A Stable Sub-Complex Between GCP4, GCP5 and GCP6 Promotes The

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RESEARCH ARTICLE A stable sub-complex between GCP4, GCP5 and GCP6 promotes the assembly of γ-tubulin ring complexes Laurence Haren, Dorian Farache*, Laurent Emorine and Andreas Merdes‡

ABSTRACT grip1 and grip2 motifs, corresponding to the N-terminal and γ-Tubulin is the main protein involved in the nucleation of C-terminal halves of GCP4, the smallest GCP (Gunawardane et al., microtubules in all eukaryotes. It forms two different complexes with 2000; Guillet et al., 2011). The crystallographic structure of GCP4 α proteins of the GCP family (γ-tubulin complex proteins): γ-tubulin shows that these domains correspond to bundles of -helices. The small complexes (γTuSCs) that contain γ-tubulin, and GCPs 2 and 3; other GCPs contain additional specific sequences, mainly at the and γ-tubulin ring complexes (γTuRCs) that contain multiple γTuSCs extreme N-terminus or in the region that links the grip1 and grip2 in addition to GCPs 4, 5 and 6. Whereas the structure and assembly motifs, as in GCPs 5 and 6 (Guillet et al., 2011; Farache et al., 2016). properties of γTuSCs have been intensively studied, little is known Depletion of GCP2 or GCP3 leads to severe spindle about the assembly of γTuRCs and the specific roles of GCPs 4, 5 abnormalities, and depleted cells are not viable. Depletion of and 6. Here, we demonstrate that two copies of GCP4 and one copy GCP4, 5 or 6 can be tolerated in fission yeast or in somatic cells of Drosophila each of GCP5 and GCP6 form a salt (KCl)-resistant sub-complex but not in vertebrates, where removal of either of these γ within the γTuRC that assembles independently of the presence of GCPs prevents the formation of the TuRC and provokes spindle γTuSCs. Incubation of this sub-complex with cytoplasmic extracts defects (Anders et al., 2006; Vérollet et al., 2006; Farache et al., containing γTuSCs leads to the reconstitution of γTuRCs that are 2016; Cota et al., 2017). Rescue experiments with chimeric proteins competent to nucleate microtubules. In addition, we investigate containing N-terminal domains fused to C-terminal domains of a sequence extensions and insertions that are specifically found at the different GCP showed that the chimeras rescued the defects as long N-terminus of GCP6, and between the GCP6 grip1 and grip2 motifs. as they carried the N-terminal domain of the depleted GCP (Farache We also demonstrate that these are involved in the assembly or et al., 2016). Thus, the GCPs are not functionally redundant, despite stabilization of the γTuRC. their structural similarities, and the function of individual GCPs are specified by their N-terminal domains. GCP2 and GCP3 interact KEY WORDS: Microtubule nucleation, γ-Tubulin ring complex, laterally through their N-terminal domains in γTuSC helices, Centrosome whereas γ-tubulin molecules are bound by the C-terminal domains (Kollman et al., 2010). FRET experiments also demonstrated a INTRODUCTION direct lateral interaction between GCP4 and GCP5 through their γ-Tubulin is a protein involved in the nucleation of microtubules. N-terminal domains (Farache et al., 2016), suggesting that the It assembles with so-called ‘gamma-tubulin complex proteins’ N-terminal domains specify lateral binding partners and, thereby, (GCPs) into multiprotein complexes of two different sizes. position the GCPs within the γTuRC helix. A ‘γ-tubulin small complex’ (γTuSC) comprises two molecules of In this context, the specific functions of GCPs 4, 5 and 6 in γ-tubulin that are bound by GCPs 2 and 3. A much larger ‘γ-tubulin γTuRC assembly need to be investigated. Because these proteins are ring complex’ (γTuRC) is formed by multiple γTuSCs that associate present only in one or two copies per complex (Murphy et al., 2001; with additional GCPs 4, 5 and 6, and several smaller accessory Choi et al., 2010), and because the rescue experiments with proteins into a helical structure of 2 MDa (Kollman et al., 2011; chimeras suggest that GCPs 4, 5 and 6 occupy non-random Farache et al., 2018). A few eukaryotes, such as Saccharomyces positions, their localization within the complex is of particular cerevisiae or Candida albicans contain only GCPs 2 and 3. In these interest. During the course of this work, three studies have described organisms, multiple γTuSCs are assembled that form a helix with the structure of native γTuRCs by cryo-electron microscopy, and the help of additional proteins, such as Spc110 or Mzt1 (Kollman have found a lateral association of four γTuSCs, bound to a lateral et al., 2010; Erlemann et al., 2012; Lyon et al., 2016; Lin et al., array of GCP4/GCP5/GCP4/GCP6, to which an additional γTuSC 2014, 2016). Most eukaryotes, however, express the full was associated (Consolati et al., 2019 preprint; Liu et al., 2020; complement of GCPs 2, 3, 4, 5 and 6, and form γTuRCs. It is Wieczorek et al., 2020). Altogether, this has raised the question believed that all these GCPs have similar structures. They are whether GCPs 4, 5 and 6 form assembly intermediates equivalent to characterized by sequence homology of two conserved regions, the γTuSCs. In this study, we demonstrate biochemically that GCPs 5 and 6, and two copies of GCP4 together form a stable, KCl-resistant γ Centre de Biologie du Développement, Centre de Biologie Intégrative, CNRS- core within the TuRC, which can be purified and drives the UniversitéToulouse III, 31062 Toulouse, France. assembly of free γTuSCs into a γTuRC that is competent to nucleate *Present address: Rosenstiel Basic Medical Sciences Research Center, Brandeis microtubules. University, Waltham, MA 02453, USA.

‡Author for correspondence ([email protected]) RESULTS γTuRC-specific GCPs 4, 5 and 6 form a core complex resistant D.F., 0000-0003-3715-9739; A.M., 0000-0002-3739-2728 to treatment with high concentrations of KCl γ Handling Editor: David Glover To determine how GCPs 4, 5 and 6 assemble within the TuRC, and

