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. 2022 Nov 7;32(21):4549-4564.e6.
doi: 10.1016/j.cub.2022.08.054. Epub 2022 Sep 13.

In vivo characterization of Drosophila golgins reveals redundancy and plasticity of vesicle capture at the Golgi apparatus

Sung Yun Park  1 , Nadine Muschalik  1 , Jessica Chadwick  1 , Sean Munro  2
Affiliations

In vivo characterization of Drosophila golgins reveals redundancy and plasticity of vesicle capture at the Golgi apparatus

Sung Yun Park et al. Curr Biol. .

Abstract

The Golgi is the central sorting station in the secretory pathway and thus the destination of transport vesicles arriving from the endoplasmic reticulum and endosomes and from within the Golgi itself. Cell viability, therefore, requires that the Golgi accurately receives multiple classes of vesicle. One set of proteins proposed to direct vesicle arrival at the Golgi are the golgins, long coiled-coil proteins localized to specific parts of the Golgi stack. In mammalian cells, three of the golgins, TMF, golgin-84, and GMAP-210, can capture intra-Golgi transport vesicles when placed in an ectopic location. However, the individual golgins are not required for cell viability, and mouse knockout mutants only have defects in specific tissues. To further illuminate this system, we examine the Drosophila orthologs of these three intra-Golgi golgins. We show that ectopic forms can capture intra-Golgi transport vesicles, but strikingly, the cargo present in the vesicles captured by each golgin varies between tissues. Loss-of-function mutants show that the golgins are individually dispensable, although the loss of TMF recapitulates the male fertility defects observed in mice. However, the deletion of multiple golgins results in defects in glycosylation and loss of viability. Examining the vesicles captured by a particular golgin when another golgin is missing reveals that the vesicle content in one tissue changes to resemble that of a different tissue. This reveals a plasticity in Golgi organization between tissues, providing an explanation for why the Golgi is sufficiently robust to tolerate the loss of many of the individual components of its membrane traffic machinery.

Keywords: Drosophila melanogaster; Golgi apparatus; ntra-Golgi transport; vesicle tether.

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Conflict of interest statement

Declaration of interests S.M. is a member of the Advisory Board of Current Biology. The authors declare no competing interests.

