronal polarity.

Figure 1

Two dGM130 truncations differentially target to Golgi in soma and dendrites. (A) Schematic of the four regions of dGM130 and the N-/C- terminal truncation constructs. Constructs are described according to the position of their first (∆N) or last (∆C) amino acid residue in dGM130. (B) Schematic of the strategy for observing the localization of dGM130 and truncations on Golgi in vivo. The medial-Golgi in red is marked by ManII–TagRFPt. (C–I) Representative confocal images show the expression of intact dGM130 and the six truncations (green) together with Golgi marker ManII–TagRFPt (red) in the dendritic shaft, branch points and soma of C3da neurons. Two truncations in red box (D, G) showed the ability to bind to Golgi in either soma or both soma and dendrites. No green fluorescence signal could be detected with truncation ∆N550 (I). Scale bars: 10 μm in dendritic shaft, 5 μm in branch point and soma.

Figure 2

The first coiled-coil region in dGM130 specifically binds to the somal Golgi. (A) Upper panels: schematic of the coiled-coil structure and domain in ∆C550. Lower panel: schematic of dGM130 truncations used in this part. (B–D) Location of the three EGFP tagged truncations (green) when expressed in C3da neurons. ∆N100-∆C550 in (B), ∆N270-∆C510 in (C) and ∆C270 in (D). The Golgi was labeled by ManII–TagRFPt (red). Scale bars: 10 μm in dendrite, 5 μm in soma.

Figure 3

Restricted Golgi-targeting of the C-terminal regions in soma and dendrites. (A) Upper panels: schematic of the coiled-coil structure and domain in the C-terminal region. Lower panel: schematic of dGM130 truncations, ∆N530, ∆N608, ∆N636 and ∆N608-∆C765 and ∆N636-∆C765. (B–D) Representative images show the diffuse and punctate distribution of ∆N530 (B), ∆N608 (C) and ∆N636 (D) in soma and dendrites (green) in different neurons. The Golgi was labeled by ManII–TagRFPt (red). (E) The pie charts represent the proportion of the two distribution states (punctate and diffuse) of the three C-terminal truncations in the soma and dendrites. (F,G) Representative images show the distributions of two C-terminal truncations with the deletion of the C-terminus. ∆N608-∆C765 in (F) and ∆N636-∆C765 in (G). Scale bars: 10 μm in dendrite, 5 μm in soma.

Figure 4

∆N530 is dynamically enriched in Golgi. (A) Schematic of the strategy for time-lapse imaging of ∆N530 in Drosophila larva. (B,C) Representative images in left show the distribution of ∆N530 (green) in soma (B) and dendrites (C) at three time points. The Golgi was labeled by ManII–TagRFPt (red). Line profiles at right show the normalized fluorescence intensities of EGFP and TagRFPt along the dotted lines at left. The arrows indicate a Golgi unit in soma (B) and a dendritic GO (C) with the enrichment of ∆N530. (D) Quantitation of neurons with Golgi-targeting ∆N530 at different imaging durations. (E) Bar chart shows the proportions of the two types of C3da neurons, ddaA and ddaF, that present Golgi-targeting ∆N530 at different imaging durations. For all quantifications, data are shown as mean ± SEM. Unpaired two-sided Student’s t-test in (E) at each time duration found no significant difference. Scale bars: 3 μm in (B), 5 μm in (C).