Received 21 January 2020; Accepted 26 March 2020 to examine whether γTuSC-like intermediates are formed by these Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs244368. doi:10.1242/jcs.244368 proteins, we destabilized the γTuRC by treating HeLa cytoplasmic The core of GCP4/5/6 forms independently of γTuRC-assembly extracts with increasing concentrations of KCl. GCPs 4, 5 or 6 were To determine whether GCPs 4, 5 and 6 associate independently of immunoprecipitated and all interacting GCPs were identified by γTuRCs, we used RNA interference (RNAi) to deplete GCP2 western blotting (Fig. 1A, Fig. S1A). Whereas the full set of GCPs (Fig. 2A). Consistent with previously published findings on the co- was immunoprecipitated at 100 mM KCl, we observed an regulation of GCPs (Vérollet et al., 2006), we noticed that siRNAs increasing loss of γTuSCs from the immunoprecipitate at higher against individual GCPs also affected the protein levels of others concentrations of KCl. At 500 mM KCl, GCPs 4, 5 and 6 remained (GCP2 siRNA also decreased levels of GCP3, and levels of GCP6 the major constituents of the immunoprecipitate, irrespective of the were slightly affected by several siRNAs). Most importantly, loss of antibody used for precipitation. This indicated that the binding GCP2 caused the disappearance of γTuRCs in sucrose gradients affinities between GCPs 4, 5 and 6 are stronger than their affinities (Fig. 2B, Fig. S2) and GCPs 4, 5 and 6 immunoprecipitated together to γTuSCs. Consistently, γTuRCs have previously been found to with γ-tubulin in intermediate fractions. Here, the sub-complex dissociate and to release γTuSCs in response to treatment with KCl peaked at a sucrose density of ∼21% (fraction 5), whereas at high concentrations (Moritz et al., 1998; Oegema et al., 1999). disassembly of γTuRCs at 500 mM KCl yielded the strongest To investigate whether GCPs 4, 5 and 6 are associated in a single peak at 17% sucrose in fraction 4 (compare Fig. 1C with Fig. 2C). sub-complex within the γTuRC, we fractionated cytoplasmic This difference might be due to the loss of interactors at high extracts on gradients of 5–40% sucrose containing KCl (100 mM concentrations of KCl. or 500 mM; Fig. 1B, Fig. S1B). We noticed that the cell line used in Since Wieczorek et al. (2020) and Liu et al. (2020) suggested the these experiments (HeLa Flp-In T-REx) contains high levels of existence of γTuSC-like structures, containing complexes GCP4/5 GCP4 protein, part of which sedimented independently of γTuRCs or GCP4/6, we tested how the depletion of individual GCPs 4, 5 or 6 in low-density-fractions (Fig. S1B; Farache et al., 2016). To test affects the composition of the GCP4/5/6 sub-complex, and whether for mutual binding of GCPs 4, 5 and 6, we performed the proposed γTuSC-like structures do, indeed, exist. We depleted immunoprecipitation from each individual fraction in the gradient each of these three GCPs individually by using RNAi, which, in by using antibodies against GCP4 (Fig. 1C) or GCP5 (Fig. 1E, left each case, resulted in the loss of γTuRCs and the accumulation of panel). At 100 mM KCl, γTuSCs and γTuRCs sedimented mainly γTuSCs, as seen in sucrose gradients (Fig. 2A,B, Fig. S2). Next, we in fractions 3 and 7, at a mean density of 13% and 28% sucrose, immunoprecipitated the proteins from the gradient fractions, using respectively. GCP2 to GCP6 co-immunoprecipitated efficiently antibodies against GCP4, GCP5 or GCP6 (Fig. 2D). GCPs 4, 5 and from fraction 7 (Fig. 1C, first lane). By contrast, at 500 mM KCl, we 6 were systematically co-precipitated in the absence of GCP2. observed the disappearance of γTuRCs, and immunoprecipitation GCP4 and GCP6 co-precipitated together even in the absence of revealed that GCPs 4, 5 and 6, and γ-tubulin associated with each GCP5. However, GCP5 bound to GCP4 or GCP6 only in the other in fractions of intermediate size, excluding GCPs 2 and 3 presence of all three proteins. This suggests a hierarchy of assembly, (Fig. 1C,E; fractions 4 and 5 at 15–23% sucrose). Interestingly, with a small and stable GCP4/6 complex enabling the association when the concentration of KCl was decreased to 200 mM or with GCP5. 100 mM KCl before gradient sedimentation, the peaks of GCP2 to GCP6 shifted back to higher fractions, and GCPs 4, 5 and 6 Excess levels of GCPs 4, 5 and 6 sequester γTuSCs and re-associated with GCPs 2 and 3 in fractions 6 and 7 at 23–30% incorporate them into γTuRCs sucrose (Fig. 1D,E, Fig. S1C). This suggests that the sub-complex We tested whether elevated amounts of GCPs 4, 5 and 6, or of GCPs 4, 5 and 6 can re-assemble with γTuSCs into γTuRCs. combinations thereof, promote the sequestration of free γTuSCs and We then purified the sub-complex comprising GCP4, GCP5 and their incorporation into γTuRCs. The Flp-In T-REx cell line allows GCP6 (hereafter referred to as GCP4/5/6) to obtain an estimation of inducible expression of transgenes in response to treatment with its size and stoichiometry. We constructed a HEK293 cell line doxycycline (Tighe et al., 2008). This cell line was used to induce expressing GCP6 with a C-terminal GST-hexa-histidine (6his) high levels of GCP5 and/or GCP6, on the high endogenous tag, by modifying the TUBGCP6 gene using CRISPR-Cas9. background of GCP4 (Fig. S1B). GCP6-inducible clones were Consecutive steps of affinity-purification over glutathione generated by stable transfection, whereas overexpression of GCP5 sepharose and over Ni-NTA agarose were carried out in the was obtained in response to transient transfection of wild-type or presence of 500 mM KCl. The eluate was analyzed by SDS–PAGE GCP6-inducible cells. This created cells producing an excess of and silver staining, and scanned signals were normalized to the either GCP4 alone, or GCPs 4 and 5, GCPs 4 and 6 or GCPs 4, 5 and molar lysine content of each protein (Dion and Pomenti, 1983; 6 (Fig. 3A). Excess of GCPs 4 and 5 did not alter the ratio between Fig. 1F). We realized that small amounts of GCP2 or GCP3 are still γTuSCs and γTuRCs (Fig. 3B,C). By contrast, excess of GCPs 4 present in the purified sub-complex, which were probably and 6 reduced the peak of γTuSCs (fraction 3), and shifted the underestimated in western blotting experiments (Fig. 1F). protein signals of GCPs 2, 3 and γ-tubulin towards high-density Quantifications from four independent experiments indicated a fractions (Fig. 3B–D; fractions 4–7, 15–30% sucrose). molar ratio of two copies of GCP4, one of GCP5 and one of GCP6, Immunoprecipitation of the gradient fractions revealed that this with three copies of γ-tubulin and one equivalent of either GCP2 or shift was due to the binding of γTuSCs to assemblies of GCPs 4 and GCP3. Although it was impossible to distinguish GCP2 and GCP3 by 6 (Fig. 3G). This is consistent with the idea of GCPs 4 and 6 forming one-dimensional electrophoresis, western blotting proved that both an intermediate that can bind γTuSC, whereas GCP5 cannot proteins were present within a single band (Fig. 1F,G). Taking this efficiently bind to GCP4 in the absence of GCP6 (Fig. 3F). When an stoichiometry into account, the estimated size of the complex should excess of all three GCPs (i.e. GCPs 4, 5 and 6) was created, be ∼750 kDa. Size-exclusion chromatography confirmed elution at a immunoprecipitation of GCP5 yielded co-precipitation of GCPs 4 size close to thyroglobulin (∼700 kDa) (Fig. 1G). These results and 6 (Fig. 3H), supporting the idea of a hierarchy of assembly, as suggest that dissociation by salt (i.e. KCl) produced a heterogeneous proposed above, with GCP4/6 as the ultimate core. Interestingly, mixture, containing a core complex made of GCPs 4, 5 and 6, high amounts of GCPs 4, 5 and 6 resulted in the disappearance of stochastically associated with either GCP2 or GCP3, and γ-tubulin. the peak of γTuSCs, and led to an increase of γTuRCs (Fig. 3B–E). Journal of Cell Science

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Fig. 1. See next page for legend. Journal of Cell Science