Figures

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Graphical abstract
Figure 1
Figure 1
Tissue-specific golgin mitochondrial relocation strategy in Drosophila melanogaster (A) Schematic of the Drosophila intra-Golgi golgins. Coiled-coil regions were predicted using a 28-residue window, other domains are as indicated. TMF is located more toward the late Golgi than are GMAP and Golgin-84. (B) Strategy for tissue-specific relocation of golgins to mitochondria. A golgin (“golgin-mito”) is relocated to the mitochondria by replacement of its C-terminal Golgi-targeting domain (yellow box) with the TMD of monoamine oxidase (MAO). (C) Confocal micrographs of the proximal cells of L3 salivary glands labeled for the V5 epitope tag in the golgin-mito (magenta), mitochondrial complex V α (green), and Golgi marker Lava lamp (Lva; blue). Insets show zooms of the boxed regions in the merge. Proximal cells were chosen as they do not contain large glue granules, and so the Golgi is more clearly visible. One golgin-mito (TMF-mito) and the negative control (no UAS) are shown. See Figure S1B for micrographs of the other golgin-mito constructs and quantification of the data. (D) Transmission electron micrographs evidencing vesicle tethering by all intra-Golgi golgin-mito chimeras in L3 salivary glands. BioID2-mito is used as a negative control. Representative micrographs are shown from 4 sections obtained from each of 8 larvae per genotype. Bottom row: zoom-in views of boxed regions in micrographs above. (C and D) Scale bars: 5 μm in (C), 1 μm in (D, top), and 100 nm in (D, bottom). See also Figure S1.
Figure 2
Figure 2
Drosophila intra-Golgi golgin-mitos capture distinct subclasses of intra-Golgi vesicles in larval salivary glands (A) Left: confocal micrographs of L3 salivary glands expressing the indicated V5-tagged golgin-mito constructs and labeled for V5 (magenta), endogenous αManII::GFP (green), and Golgi marker Lva (blue). The endosome-to-Golgi tether Golgin-245-mito is used as an additional negative control to demonstrate that capture of Golgi-derived vesicles is specific to intra-Golgi golgins. Insets show zooms of the boxed regions in the merge. (A) Right: quantification of αManII::GFP relocation using ratios between area of cargo in mitochondria and total area of mitochondria. (B) As in (A), except labeled for endogenous Golgin-84 (green) and trans-Golgi marker Arl1 (blue). Quantifications were obtained by calculating the ratio between area of cargo in mitochondria to total area of cargo. GMAP-mito captures Golgin-84 more efficiently than TMF-mito. Golgin-84-mito was not tested as it is labeled by the Golgin-84 antibody. (C) As in (A), except labeled for endogenous Glg1 (green) and the Golgi marker Lva (blue). Quantifications were obtained by calculating the ratio between area of cargo in mitochondria to total area of cargo, demonstrating specificity of TMF-mito for Glg1-containing vesicles among the intra-Golgi golgin subset (left). Scale bars: 5 μm. For quantifications in (A)–(C), five larvae were examined with 2–17 cells segmented in each and quantified per cell. The individual cell ratios are shown with small, partially transparent, symbols, and the mean for each of the five larvae shown as large opaque points. Points are shaped and colored according to larval replicate, with the coding scheme of cell-by-cell points matching that of their corresponding larval points. The mean larval ratio (line) and SEM (bars) are plotted for each genotype. Data analyzed by one-way nested ANOVA using Tukey’s multiple comparisons, ns, not significant, ∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗∗p ≤ 0.0001. See also Figure S2 and Data S1.
Figure 3
Figure 3
Intra-Golgi golgin vesicle tethering activities are altered in larval wing imaginal discs (A) Confocal micrographs of L3 wing imaginal discs expressing the indicated V5-tagged golgin-mito constructs and labeled for V5 (magenta), endogenous αManII::GFP (green), and Golgi marker Lva (blue). Insets show enlargements of the boxed regions in the merge, and quantification of experiment is shown on the right. (B) As in (A), except labeled for V5 (magenta), endogenous Golgin-84 (green), and the Golgi marker Lva (blue). TMF-mito efficiently captures Golgin-84 cargo in contrast to what was observed in larval salivary glands. (C) As in (A), except labeled for V5 (magenta), endogenous Glg1 (green), and Golgi marker Lva (blue). Scale bars: 5 μm. Quantifications in (A)–(C) were obtained by calculating the ratio between area of cargo in mitochondria to total area of mitochondria. Cell-by-cell ratios from five discs each from a different larva were pooled and are shown as truncated violin plots. Ratios for discs (averaged cell-by-cell ratios) are plotted as large opaque points. The cells in discs were segmented and quantified with 68–495 cells depending on the disc. The mean larval ratio (line) and SEM (bars) are plotted for each genotype. Data analyzed by one-way nested ANOVA using Tukey’s multiple comparisons, ns, not significant, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0.0001. Full data in Data S1 and see also Figure S2.
Figure 4
Figure 4
Generation of mutants in the intra-Golgi golgins using CRISPR-Cas9 (A) Schematic of the Drosophila GMAP genomic locus. A gRNA targets the 5′ end of exon 3 to mutate all potential isoforms. The resulting allele GMAP_2.2.5 carries a 4-bp deletion. (B) Schematic of the Drosophila TMF genomic locus. A pair of gRNAs was designed to delete the coding region with small parts of both UTRs remaining. Allele TMF_Δ19 carries a precise deletion of 3,239 bp. (C) Schematic of Drosophila golgin-84. Two gRNAs were designed to delete the coding region, with the resulting allele golgin-84_ Δ13.3 having a +1 bp/−614 bp indel resulting in a protein lacking the N-terminal 178 residues, including the vesicle tethering motif. (D) Immunoblot to confirm that GMAP_2.2.5 is a strong hypomorph. The arrow highlights a weak band of ∼150 kDa that appears in the mutant. The GMAP antibody was raised against residues 778–1,057. (E) Immunoblot to show the loss of Golgin-84 in the golgin-84_ Δ13.3 mutant, confirming that it is a null allele. The antibody was raised against residues 1–300. See also Figure S3.
