Gorab is a Golgi protein required for structure and duplication of Drosophila centrioles

Abstract

We demonstrate that a Drosophila Golgi protein, Gorab, is present not only in the trans-Golgi but also in the centriole cartwheel where, complexed to Sas6, it is required for centriole duplication. In addition to centriole defects, flies lacking Gorab are uncoordinated due to defects in sensory cilia, which lose their nine-fold symmetry. We demonstrate the separation of centriole and Golgi functions of Drosophila Gorab in two ways: first, we have created Gorab variants that are unable to localize to trans-Golgi but can still rescue the centriole and cilia defects of gorab null flies; second, we show that expression of C-terminally tagged Gorab disrupts Golgi functions in cytokinesis of male meiosis, a dominant phenotype overcome by mutations preventing Golgi targeting. Our findings suggest that during animal evolution, a Golgi protein has arisen with a second, apparently independent, role in centriole duplication.

Fig. 1. Gorab associates with both Centrosomes and Golgi

(a,b) Syncytial embryos expressing poly-Ubiquitin-GFP-Gorab and stained to reveal Asterless (Asl) and the trans-Golgi Golgin245 in a field of interphase (a) and mitotic (b) nuclei. Experiments repeated 3 times with similar results. Main scale bar, 5 µm; Inset scale bar, 1 µm(c) Cultured D.Mel-2 cells immuno-stained to reveal Gorab, GM130 (cis-Golgi), and Golgin245 (trans-Golgi).Experiment repeated 3 times with similar results. Main scale bar, 5 µm; Inset scale bar, 0.5 µm.(d) Cultured D.Mel-2 cells immunostained to reveal Gorab, dPLP (centrosome), and Golgin245 (trans-Golgi). Arrowheads indicate centrosomes. Experiment repeated 4 times with similar results. Main scale bar,5 µm; Inset scale bar, 0.5 µm.

Fig. 2. Gorab directly interacts and colocalises with Sas6.

(a)In vitro assay of the Gorab-Sas6 interaction. GST-Gorab was incubated with ³⁵S-Methionine-labelled Sas6 and resulting complexes subjected to SDS-PAGE and autoradiography. Experiment repeated 3 times with similar results (b) Schematic of Sas6 indicating fragments interacting (blue) or not interacting with Gorab (yellow). Arrow, minimal interacting fragment (GIR: Gorab Interacting Region). (c) 3D-SIM localization of endogenous Gorab (green) and Sas6 (red) throughout the D-Mel2 cell cycle relative to zone III marker, dPLP (blue). Arrowheads, site of procentriole formation. Experiment repeated 2 times with similar results. Scale bars,250 nm.

Fig. 3. gorab null mutant flies show female sterility and coordination defects.

(a) Schematic of gorab gene showing guide RNA binding sites (red) for CRISPR/Cas9 mutagenesis and primer binding sites for detection and sequencing of indicated gorab¹ and gorab² deletion mutants (blue). (b) Complementation tests for female fertility and coordination. 200 individuals tested per genotype. (c) Western blot of extracts from 5 adult females of indicated genotypes. Gorab revealed by antibody raised against its N terminal fragment (see Online Methods). α-tubulin, loading control. Experiment repeated once with similar result. (d) Climbing ability of wild type, gorab¹and rescued (N-terminal-GFP-tagged gorab^WT cDNA expressed from ubiqutin promoter in gorab¹ background) flies raised at 29 °C. Cohorts of 15 flies scored for number climbing 5 cm in 1 min.); Means ± s.e.m are shown for N=3 independent experiments, n= 15 flies/genotype investigated in each experiment. p values of two tailed unpaired t-tests are shown. p value in blue indicates significant difference (99% confidence interval) (e) Fertility of wild type, gorab¹and rescued (as in d) females individually mated with wild type males at 25°C over 6 days. Data points represent number of progeny of individual females. Means ± s.e.m are shown for n=15 females per genotype. p values of two tailed unpaired t-tests are shown. p value in blue indicates significant difference (99% confidence interval). Experiment repeated once with similar result. (f) Embryos from wild-type and gorab¹mutant mothers stained to reveal α-tubulin (green), centrosomin (CNN, red), and DNA (blue).; n=100 embryos were observed per genotype in two independent replicate experiments with similar results. Arrowhead, single centrosome of monopolar spindle. Scale bar, 100 µm (upper panel); 35 µm (lower panel). (g) Embryos from elav>gorab^WT,gorab¹ mothers stained to reveal α-tubulin (green), asterless (Asl, red), and DNA (blue). Arrowhead, third pole of a multipolar spindle; asterisks, spindle poles lacking centrioles. Scale bar, 10 µm. (h) Wing discs from wild type and gorab¹ larvae immunostained against dPLP to reveal centrosomes (red).Experiment repeated 3 times with similar results. Dashed lines, mitotic cells; Arrows, mitotic centrosomes. Scale bar,10 µm.