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Fig. 1. Isolation of a GCP4/5/6 sub-complex at high KCl levels. (A) Western failed to nucleate any microtubules. Quantification of the number of blots showing co-immunoprecipitation experiments of GCPs 4, 5 and 6 from microtubules nucleated per bead showed that reconstituted GCP4/5/ HeLa cell cytoplasmic extracts. First panel: Soluble extract. * indicates a 6-beads with γTuSCs reached up to 78% of the nucleation capacity degradation product of GCP6. Second to fourth panels: Extracts were γ supplemented with increasing concentrations of KCl (100–750 mM) and of the positive control, i.e. beads bound to native TuRC (Fig. 4F). incubated with antibodies against GCP4 (IPαGCP4), GCP5 (IPαGCP5) or The difference in nucleation capacity compared to the positive GCP6 (IPαGCP6). Immunoprecipitated proteins were separated by control might reflect a decrease in the number of bound complexes SDS–PAGE, blotted and probed with antibodies against the different GCPs (in particular after GCP2-depletion). Altogether, our results showed and γ-tubulin. Brackets on the right indicate the KCl resistance of GCPs 4, 5 that the GCP4/5/6 sub-complex can trigger the assembly of and 6 in the co-immunoprecipitate. (B) Sucrose-gradient fractionation of HeLa nucleation-competent γTuRCs. cell cytoplasmic extract, prepared with 100 mM KCl and centrifuged (top) or adjusted to a final concentration of 500 mM KCl before centrifugation (bottom). γ Levels of γTuSCs and γTuRCs in the fractions were visualized by western Role of the GCP6-specific insertions in TuRC assembly blotting using anti-γ-tubulin antibody. At 100 mM KCl, fraction 3 (13% sucrose) Since GCP6 appears to play a central role in the assembly of the contains the majority of γTuSCs, fraction 7 (28% sucrose) the majority of γTuRC, and since this protein is characterized by extensive non-grip γTuRCs. At 500 mM KCl, the peak of γTuRCs was lost. Thyroglobulin (Tg, 19.4 sequences in its N-terminal region and in the domain connecting the S) sediments in fraction 5 (21% sucrose). (C) Immunoprecipitation of GCP4 grip1 and grip2 motifs (Figs 5A and 6A), we constructed deletion – from fractions 2 7 of the sucrose gradient at 500 mM KCl. GCP4 mutants, to evaluate the importance of these sequences. The co-precipitated with GCP5, GCP6 and γ-tubulin in fractions 4–5 (between 15% and 23% sucrose). In the first lane, co-precipitation of all GCPs from fraction 7 deletion mutants were expressed in HeLa cell lines in an inducible of the 100 mM KCl gradient is shown as a positive control. (D) Sucrose- manner, following depletion of endogenous GCP6 by using RNAi. gradient fractionation of a HeLa cell cytoplasmic extract adjusted to 500 mM We measured the potential of the mutants to rescue the assembly and KCl before centrifugation on a sucrose gradient containing 200 or 100 mM KCl. function of γTuRCs by quantifying the amount of γTuRCs in The gradient containing 500 mM KCl is shown in B, bottom row. sucrose gradients, and the formation of bipolar spindles in mitotic (E) Immunoprecipitation of GCP5 from fractions 3–7 of gradients containing γ cells. In agreement with published data, depletion of GCP6 induces 500, 200 or 100 mM KCl. Co-precipitation of GCP4, GCP6 and -tubulin can be γ γ observed to peak in fractions 4 and 5 (15–23% sucrose) at 500 mM KCl, in the loss of TuRCs, the delocalization of -tubulin from fractions 5 and 6 (19–26% sucrose) at 200 mM KCl, and in fraction 6 and centrosomes and from the mitotic spindle, and inhibits the 7 (23–30% sucrose) at 100 mM KCl. The shift of the peaks towards later separation of the spindle poles (Bahtz et al., 2012; Farache et al., fractions correlates with the co-precipitation of increasing amounts of GCP2 2016; Cota et al., 2017). Deletion mutants were designed on the and GCP3. (F) Analysis of the purified GCP4/5/6 complex from cells basis of structure prediction. The wide central insertion (residues expressing endogenous GST-hexa-histidine (GST-6his)-tagged GCP6, using 675–1501) can be divided into three main amino acid regions. The SDS–PAGE and silver staining. The position of the core subunits was verified first region (675–816) is predicted to be a continuum of the helix by western blotting (see G). The graph on the right shows the stoichiometry of α the proteins, calculated from the intensities of the protein bands relative to 11 seen in GCP4, with a potential coiled-coil structure (residues GCP6 (mean±s.d., n=4 independent experiments). (G) Bottom: western blot 730–760). The middle region (816–1400) is unstructured, and showing the fractionation of the purified GCP4/5/6 complex by gel filtration. contains a repeated sequence described to be phosphorylated by Top: size-exclusion chromatography. The majority of the complex eluates with Plk4 (residues 1027–1269, Bahtz et al., 2012). The third region thyroglobulin (Tg) at 669 kDa and above ferritin (440 kDa). (1400–1501) is composed of multiple small helices (Fig. 5A). Combined deletions of these domains were evaluated for rescue of These results indicate that the GCP4/5/6 sub-complex binds spindle bipolarity (Fig. 5B–D). Depletion of GCP6 in wild-type γTuSCs, thereby leading to their assembly into γTuRCs. cells resulted in >70% of mitotic cells with a monopolar spindle. The deletion mutants rescued the bipolarity of spindles, except Reconstituted γTuRCs from the GCP4/5/6 core are able to when the third region (1400–1501) was absent. GCP6Δ675-1400 nucleate microtubules fully rescued spindle bipolarity, whereas GCP6Δ675-1501 showed To evaluate the capacity of the GCP4/5/6 sub-complex to promote no rescue at all (Fig. 5D). Moreover, GCP6Δ675-1400 restored the the assembly of γTuRCs, we loaded the sub-complex onto beads recruitment of γ-tubulin to the centrosomes and to the mitotic and complemented these beads with γTuSCs, using microtubule spindle (Fig. 5C), and rescue was also observed in the absence of nucleation assays and western blotting to assess the presence and induction, due to a leak of the promoter (Fig. 5C–E). Sucrose activity of reconstituted γTuRCs (Fig. 4A). Dynabeads coupled to gradient profiles confirmed that γTuRCs were fully recovered when anti-GCP5 antibody were used to immunoprecipitate either γTuRC only region 675–1400 was deleted (Fig. 5F,G). These results show or the GCP4/5/6 sub-complex (using 100 mM or 600 mM KCl, that most of the central insertion of GCP6 is not required for γTuRC respectively) from a HEK293 cytoplasmic extract (hereafter referred assembly, except for the third region containing the last 100 amino to as γTuRC extract). We also used cells depleted of GCP2 to acids. We immunoprecipitated the GCP4/5/6 complex at 500 mM prepare extracts at 600mM KCl, to ensure that the sub-complex was KCl from cells expressing GCP6Δ675-1400 in low amounts efficiently stripped of γTuSCs. The obtained beads bound to GCP4/ (without addition of doxycycline). This deletion mutant of GCP6 5/6 were washed and incubated at 100 mM KCl with a second still associated with GCP4 and GCP5, thus, maintaining stable extract, prepared from cells depleted of GCPs 4, 5 and 6 (hereafter interactions at high concentrations of KCl (Fig. 5G). referred to as γTuSC extract, Fig. 4B). Reconstitution of γTuRCs To investigate the role of the N-terminal extension of GCP6, it was monitored by western blot (Fig. 4C), and by measuring the was gradually shortened by cutting between its multiple predicted nucleation activity of the beads following incubation with pure helices (Fig. 6A). Deletion of the entire extension (GCP6 352-1819) tubulin (Fig. 4D–F). Compared with γTuRC beads, GCP4/5/6- abolished the capacity of GCP6 to rescue spindle bipolarity and beads were lacking GCPs 2 and 3, and were unable to nucleate prevented the assembly of γTuRCs. All other mutants rescued to microtubules. Addition of the γTuSC extract resulted in re- various extents with rescue efficiency gradually decreasing with the incorporation of γTuSCs, and in recovery of the nucleation length of the deletion (Fig. 6B–D; Fig. S3). GCP6 280-1819 capacity of the beads. Used as a control, the γTuSC extract did generated low amounts of γTuRCs, and complexes of intermediate not show any binding to beads without GCP4/5/6, and these beads size accumulated on sucrose gradients. These complexes may Journal of Cell Science