Figure 5
Figure 5
Phenotypic analyses of intra-Golgi golgin single, double, and triple mutants (A) Graph showing the total number of offspring from wild type (WT) and TMF mutant males. 20 crosses with two wild-type virgins and one individual male were set up for each genotype (means with error bars showing SEM). Statistical significance determined by unpaired Mann-Whitney test. Data in Data S1. (B–E) Confocal micrographs of the acrosome markers Snky-GFP (B and C) and GFP-LAMP (D and E) in green and DNA (DAPI) in magenta in developing spermatids of wild type (WT) and TMF mutant males. Acrosomes form in TMF mutants. (F and G) As in (B)–(E), but a Z-projection over a larger field of view to show that GFP-LAMP is partially mislocalized in the TMF mutant spermatids. (H and I) Schematics of testis development in the pupa (H) and a mature testis in the adult (I). APF, after puparium formation. (J and K) Images of testes (te) from wild type (J) and TMF mutants (K and K’) with morphological defects found in 4/27 cases. (L–N) Images of flies/pupae of WT (L), TMF,GMAP double (M and M’), and TMF,GMAP;;golgin-84 triple (N–N”) mutants. TMF,GMAP double mutants die during or shortly after eclosion, while the majority of TMF,GMAP;;golgin-84 triple mutants die as pupae. (O and P) Images of testes from TMF,GMAP double (O–O”) and TMF,GMAP;;golgin-84 triple (P–P”) mutants. These mutants completely fail to form normal testes (te) and exhibit striking morphological defects. (Q–U) Confocal micrographs of F-actin (phalloidin; gray scale) and DNA (DAPI; cyan) in testes from wild type (WT) and intra-Golgi golgin single, double, and triple mutants. Images were taken at low magnification to show the whole testes (low) or higher magnification to show the actin in the muscle sheath surrounding the testes (high). The muscle sheath is affected by loss of intra-Golgi golgin function. TMF single mutants occasionally lack muscles at the tip of the testis and show muscle irregularities. The muscle sheath is strongly affected in TMF,GMAP double and TMF,GMAP;;golgin-84 triple mutants and often fails to cover all of the testes. Occasionally, the muscle is striated instead of smooth (arrows in S”). Scale bars: (B–G) 10 μm. Scale bars: (Q–T) low magnification: 100 μm, high magnification: 10 μm. See also Figure S3.
Figure 6
Figure 6
Intra-Golgi golgin mutants retain Golgi polarity but show strong glycosylation defects (A–E) Confocal micrographs of L3 salivary gland cells labeled for the cis-Golgi marker GM130 (magenta) and the trans-Golgi marker GCC88 (green). Glands prepared from wild type (WT) or the indicated intra-Golgi golgin double and triple mutants. The insets show a higher magnification of the Golgi highlighted in the image. The dotted line illustrates a typical line profile used for quantification, with the graphs showing the normalized means of 10 line profiles of GM130 and GCC88 across the Golgi stack. Error bars, SD. Full data in Data S2. (F) Immunoblots of total protein extracts from L3 salivary glands from wild type (WT) and intra-Golgi golgin mutants, probed for the heavily glycosylated mucin Sgs3. (G) Lectin Vicia villosa agglutinin (VVA) staining of L3 salivary gland cells of wild type and a mutant lacking GMAP, Golgin-84, and TMF. VVA stains the Golgi in wild-type glands, with specificity confirmed by use of the competing sugar N-acetyl-D-galactosamine (GalNAc) at 0.3 M. Scale bars: 2 μm in (A)–(E) and 5 μm in (G). See also Figures S4–S6.
Figure 7
Figure 7
TMF-mito acquires Golgin-84 vesicle capture function in GMAP mutant larval salivary glands (A) Confocal micrographs of L3 salivary glands expressing golgin-mito constructs, or the BioID2-mito negative control, and labeled for the V5 tag (magenta), endogenous Golgin-84 (green), and trans-Golgi marker Golgin-245 (blue). Glands are from wild type (first three rows) and GMAP mutants (bottom row). Insets are zooms of the boxed regions in the merge. Scale bars: 5 μm. (B) Quantification of the mitochondrial relocalization of Golgin-84 in (A), using the ratio between the area of cargo in mitochondria and total area of cargo. Cell-by-cell ratios are plotted as small, partially transparent, points; larval ratios (averaged cell-by-cell ratios) are plotted as large opaque points. Points are shaped and colored according to larval replicate, with the format of cell-by-cell points matching that of their corresponding larval means. The mean larval ratio (line) and SEM (bars) are plotted for each genotype. Data analyzed by one-way nested ANOVA using Tukey’s multiple comparisons, ns, not significant, ∗∗p ≤ 0.01 and ∗∗∗∗p ≤ 0.0001. n = 5 larvae (4 for BioID2-mito), with 6–19 cells segmented per larvae. Full data in Data S1. (C) Confocal micrographs of L3 salivary gland cells prepared from GFP::TMF control or GMAP,GFP::TMF mutants and labeled for GFP::TMF (green) and the medial-Golgi marker Golgin-84 (magenta). Insets show a zoom of the Golgi highlighted in the image. The dotted line illustrates a typical line profile used for quantification, with the graphs showing the normalized means of 15–30 line profiles of the marker proteins across the Golgi stack. Error bars, SD. Scale bars: 2 μm, full data in Data S2. See also Figure S7.
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References

    1. Schjoldager K.T., Narimatsu Y., Joshi H.J., Clausen H. Global view of human protein glycosylation pathways and functions. Nat. Rev. Mol. Cell Biol. 2020;21:729–749. - PubMed
    1. Sandhoff R., Sandhoff K. Emerging concepts of ganglioside metabolism. FEBS Lett. 2018;592:3835–3864. - PubMed
    1. Moremen K.W., Tiemeyer M., Nairn A.V. Vertebrate protein glycosylation: diversity, synthesis and function. Nat. Rev. Mol. Cell Biol. 2012;13:448–462. - PMC - PubMed
    1. Glick B.S., Luini A. Models for Golgi traffic: a critical assessment. Cold Spring Harb. Perspect. Biol. 2011;3:a005215. - PMC - PubMed
    1. Munro S. What is the Golgi apparatus, and why are we asking? BMC Biol. 2011;9:63. - PMC - PubMed

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