Fig. 4. Loss of daughter centriole and asymmetrical mother centrioles in gorab mutant ciliated neurons

(a)Schematic of femoral chordotonal organs (fChO) and stained, in wild type and gorab¹ mutant, to reveal transgenic Rootletin-GFP (green), dPLP in basal bodies (red), and actin in scolopale rods (white). Experiment repeated twice with similar results. Scale bar,10 µm. (b) Detail of basal body organisation in wild-type and gorab¹ stained as in a).Experiment repeated twice with similar results. Scale bar,10 µm. (c) Localisation of daughter centriole specific YFP-Centrobin (Cnb, green) in wild-type and gorab¹fChO basal bodies also stained to reveal dPLP (red) and Actin (grey). Experiment repeated twice with similar results. Scale bar, 10 µm. (d) Localization of GFP-tagged Gorab expressed in gorab¹ mutant background. Arrowheads, GFP-Gorab at basal bodies. Experiment repeated with similar result. Scale bar, 10 µm. (e) EM images of longitudinal sections wild-type and gorab¹fChOs. n= 11 ciliated cells were scored. Arrowheads, distal (DBB) and proximal (PBB) basal bodies. Scale bar, 0.2 µm. (f) Transverse sections of basal bodies, transition zones and cilia in wild-type and gorab¹imaged by TEM. n=16 ciliated cells were scored.Scale bar, 0.1 µm.

Fig. 5. Domain structure of Gorab.

(a)In vitro interaction of GST-Sas6 and ³⁵S-Methionine-labelled Gorab. Experiment repeated 2 times by different investigators with similar result. (b) Identification of minimal Sas6 interacting region (SIR) in Gorab (upper panel). Green bars, Gorab fragments interacting with Sas6. Red bars, Gorab fragments not interacting with Sas6. Green and red stripes, weak interaction. Arrow indicates minimal interacting fragment. Minimal Gorab deletion that abolishes Sas6 interaction (lower panel). Green bars, Sas6 interacting Gorab constructs; red bars, non-interacting constructs. Arrow, minimal deletion abolishing the interaction with Sas6, the Sas6 interacting domain (SID). (c) Sas6 interaction with ³⁵S-Methionine-labelled full length (FL) Gorab and indicated deletion variants. Experiment repeated independently by different investigator with similar result.(d) Prediction of coiled-coil region in Gorab, by Coils server by scanning windows of 14, 21 and 28 residues.(f) Comparison of domain topologies of Drosophila and human Gorab. Sas6 interacting domain (SID, purple), Golgi targeting domain (Goldi TD, green), and IGRAB domain are indicated. (g) Alignment of predicted coiled-coil region of Drosophila Gorab and five vertebrate species homologues. Pink, amino acids conserved between all; grey, similar amino acid groups; dark grey, single divergent amino acids; and yellow, a single divergent amino acid in Drosophila. Alignment generated with Clustal Omega. Purplebox, SID.

Fig. 6. Functional Domains of Gorab.

(a)Schematic of mutant gorab transgenes used in localization and rescue experiments. Asterisk, position of Val266 to Pro substitution. Gaps connected with thin line indicate extents of deletions. Tables summarizes rescue and localization experiments alongside indicated mutant forms. (b)Intracellular localization of Gorab mutant transgene products, Wing discs from larvae expressing the indicated GFP-tagged Gorab mutant protein stained to reveal dPLP (red) and Golgin245 (grey). White dashed lines, mitotic cells; yellow dashed lines, interphase cells with assembled Golgi. Experiments repeated 3 times with similar results. Arrowheads, centrosomes of mitotic cells. Scale bar, 5µm. (c) Rescue of climbing ability by ubiquitous expression of indicated N-terminally GFP-tagged transgene in gorab¹ flies raised at 29 °C. Means ± s.e.m for N=3 experiments, n= 15 flies/genotype per experiment. p values of two tailed unpaired t-tests are shown. p value in blue indicates significant difference (99% confidence interval) (d) Rescue of female sterility by ubiquitous expression of indicated N-terminally GFP-tagged transgenes in gorab¹ background.Data points represent the number of progeny of individual females. Mean ± s.e.m are shown for n=15 females per genotype. p values of two tailed unpaired t-tests are shown. p value in blue indicates significant difference (99% confidence interval). Experiment repeated once with similar result.

Fig. 7. Dominant male sterility and cytokinesis defects upon expression of C-terminally GFP tagged Gorab.

(a) Fertility of wild type, gorab¹ males and males expressing indicated transgenes. Data points represent the number of progeny of individual males. Means ± s.e.m are shown for n=15 males per genotype. p values of two tailed unpaired t-tests are shown. p value in blue indicates significant difference (99% confidence interval). Experiment repeated once with similar result(b) Phase contrast micrographs of spermatids from males expressing indicated transgenes. Arrowheads indicate nuclei; asterisk, the mitochondrial derivative Nebenkern. Experiment repeated 3 times with similar results. Scale bar, 10 µm. (c) Apical parts of testes from males expressing N- or C-terminally GFP-tagged Gorab and stained to reveal dPLP (red) and Golgin245 (grey). Experiment repeated 2 times with similar results. Scale bars, 20 µm and 5µm (inset). (d) Spermatocytes in meiotic telophase from of males expressing N- or C-terminally GFP-tagged Gorab (red) and stained to reveal tubulin (green), anillin (white) and DNA (blue). Experiment repeated 2 times with similar results. Scale bar, 10 µm.