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Fig. 2. The GCP4/5/6 complex forms independently of γTuRCs. (A) Western blot analysis of cytoplasmic extracts from control HeLa cells and HeLa cells treated with siRNAs targeting GCPs 2, 4, 5 or 6. Lane labeling indicates siRNAs (inputs) as used in B; cont, control siRNA. Notice that certain siRNAs specifically targeting one GCP may also co-regulate other GCPs (B) Sucrose-gradient fractionation of cell extracts as shown in A, visualized with anti-γ-tubulin staining. The peak of γTuRC in fraction 7 of control extracts is lost upon treatment with siRNAs against different GCPs. (C) Immunoprecipitation of GCP4 from fractions 2–7, after sucrose gradient centrifugation of the cytoplasmic extract from GCP2 siRNA-treated cells. GCP4 co-precipitated with GCP5, GCP6 and γ-tubulin, mainly in fraction 5 (21% sucrose). First lane (from left): positive control, showing co-precipitation of all GCPs from fraction 7 of a gradient from untreated cells. (D) Immunoprecipitation of GCPs 4, 5 or 6 from fractions 2–5, after sucrose gradient centrifugation of extracts from HeLa cells treated with siRNAs against GCPs 2, 4, 5 or 6. In the absence of GCP2, GCPs 4, 5 and 6 co-precipitated, with a peak in fraction 5 (21% sucrose). GCPs 4 and 6 co-precipitated in the absence of GCP5, with a peak in fraction 4 (17% sucrose). The absence of GCPs 2 and 3 from the complexes was verified as shown in C but, for simplicity, the corresponding western blots are omitted in D. represent partially assembled γTuRCs, or unstable γTuRCs that with γTuSCs, to a degree similar to that of wild-type protein, started to disassemble during sedimentation. To evaluate whether showing that the protein interactions were maintained. By contrast, GCP6 mutants 280-1819 or 352-1819 affected the interactions with the majority of GCP6 352-1819 failed to interact with the other other GCPs of the γTuRC, we overexpressed them and performed GCPs (Fig. 6E,F). We then immunoprecipitated GCP6 280-1819 immunoprecipitations from gradient fractions as described for from cytoplasmic extracts at increasing concentrations of KCl.

Fig. 3. GCP6 280-1819 co-immunoprecipitated with GCP4 and Although GCPs 2, 3, 4 and 5 efficiently co-precipitated at 100 mM Journal of Cell Science

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Fig. 3. Overexpression of GCPs 4, 5 and 6 drives incorporation of free γTuSCs into γTuRCs. (A) Western blot analysis of cytoplasmic extracts from HeLa cells, showing excessive amounts of GCPs 4, 5 and/or 6 (inputs for B–E). Protein levels of GCP4 are present in excess in these cells (see Fig. S1B). Excess levels of GCP5 and GCP6 protein were obtained by inducing overexpression of the proteins in transiently transfected cells (OE GCP5) or in stably transfected cells (OE GCP6). (B) Sucrose-gradient fractionation of the extracts shown in A, using anti-γ-tubulin staining. Excess of GCPs 4 and 5 (GCP4+5) had no effect on the profile of the gradient, compared to control (excess of GCP4 only), whereas excess of GCPs 4 and 6 (GCP4+6) led to the formation of higher-order complexes. Excess of GCPs 4, 5 and 6 (GCP4+5+6) resulted in complete sequestration of γTuSCs and incorporation of the latter into γTuRCs. (C–E) Sucrose- gradient fractionation of extracts containing excess levels of GCPs 4 and 5 (C); GCPs 4 and 6 (D); and GCPs 4, 5 and 6 (E). Fractions were separated by SDS– PAGE, blotted, and probed with antibodies against the different GCPs and γ-tubulin, to visualize displacement of γTuSCs and proteins in excess (arrows). (F–H) Immunoprecipitation using fractions 2–7 of the gradients shown in C–E, using anti-GCP5 (F,H) or anti-GCP6 (G) antibodies. The different immunoprecipitates were western blotted with antibodies as indicated. (F) GCP5 in excess failed to co-precipitate with significant amounts of other GCPs in fractions 2–5. (G) By contrast, GCP6 in excess precipitated together with GCPs 2, 3, 4 and γ-tubulin in intermediate-sized complexes in fractions 4 and 5 (15–23% sucrose). (H) Excess of GCPs 4, 5 and 6 resulted in co-precipitation of the three proteins with γ-tubulin in fraction 5 (21% sucrose). Journal of Cell Science

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Fig. 4. See next page for legend. Journal of Cell Science

