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• W4320073612 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract To establish the microtubule cytoskeleton, the cell must tightly regulate when and where microtubules are nucleated. This regulation involves controlling the initial nucleation template, the γ-tubulin ring complex (γTuRC). Although γTuRC is present throughout the cytoplasm, its activity is restricted to specific sites including the centrosome and Golgi. The well-conserved γ-tubulin nucleation activator (γTuNA) domain has been reported to increase the number of microtubules (MTs) generated by γTuRCs. However, previously we and others observed that γTuNA had a minimal effect on the activity of antibody-purified Xenopus γTuRCs in vitro (Thawani et al., eLife, 2020; Liu et al., 2020). Here, we instead report, based on improved versions of γTuRC, γTuNA, and our TIRF assay, the first real-time observation that γTuNA directly increases γTuRC activity in vitro, which is thus a bona fide γTuRC activator. We further validate this effect in Xenopus egg extract. Via mutation analysis, we find that γTuNA is an obligate dimer. Moreover, efficient dimerization as well as γTuNA’s L70, F75, and L77 residues are required for binding to and activation of γTuRC. Finally, we find that γTuNA’s activating effect opposes inhibitory regulation by stathmin. In sum, our improved assays prove that direct γTuNA binding strongly activates γTuRCs, explaining previously observed effects of γTuNA expression in cells and illuminating how γTuRC-mediated microtubule nucleation is regulated. Editor's evaluation This fundamental Research Advance is of interest to cell biologists studying the mechanisms and control of microtubule nucleation. Rale et al. convincingly establish the regulatory role of the γ-TuNA motif in microtubule nucleation and settle prior conflicting results in the literature. They show that γ-TuNA binds to and activates γ-TuRC-based microtubule nucleation both in Xenopus extracts and in vitro. https://doi.org/10.7554/eLife.80053.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Microtubule (MT) assembly is a critical cellular process tightly regulated in both space and time. Spatiotemporal control of MT nucleation allows cells to use the same pool of soluble tubulin to generate different intracellular structures, from the interphase cytoskeletal transport network to the complex mitotic spindle. Yet, while the core MT nucleation machinery has been well characterized, how MT nucleation is locally activated remains poorly understood. The key MT nucleator is the γ-tubulin ring complex (γTuRC). γTuRC is a large, 2.2 MDa complex that forms an asymmetric ring of γ-tubulin subunits (Zheng et al., 1995; Moritz et al., 1998). This ring is thought to act as an initial template for the MT (Moritz et al., 2000). As α/β-tubulin subunits bind to the ring of γ-tubulin, they form the nucleus of a new MT, rapidly transitioning from nucleation toward the more favorable regime of MT polymerization (Jackson and Berkowitz, 1980; Mitchison and Kirschner, 1984). In vitro studies with purified human and Xenopus γTuRCs have shown that these can indeed catalyze the nucleation of new MTs (Choi et al., 2010; Thawani et al., 2020; Liu et al., 2020). Recent studies have also shown that γTuRC acts with the MT polymerase, XMAP215/ch-TOG, to nucleate MTs (Thawani et al., 2018; Flor-Parra et al., 2018; Gunzelmann et al., 2018; King et al., 2020a). Structural studies of γTuRCs from yeast, frogs (Xenopus laevis), and humans revealed remarkable conservation of the γ-tubulin ring structure, although the composition of γTuRC differs substantially across these organisms (Kollman et al., 2015; Liu et al., 2020; Wieczorek et al., 2020a; Wieczorek et al., 2020b; Consolati et al., 2020). Intriguingly, the pitch and diameter of the γ-tubulin ring appears to be incompatible with that of the assembled MT lattice. This suggests that γTuRC