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Fig. 4. Reconstitution of functional γTuRCs from the purified GCP4/5/6 factors have been identified in different species of yeast, and include complex. (A) Experimental design. Dynabeads coupled with antibody against Spc72, Mozart1 and Mto1/2 (Lin et al., 2016; Masuda and Toda, γ GCP5 were incubated with TuRC extract: cytoplasmic extract from HEK293 2016; Lynch et al., 2014; Leong et al., 2019). In most eukaryotes, cells lysed in the presence of 100 mM KCl, with or without adjustment to a final concentration of 600 mM KCl. Beads incubated with extracts at 600 mM KCl fully assembled, helically shaped complexes are already present as were subsequently rinsed and incubated with γTuSC extract: cytoplasmic soluble entities in the cytoplasm in the form of γTuRCs. extract from HEK293 cells transfected with siRNAs against GCPs 4, 5 and 6, Nevertheless, these soluble γTuRCs remain inactive unless and lysed in the presence of 100 mM KCl. Following incubation, rinsed beads recruited to specific sites of microtubule nucleation, thus, were used in a microtubule nucleation assay or analysed by western blotting. allowing tight spatial and temporal control of microtubule γ γ (B) Western Blot analysis of TuRC and TuSC extracts. * indicates a formation (Farache et al., 2018). GCPs 4, 5 and 6 are essential for degradation product of GCP6. (C) Left: western blot analysis of the eluates γ γ γ the assembly and/or stabilization of TuRCs, as depletion of either from the anti-GCP5 beads loaded with TuRCs ( TuRC extract at 100 mM γ KCl), with the GCP4/5/6 complex (γTuRC extract at 600 mM KCl), with the component causes a reduction of TuRCs both at the centrosome GCP4/5/6 complex and in a second step with the γTuSC extract, or with the and in the cytoplasm (Izumi et al., 2008; Bahtz et al., 2012; γTuSC extract alone. Right: western blot of anti-GCP5 beads loaded with the Scheidecker et al., 2015; Farache et al., 2016; Cota et al., 2017). GCP4/5/6 complex from GCP2-depleted cells (+RNAi GCP2), with (+) or Besides GCPs 4, 5 and 6, additional factors might also be needed, without (−) supplementation of γTuSC extract. (D) Microtubule nucleation such as Mozart1, actin or other proteins that correspond to assay using the anti-GCP5 beads as described in C, incubated for 3 min at unassigned densities in cryo-electron microscopy-obtained 37°C with pure TAMRA-labeled tubulin. Representative images are shown γ (beads are autofluorescent). Scale bar: 5 μm. (E) Quantification (in %) of beads structures of native TuRCs (Lin et al., 2016; Cota et al., 2017; showing radial microtubule arrays, i.e. nucleating beads; mean±s.d., n=3 Consolati et al., 2019 preprint; Liu et al., 2020; Wieczorek et al., independent experiments, >100 beads counted per experiment. 2020). It is now clear that GCPs 4, 5 and 6 integrate into the helical (F) Number of microtubules associated per bead (25 beads scored per wall of the γTuRC, and that they are laterally bound to γTuSCs, but condition, mean±s.d. is indicated). their specific role within the γTuRC still remains to be determined (Consolati et al., 2019; Liu et al., 2020; Wieczorek et al., 2020). Our KCl, their interaction was lost at 500 mM KCl. At 200 mM KCl, results show that they form a nucleus promoting the stable assembly GCP2 and GCP3 still interacted with wild-type GCP6; however, of the 2 MDa complex. During the formation of γTuRCs, their interaction with the 280-1819 mutant was strongly reduced complexes comprising GCPs 4, 5 and 6 may act as building (Fig. 6G). Taken together, these results suggest that the GCP6 blocks that recruit γTuSCs by lateral association and thereby initiate amino acid region 280–352 is necessary for interactions with GCP4 γTuRC-assembly. This hypothesis is directly supported by our and γTuSCs, and that the first 280 residues of GCP6 stabilize its experiments with salt-stripped (KCl-treated) sub-complexes of interactions with the γTuRC. GCPs 4, 5 and 6 that can drive the formation of functional γTuRCs, In analogy to our deletion experiments on GCP6, we also after incubation with γTuSC-containing cytoplasm (Fig. 4). attempted to delete the N-terminal extension of GCP5. However, The assembly of γTuRCs mediated by GCPs 4, 5 and 6 may be due to the unavailability of antibodies recognizing GCP5 outside its important for the regulation of its microtubule-nucleation activity: N-terminal domain, this experiment was not controllable. We cryo-electron microscopy structures show an asymmetric successfully deleted the internal insertion of GCP5 (residues 627– architecture, incompatible with the geometry of microtubules 704), and noticed that the resulting mutant was still able to rescue (Consolati et al., 2019 preprint; Liu et al., 2020; Wieczorek et al., spindle bipolarity and γTuRC assembly in GCP5-depleted cells 2020). The presence of GCPs 4, 5 and 6 might, therefore, prevent (data not shown). soluble γTuRCs from acquiring an active conformation, unless bound to additional activating factors, such as Cdk5rap2/Cep215, at DISCUSSION designated microtubule-organizing centers. Consistently, the GCP5 We demonstrate that GCPs 4, 5 and 6 form a stable sub-complex that subunit was identified in two different conformations at position 10 permits the association with γTuSCs into a functional γTuRC. within the γTuRC, and conformational changes propagated towards Within the sub-complex, the stoichiometric ratio of these GCPs positions 11–14 of the γTuRC-helix, suggesting that these regulate matches the values found in recently reported structures of native the overall structure and the activation of the γTuRC (Wieczorek γTuRCs (Consolati et al., 2019 preprint; Liu et al., 2020; Wieczorek et al., 2020). In addition, specific non-grip domains in GCP6 might et al., 2020). We find that GCPs 2 and 3 are also present in our also be involved in the binding to regulatory factors. The large preparations of the sub-complex, albeit at a molar ratio equivalent to central insertion in GCP6, including nine tandem repeats of 27 half of γTuSC. We believe that this reflects stochastic binding of amino acids, has been proposed to be regulated by Plk4-dependent remnant γTuSCs after incomplete salt-stripping at 500 mM KCl. phosphorylation (Bahtz et al., 2012). Contrary to this view, More-stringent conditions with even higher concentrations of KCl our deletion experiments show that the majority of the central failed to increase the purity of the sub-complexes but, rather, led to insertion – i.e. residues 675–1400 that comprise the tandem repeats their disassembly (Fig. 1A). – can be removed from GCP6 without impacting the assembly or the activity of the γTuRC because a Δ675-1400 deletion mutant permits Role of the GCP4/5/6 sub-complex in the assembly and the assembly of regular mitotic spindles. Moreover, neither the stabilization of γTuRC recruitment of γTuRCs to the centrosome nor to the mitotic spindle Structural and biochemical work has shown that γ-tubulin were affected by this deletion. Eventually, this GCP6 region might complexes in budding yeast are formed by lateral assembly of be involved in recruitment to specific non-centrosomal microtubule- γTuSCs (Kollman et al., 2010). Lateral association and organizing centers (Oriolo et al., 2007). oligomerization into a helically shaped template are supported by Besides any regulatory role, the GCP4/5/6 sub-complex might targeting factors, such as Spc110, that promote the recruitment of contribute to the stabilization of γTuRCs post assembly by γTuSCs to the spindle pole body and, thus, couple localization to the preventing the loss of γTuSCs from the helical complex. In assembly into a nucleation-competent complex (Kollman et al., particular, the N-terminal extension of GCP5 might be part of a

2010; Lyon et al., 2016; Lin et al., 2014, 2016). Other targeting lumenal bridge within the γTuRC and might thereby fulfil a Journal of Cell Science

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Fig. 5. See next page for legend. Journal of Cell Science

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Fig. 5. Structure and function of the GCP6-specific central insertion. form in the absence of GCP5, and that these are sufficient to (A) Schematic alignment of GCP4 and GCP6 protein sequences. Dark blue, establish lateral contacts with because excess protein levels of GCPs conserved N-terminal domain; red, C-terminal domain; light blue, specific 4 and 6 are able to immunoprecipitate together with GCPs 2 and 3, extension and insertion within GCP6. Numbers indicate amino acid positions at junctions between domains and of the deletions. Predicted structural without GCP5 (lanes 4, 5 in Fig. 3G). By contrast, GCP5 needs the features are in indicated as gray bars. (B) Overview of the functionality of GCP6 presence of both GCPs 4 and 6 to form any higher-order complex, deletion mutants to rescue spindle bipolarity in mitotic cells. Deletion mutants suggesting that, in human cells, a γ-TuG4/5 does not exist (Fig. 2). were expressed from stably transfected cells, with or without induction, after The situation might be different in other species, such as fission treatment with GCP6 siRNA. Constructs were resistant to the siRNA yeast, where the GCPs 4 and 5 orthologs Gfh1p and Mod21p, treatment. (C) Comparison of mitotic spindles in control HeLa cells and in cells respectively, can be co-immunoprecipitated in the absence of the expressing GCP6Δ675-1501 or GCP6Δ675-1400 in response to transfection with GCP6 siRNA. γ-tubulin is stained in red, microtubules are stained in green, GCP6 ortholog Alp16 (Anders et al., 2006). In both experimental DNA is stained in blue. Staining of γ-tubulin is shown in the images on the right. systems, though, the GCP6 ortholog needs GCP4 to associate with Images in the first row show an untreated control cell (no siRNA). Scale bar: GCP5, suggesting that the spatial arrangement of GCPs 4, 5 and 6 5 μm. (D) Number of mitotic cells with monopolar spindles (in %), determined in within γ-tubulin complexes is conserved across species. On the control cells and in cells expressing GCP6Δ675-1501 or GCP6Δ675-1400, basis of our experiments, using depletion or overexpression of with or without GCP6 siRNA transfection, with or without overexpression (OE) individual GCPs (Figs 2 and 3), we propose a hierarchy of assembly, Δ of the indicated constructs following their induction by doxycycline. GCP6 675- in which GCPs 4 and 6 together enable the recruitment of GCP5 into 1400 rescues spindle bipolarity, even without doxycycline treatment, due to γ leaky expression (mean±s.d., n=3 independent experiments, 100 cells counted a stable sub-complex that drives lateral association with TuSCs per experiment). (E) Western blot analysis of cytoplasmic extracts from cell lines into a full-sized γTuRC (Fig. 7). Studies by Cota et al. (2017) and expressing GCP6Δ675-1501 or GCP6Δ675-1400, transfected or not with GCP6 Farache et al. (2016) have shown that depletion of either GCP4 or siRNA, with or without doxycycline-induced overexpression of the respective GCP5 leads to milder defects than depletion of GCP6, confirming a mutant GCP6 (the corresponding cytoplasmic extracts were used as inputs for central role of GCP6 in γTuRC-assembly. This raises the possibility F,G). Blots were probed with antibodies against GCP6 (top and middle rows) γ γ that partial or unstable -tubulin complexes can still form through and an antibody against -tubulin (bottom rows). (F) Sucrose-gradient γ γ fractionation of extracts from control HeLa cells or cells expressing GCP6Δ675- lateral interactions between GCP6 and TuSCs, or that TuSCs 1501 or GCP6Δ675-1400 after GCP6 siRNA transfection and overexpression of assemble into larger complexes in situ, e.g. at centrosomes or the mutants. Top row shows untreated control (no siRNA). Fractions were spindle pole bodies, partially stabilized by GCP6 and with restricted separated by SDS–PAGE and analysed by western blotting, using an antibody nucleation capacity (Vérollet et al., 2006; Xiong and Oakley, 2009; against γ-tubulin. The graph on the right shows the quantification of the γ-tubulin Masuda and Toda, 2016). signal in fraction 7 of the gels displayed, relative to the untreated control cells. To gain further insights into the assembly of γTuRCs, it would be (G) Immunoprecipitation of GCP5 from a cytoplasmic extract prepared from the cell line expressing GCP6Δ675-1400, without overexpression, at 100 mM or of interest to perform controlled reconstitution experiments of the 500 mM KCl. Co-precipitation of the mutant (arrow) is similar at both multiprotein complex using purified recombinant components. concentrations. MATERIALS AND METHODS stabilizing role (Wieczorek et al., 2020). Moreover, the N-terminal Cell culture and generation of stable cell lines extension of GCP6 (amino acids 1–352) might correspond, at least HeLa Flp-In T-REx (a gift from Stephen Taylor, University of Manchester, in part, to a stabilizing ‘plug’ seen in the cryo-electron microscopy UK; Tighe et al., 2008), and HEK293 FT (Invitrogen, Carlsbad, CA) cells ’ ’ structure of the γTuRC (Wieczorek et al., 2020). In accordance, we were grown in Dulbecco s modified Eagle smedium(DMEM), observed that progressive shortening of the N-terminal extension of supplemented with 10% fetal bovine serum at 37°C, under 5% CO2. Cells γ were authenticated and tested for contamination. For CRISPR/Cas9 targeting GCP6 destabilizes the TuRC and renders it more sensitive to of GCP6 in HEK293 FT cells, a target guide RNA overlapping the stop codon treatment with KCl. For example, the deletion of the first 279 amino (5′-AACTACTACCAGGACGCCTG-3′, computed from http://crispr.mit. acids leads to the loss of γTuSCs at 200 mM KCl, and to the loss of edu/) was inserted into pSpCas9(BB)-2A-Puro (Addgene plasmid #48139). GCPs 4 and 5 at 400 mM KCl, whereas wild-type γTuRCs remain The homologous recombination donor was a DNA fragment consisting of a stable under these conditions (Fig. 6G). Additional stabilization of 1445 bp left homology arm (GCP6 exon 20–25), a 2244 bp insertion the γTuRC might be provided by contacts between the central sequence (i.e. GST-6his coding sequence in-frame with exon 25 of GCP6, insertion in GCP6 (amino acids 675–1501) and GCP2 in position followed by a puromycin-resistance cassette), and a 1577 bp right-homology 13, since GCP6 deletion mutants that lack this insertion (Δ675- arm (GCP6 exon 25 and 3′UTR sequence). Cells were transfected using 1501) prevent the formation of stable γTuRCs (Fig. 5F; Wieczorek Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Limiting dilution cloning et al., 2020). was performed 3 days post transfection, in conditioned medium supplemented with 2 μg/ml puromycin. Targeted clones were identified by Overall, the sub-complex of GCPs 4, 5 and 6 might promote γ PCR, and verified by sequence analysis and by western blotting using an anti- lateral associations with TuSCs through the grip1 domains of its GST antibody (Roche Applied Science, Basel, Switzerland). peripheral constituents GCP4 and GCP6, and might stabilize HeLa Flp-In T-REx overexpressing cell lines were obtained as interactions within a larger γTuRC through the N-terminal described (Farache et al., 2016). GCP5 and GCP6 (resistant to siRNA) extensions of GCPs 5 and 6. were expressed from pCDNA5/FRT/TO (Invitrogen). Internal deletions of The question has been raised whether γ-tubulin and one copy of GCP6 were constructed using the Gibson kit (NEB, Ipswich, MA), GCP4 assemble with either GCP5 or GCP6 into intermediate whereas N-terminal deletions were generated by PCR (using restriction heterotetramers, such as ‘γ-TuG4/5’ and ‘γ-TuG4/6’, equivalent to sites BamH1 and Ale1 in the sequences of the vector and the GCP6 DNA, γTuSCs (Liu et al., 2020; Wieczorek et al., 2020). Our data do not respectively). Briefly, GCP6 constructs were co-transfected with pOG44 (Invitrogen) expressing the Flp recombinase by using CaCl2. provide direct evidence for this mechanism, since extraction using μ ‘ ’ Resistant clones at 200 g/ml hygromycin B were picked and expanded to KCl or GCP2 depletion yields monolithic sub-complexes, in obtain clonal cell lines. The phenotypes of at least two independent clones which GCPs 4, 5 and 6 are stably bound to each other. In fact, were compared for each construct. Transient transfection of the GCP5 extraction by using KCl demonstrates that the affinities between the construct was performed using Lipofectamine 2000. Transgene GCPs of this sub-complex are higher than the affinities between expression was induced using 1 μg/ml doxycycline. For RNA

γTuSCs. Nevertheless, we have seen that γ-TuG4/6 intermediates interference (RNAi), 10 nM siRNA were transfected into HeLa Flp-In Journal of Cell Science

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Fig. 6. See next page for legend.

T-REx cells, using Lipofectamine RNAi max (Invitrogen; Farache et al., Purification of the GCP4/5/6 sub-complex 2016). After 24 h, medium was replaced with medium containing or not Cells homozygous for GCP6-GST-6his were harvested by trypsinization, containing doxycycline. Cells were harvested or fixed 72 h post rinsed with PBS and stored at −80°C. Pellets were resuspended in 50 mM transfection. HEPES-KOH pH 7.2, containing 1 mM MgCl2 and 1 mM EGTA (HB) plus Journal of Cell Science

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Fig. 6. Structure/function of the GCP6-specific N-terminal extension. (A) Schematic alignment of GCP4 and GCP6 protein sequences. Dark blue, conserved N-terminal domain; red, C-terminal domain; light blue, specific extension and insertion within GCP6. Numbers indicate amino acid positions of the deletions within the N-terminal extension of GCP6. Predicted helical regions are indicated as gray bars. (B) Number of of monopolar spindles (in %) within stable cell lines expressing different GCP6 deletion mutants, after siRNA transfection and overexpression of the mutants (mean±s.d., n=3 independent experiments, 100 cells counted per experiment). (C) Western blot analysis of cytoplasmic extracts from cell lines as described in B, transfected or not with GCP6 siRNA, with or without overexpression of the mutants (inputs for D–G). Blots were probed with antibodies against GCP6 (top rows) or γ-tubulin (bottom rows). (D) Sucrose-gradient fractionation of extracts from cell lines as described in B, after GCP6 siRNA transfection and overexpression of the mutants. The graph on the right shows the quantification of γ-tubulin signal in fraction 7 of the blots displayed, relative to the untreated control shown in Fig. 5F. (E) Sucrose-gradient fractionation of extracts from cell lines overexpressing GCP6 280-1819 and GCP6 352-1819. Western blots were probed with an antibody against γ-tubulin. (F) Immunoprecipitation of the overexpressed mutants from fractions 3–7 of the gradients described in E. GCP6 280-1819 co-precipitates with GCPs 2, 3 and 4, and with γ-tubulin (fractions 4, 5) but GCP6 352-1819 does not. (G) Immunoprecipitation of GCP6 280-1819 from extracts of untreated control cells (left), or cells treated with GCP6 siRNA, and induced to overexpress of the mutant (right). Extracts were supplemented with increasing concentrations of KCl (100–600 mM). The graphs below show the quantification of co-immunoprecipitated GCPs (in %) at the different KCl concentrations, by analyzing the band intensities relative to the amounts of protein precipitated at 100 mM KCl.