undergoes a conformational change to reduce its diameter before it can nucleate MTs (Thawani et al., 2020; Liu et al., 2020). One possibility is that this activating conformational change is stimulated by direct binding of ‘activation’ factors. At the same time, other modes of activation are also plausible. The centrosomal scaffold protein Cdk5rap2, which recruits γTuRC to the centrosome and Golgi (Andersen et al., 2003; Bond et al., 2005; Fong et al., 2008; Choi et al., 2010; Mennella et al., 2012; Lawo et al., 2012), has been shown to increase γTuRC’s nucleation activity (Fong et al., 2008; Choi et al., 2010; Roubin et al., 2013). Previous domain-mapping studies found that the γ-tubulin nucleation activator (γTuNA or CM1) sequence in Cdk5rap2’s N-terminus is critical to bind and activate γTuRC (Figure 1A; Fong et al., 2008; Choi et al., 2010). The γTuNA sequence is well-conserved across yeast, nematodes, flies, frogs, and humans (Samejima et al., 2010; Conduit et al., 2014; Feng et al., 2017; Fong et al., 2008; Choi et al., 2010), and identical γTuNA domains have been identified in related centrosomal and Golgi proteins such as myomegalin (Roubin et al., 2013). A bipartite version of γTuNA is also present in the microtubule branching factor, TPX2 (Alfaro-Aco et al., 2017; King and Petry, 2020b). Thus, understanding how the γTuNA domain interacts with γTuRC might bring insights into the regulation of MT assembly in a wide variety of organisms and contexts. Figure 1 with 2 supplements see all Download asset Open asset Cdk5rap2’s γTuNA domain increases MT nucleation in Xenopus egg extract and requires the universal MT template, the γ-tubulin ring complex (γTuRC). (A) Schematic of Xenopus Cdk5rap2’s domains. The γTuRC nucleation activator domain, γTuNA, is located from amino acids 56–86 in Xenopus laevis isoform X1 (905 aa) or 60–90 in human CDK5RAP2 isoform A (1893 aa). Predictions of disorder (PONDR-FIT; Xue et al., 2010) and coiled-coil regions (COILS) are shown as a red/yellow gradient or blue boxes, respectively. (B) Alignment of wildtype human and Xenopus γTuNAs. Identical residues are red. The human F75 residue (first mutated in Fong et al., 2008) is equivalent to residue F71 in Xenopus. In this study, mutations of well-conserved, identical residues are designated according to the human residue number (e.g. human F75A is equivalent to Xenopus F71A; both hereafter referred to as “F75A”). (C) TIRF assay of MT nucleation in Xenopus egg extract. A titration series of wildtype or ‘F75A’ versions of Xenopus γTuNA (Strep-His-Xen. γTuNA-aa 56–89) were added to extract as shown. EB1-mCherry was used to mark growing MT plus-ends (pseudo-colored green in images). Bar = 5 µm. (D) Quantification of the number of EB1 spots in C. The data were normalized by the buffer controls, and are shown as fold-changes. Black error bars are the standard error of the mean (SEM) for three independent extracts. Thin colored lines on either side of the central trendline represent 95% confidence intervals. (E) Western blot of γ-tubulin levels before and after mock-treatment or incubation with Strep-His-Halo-Xenopus γTuNA-coupled beads. After a single pulldown, the majority of γ-tubulin signal is lost. (F) TIRF assay of mock- and γTuRC-depleted extract. Alexa-488 labeled tubulin (green) and EB1-mCherry (red) were used to visualize microtubules in extract with or without 2 µM Strep-His-Xenopus γTuNA. See “Figure 1—source data 1” and “Figure 1—source data 2” for numerical data and raw blot. Figure 1—source data 1 Numerical data for Figure 1. https://cdn.elifesciences.org/articles/80053/elife-80053-fig1-data1-v2.zip Download elife-80053-fig1-data1-v2.zip Figure 1—source data 2 Labeled and raw blots used in Figure 1. https://cdn.elifesciences.org/articles/80053/elife-80053-fig1-data2-v2.zip Download elife-80053-fig1-data2-v2.zip Direct binding of γTuNA has been proposed to activate γTuRC, as addition of γTuNA increases γTuRC