100 mM KCl (HB100), and supplemented with 0.1 mM GTP, 1 mM DTT, 1 mM PMSF and complete protease inhibitor cocktail (Roche, Basel, CH). Cells were disrupted by sonication and centrifuged for 30 min at 30,000 g. Protein precipitation from the supernatant was performed by adding 30% of a saturated (NH4)2SO4 solution, followed by solubilization in HB plus 500 mM KCl (HB500), supplemented with 1 mM DTT, 1 mM PMSF, protease inhibitors, 15 mM imidazole and 0.05% IGEPAL CA-630 detergent. The solution was added to glutathione Sepharose 4B (GE Healthcare, Chicago, IL) and incubated 4 h at 4°C under agitation. Beads were washed twice with HB500 containing supplements, and twice with the same buffer without PMSF and protease inhibitors. Elution was performed with the same buffer containing 40 mM reduced glutathione, pH 7.2. Buffer was exchanged by passing the eluate through a desalting PD-10 column (GE Healthcare, Chicago, IL), equilibrated with 50 mM HEPES- KOH pH 7.2, containing 1 mM MgCl2,500mMKCl,0.5mMDTTand 0.05% IGEPAL CA-630. The solution was then incubated overnight with Fig. 7. Model of γTuRC assembly. We propose that lateral alignment of GCP6 Ni-NTA agarose (Qiagen, Hilden, Germany) at 4°C, washed twicewith the and GCP4 enables binding of GCP5, and of an additional copy of GCP4, same equilibration buffer, and twice with equilibration buffer thereby forming a stable intermediary, the GCP4/5/6 sub-complex. This supplemented with 15 mM imidazole. Elution was performed using sub-complex then drives and stabilizes association with γTuSCs until one 200 mM imidazole. complete helical turn is reached. Sequence extensions within the N-terminal Eluted proteins were analyzed on western blots or polyacrylamide gels regions of GCP5 and GCP 6, as well as a sequence insertion between the and stained using the PlusOne silver staining kit (GE Healthcare, Chicago, grip1 and grip2 motifs of GCP6 might contribute to the stabilization of IL). Size-exclusion chromatography was performed on Superdex™ 200 interactions between neighboring γTuSCs and across the lumen of the γTuRC. Increase 5/150 GL columns (GE Healthcare, Chicago, IL), using thyroglobulin and ferritin as markers for calibration. added for 1 h at 4°C, and proteins were eluted in gel-loading buffer, after two washes with HB100 containing 10% glycerol. Sucrose gradient sedimentation Immunoprecipitation from the gradient fractions was performed as above, Sucrose gradients were performed as described by Farache et al. (2016) in i.e. by addition of antibodies to the fractions and incubation for 2 h at 4°C, HB100 or by using varying concentrations of KCl. followed by incubation with protein A-dynabeads for 1 h. The beads were then washed twice in HB100 containing 10% glycerol and samples were Immunoprecipitation eluted in gel-loading buffer. Cytoplasmic lysates were produced from trypsinized HeLa Flp-In T-Rex cells, lysed in HB100 supplemented with 1 mM GTP, 1 mM DTT, 1 mM Microtubule nucleation from beads PMSF, protease inhibitors, 1% IGEPAL CA-630 and 10% glycerol. After Dynabeads were washed and incubated with the anti-GCP5 antibody in 5 min on ice, cells were centrifuged for 10 min at 16,000 g. Aliquots of the PBS, containing 0.02% Tween-20, for 2.5 h at 4°C. After three additional supernatant containing 500 μg of protein were diluted in 100 μl HB100 washes, HEK293 FT cytoplasmic extracts (γTuRC extracts) were added to containing supplements. These cytoplasmic lysates where then the beads and incubated for 2 h at 4°C. Extracts were prepared as described supplemented with KCl to increase the concentration as needed, before in the immunoprecipitation protocol, in the presence of 100 or 600 mM KCl. incubation with 1–2 μg of anti-GCP4, anti-GCP5 or anti-GCP6 antibodies For each reaction, we used cells grown to confluence on a 100-mm dish for 2 h at 4°C. A50 μl aliquot of protein A-dynabeads (Invitrogen) was (yielding 4 mg of protein) that were lysed in 100 μl buffer per 5 μl beads. Journal of Cell Science

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After three washes with HB100+10% glycerol, a freshly prepared Competing interests cytoplasmic extract depleted of GCPs 4, 5 and 6 (γTuSC extract) was The authors declare no competing or financial interests. added to the beads. Again, cells from one 100 mm dish were lysed in 100 μl HB100 containing supplements. After a 2 h incubation at 4°C, beads were Author contributions washed three times with Brinkley renaturing buffer (BRB80; 80 mM PIPES Conceptualization: L.H., D.F., L.E., A.M.; Methodology: L.H., D.F., L.E.; Validation: pH 6.9, 1 mM EGTA, 1 mM MgCl ) and resuspended in 12.5 μl L.H., D.F., L.E.; Formal analysis: L.H., D.F., L.E.; Investigation: L.H., D.F., L.E.; Data 2 curation: L.H., D.F., L.E.; Writing - original draft: L.H., A.M.; Writing - review & editing: BRB80. Nucleation was tested by incubating 2.5 μl beads for 3 min at μ μ L.H., D.F., A.M.; Supervision: L.H., A.M.; Project administration: A.M.; Funding 37°C with a solution containing 1 l tubulin at 10 mg/ml, 1 l acquisition: A.M. carboxytetramethylrhodamine (TAMRA)-labeled tubulin (a gift from Nathalie Morin, Centre de Recherche en Biologie cellulaire de Funding Montpellier, Montpellier, France) at 2 mg/ml, and 0.5 μl 10 mM GTP The project was in part supported by Agence Nationale de la Recherche (grant (modified from Choi and Qi, 2014). The reaction was stopped by adding number: 13-BSV8-0007-01), and by Fondation ARC pour la recherche sur le cancer 45 μl of 1% glutaraldehyde in BRB80, for 5 min at 37°C. Beads were (grant number: SFI20121205511). diluted in BRB80 and layered onto a cushion of 10% glycerol in BRB80, centrifuged onto coverslips and mounted in Vectashield solution (Vector Supplementary information Laboratories, Peterborough, UK). Proteins were eluted from the remaining Supplementary information available online at beads with gel-loading buffer and analyzed by western blotting. http://jcs.biologists.org/lookup/doi/10.1242/jcs.244368.supplemental

Peer review history Western blot analysis The peer review history is available online at Proteins were detected using an Odyssey imaging system (Li-cor https://jcs.biologists.org/lookup/doi/10.1242/jcs.244368.reviewer-comments.pdf Biosciences, Lincoln, NE) according to the manufacturer’s protocol, with IRDye 800CW- and 680CW-conjugated secondary antibodies (Invitrogen). References Protein levels were quantified using Odyssey 2.1 software. The Odyssey Anders, A., Lourenço, P. C. C. and Sawin, K. E. (2006). Noncore components of fluorescence system provides a linear relationship between signal intensity the fission yeast γ-tubulin complex. Mol. Biol. Cell 17, 5075-5093. doi:10.1091/ and antigen loading. Band intensities were measured after background mbc.e05-11-1009 subtraction. For the quantification of γ-tubulin in fractions of sucrose Bahtz, R., Seidler, J., Arnold, M., Haselmann-Weiss, U., Antony, C., Lehmann, gradients, the band intensities of individual fractions were normalized to the W. D. and Hoffmann, I. (2012). GCP6 is a substrate of Plk4 and required for γ centriole duplication. J. Cell Sci. 125, 486-496. doi:10.1242/jcs.093930 total amount of -tubulin in the experiment, corresponding to the sum of the Choi, Y.-K. and Qi, R. Z. (2014). Assaying microtubule nucleation by the gamma- intensities of all fractions of the sucrose gradient. tubulin ring complex. Methods Enzymol. 540, 119-130. doi:10.1016/B978-0-12- 397924-7.00007-8 Fluorescence microscopy Choi, Y.-K., Liu, P., Sze, S. K., Dai, C. and Qi, R. Z. (2010). CDK5RAP2 stimulates Cells grown on coverslips were fixed in methanol at −20°C, and processed microtubule nucleation by the γ-tubulin ring complex. J. Cell Biol. 191, 1089-1095. for immunofluorescence, following standard protocols. Fluorescence doi:10.1083/jcb.201007030 Consolati, T., Locke, J., Roostalu, J., Asthana, J., Lim, W. M., Gannon, J., microscopy was performed on a wide-field microscope (Axiovert 200M; Martino, F., Costa, A. and Surrey, T. (2019). Microtubule nucleation by single Carl Zeiss, Oberkochen, Germany) equipped with a Z motor, using a 63× human γTuRC in a partly open asymmetric conformation. bioRxiv. doi:10.1101/ (Plan Apo, 1.4 NA) objective. Images were acquired with an MRm camera 853218 and Axiovision software. Images of microtubule-nucleating beads were Cota, R. R., Teixidó-Travesa, N., Ezquerra, A., Eibes, S., Lacasa, C., Roig, J. and obtained with a LSM 710 confocal microscope (Carl Zeiss), using a 63× Lüders, J. (2017). MZT1 regulates microtubule nucleation by linking γTuRC (Apo, 1.4 NA) objective, and an excitation wavelength of 561 nm. Images assembly to adapter-mediated targeting and activation. J. Cell Sci. 130, 406-419. shown in Figs 4 and 5 correspond to single sections. doi:10.1242/jcs.195321 Dion, A. S. and Pomenti, A. A. (1983). Ammoniacal silver staining of proteins: Image processing was performed using Adobe Photoshop. Identical mechanism of glutaraldehyde enhancement. Anal. Biochem. 129, 490-496. settings of exposure and contrast were used for corresponding experiments doi:10.1016/0003-2697(83)90582-1 and controls. Erlemann, S., Neuner, A., Gombos, L., Gibeaux, R., Antony, C. and Schiebel, E. (2012). An extended γ-tubulin ring functions as a stable platform in microtubule Antibodies nucleation. J. Cell Biol. 197, 59-74. doi:10.1083/jcb.201111123 ́ Rabbit anti-GCP6 (for immunoprecipitation, Abcam, Cambridge, UK); Farache, D., Jauneau, A., Chemin, C., Chartrain, M., Remy, M.-H., Merdes, A. γ mouse anti-γ-tubulin TU-30 (for western blotting 1:5000, Exbio, Vestec, and Haren, L. (2016). Functional analysis of -tubulin complex proteins indicates specific lateral association via their N-terminal domains. J. Biol. Chem. 291, CZ); rabbit anti-GCP4 (for western blotting 1:1000; Fava et al., 1999); 23112-23125. doi:10.1074/jbc.M116.744862 rabbit anti-GCP2 (for western blotting 1:700; Haren et al., 2006); rabbit Farache, D., Emorine, L., Haren, L. and Merdes, A. (2018). Assembly and anti-γ-tubulin R75 (for immunofluorescence 1:1000; Julian et al., 1993); regulation of γ-tubulin complexes. Open Biol. 8, 170266. doi:10.1098/rsob. mouse anti-GCP3 C3 (for western blotting 1:700), mouse anti-GCP5 E1 170266 (for immunoprecipitation), rabbit anti-GCP5 H300 (for western blotting Fava, F., Raynaud-Messina, B., Leung-Tack, J., Mazzolini, L., Li, M., Guillemot, 1:500), mouse anti-GCP6 H9 (for western blotting, 1:1000, Santa Cruz J. C., Cachot, D., Tollon, Y., Ferrara, P. and Wright, M. (1999). Human 76p: a γ J. Cell Biol. Biotechnology, Santa Cruz, CA); mouse anti-α-tubulin (for new member of the -tubulin-associated protein family. 147, 857-868. doi:10.1083/jcb.147.4.857 immunofluorescence 1:1000, Sigma Aldrich, St Louis, MO). Quantities Guillet, V., Knibiehler, M., Gregory-Pauron, L., Remy, M.-H., Chemin, C., of antibodies used for immunoprecipitation were as reported in the Raynaud-Messina, B., Bon, C., Kollman, J. M., Agard, D. A., Merdes, A. et al. Immunoprecipitation section above. (2011). Crystal structure of γ-tubulin complex protein GCP4 provides insight into microtubule nucleation. Nat. Struct. Mol. Biol. 18, 915-919. doi:10.1038/nsmb. Acknowledgements 2083 We thank Valérie Guillet (Toulouse) for help with size exclusion chromatography, Gunawardane, R. N., Martin, O. C., Cao, K., Zhang, L., Dej, K., Iwamatsu, A. and Stephen Taylor (Faculty of Life Sciences, University of Manchester, Manchester, Zheng, Y. (2000). Characterization and reconstitution of Drosophila γ-tubulin ring UK) for the gift of HeLa Flp-in T-Rex, Nathalie Morin (Centre de Recherche en complex subunits. J. Cell Biol. 151, 1513-1524. doi:10.1083/jcb.151.7.1513 Biologie cellulaire de Montpellier, Montpellier, France) for the gift of TAMRA-tubulin Haren, L., Remy, M.-H., Bazin, I., Callebaut, I., Wright, M. and Merdes, A. (2006). and Jens Lüders (Institute for Research in Biomedicine, Barcelona, Spain) for fruitful NEDD1-dependent recruitment of the γ-tubulin ring complex to the centrosome is discussions during the course of this work. We thank all our colleagues at the necessary for centriole duplication and spindle assembly. J. Cell Biol. 172, UniversitéPaul Sabatier for critical discussion and for technical help. The project 505-515. doi:10.1083/jcb.200510028 was in part supported by grant 13-BSV8-0007-01 from Agence Nationale de la Izumi, N., Fumoto, K., Izumi, S. and Kikuchi, A. (2008). GSK-3β regulates proper Recherche, and by grant SFI20121205511 from Fondation ARC pour la recherche mitotic spindle formation in cooperation with a component of the γ-tubulin ring sur le cancer. complex, GCP5. J. Biol. Chem. 283, 12981-12991. doi:10.1074/jbc.M710282200 Journal of Cell Science

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