activity in human cells (Choi et al., 2010; Cota et al., 2017). This activation effect in human cells is, in fact, also well-conserved across the phylogenetic tree with ectopic γTuNA expression triggering increased MT nucleation in fission yeast (Lynch et al., 2014), Drosophila (Tovey et al., 2021), and mice (Muroyama et al., 2016). Prior work has also identified a key hydrophobic residue in γTuNA, F75, that is critical for γTuNA’s activation effect, suggesting a direct interaction with γTuRC involving this central region (Fong et al., 2008; Choi et al., 2010). Whether this activation effect is due to a direct increase in γTuRC activity has been an open question, although in vitro results with purified γTuRC and γTuNA suggest that this is the case (Choi et al., 2010; Muroyama et al., 2016). While these fixed endpoint results are suggestive, the field has been lacking a real-time, high-resolution observation of a direct γTuNA-mediated increase in γTuRC activity. Previously we reported that γTuNA had little effect on the activity of antibody-purified Xenopus γTuRC (Thawani et al., 2020). Our observation was seemingly corroborated by independent in vitro and structural data published that same year (Liu et al., 2020). However, after substantial improvements in our γTuRC purification protocol, we now report the first real-time observation that the γTuNA domain directly increases γTuRC’s nucleation ability. Using mutation analysis, we find that the γTuNA domain binds γTuRC as a dimer, providing the first biochemical validation of a recent γTuRC structural model containing a parallel coiled-coil binding partner presumed to be γTuNA (Wieczorek et al., 2020a). Critically, we show that complete dimerization of the γTuNA domain is required for binding and activation of γTuRC in extract and in vitro. Finally, we reveal that γTuNA-mediated activation of γTuRC is sufficient to counteract indirect regulation by the tubulin-sequestering protein, stathmin. In sum, our study provides a direct observation of γTuNA domains as bona fide γTuRC activators. Results Cdk5rap2’s γTuNA domain increases MT nucleation in Xenopus egg extract To study how Xenopus Cdk5rap2 affects γTuRC’s activity, we added its purified γTuNA domain (Figure 1A–B; Figure 1—figure supplement 1; aa 56–89, isoform X1) to Xenopus laevis egg extract and assessed its impact on microtubule (MT) nucleation (Figure 1C). Using total internal reflection (TIRF) microscopy and fluorescent end binding protein 1 (EB1) to label growing MT plus ends, we quantified individual MT nucleation events (Figure 1C–D). In the control reaction, the egg extract showed a typical low level of MT nucleation (Figure 1C, ‘buffer’,~3 MTs per field). In contrast, addition of wildtype γTuNA triggered an increase in MT nucleation of up to ~75-fold in a titration series (Figure 1C–D). The Xenopus F71A mutant equivalent to the human F75A mutant (Figure 1B), hereafter referred to as ‘F75A’, did not significantly increase MT number even at the highest concentration (3.6 µM, Figure 1C–D). Thus, the γTuNA domain activates MT nucleation in extract and requires the F75 residue, validating prior studies (Fong et al., 2008; Choi et al., 2010). Using sucrose gradients to fractionate mock and γTuNA-treated extracts, we also conclude that the γTuNA domain has no effect on γTuRC assembly, ruling out one possible explanation for this increase in MT number (Figure 1—figure supplement 2). While we cannot rule out that full-length Cdk5RAP2 might affect γTuRC assembly, we believe this is also unlikely as recent work has demonstrated that γTuRC can be assembled via heterologous expression of just γTuRC components and the RUVBL1-RUVBL2 AAA ATPase complex, without addition of a CM1-containing protein (Zimmermann et al., 2020). The γTuNA domain requires the universal MT template, the γ-tubulin ring complex (γTuRC) We next confirmed whether the γTuNA domain’s ability to increase MT nucleation in extract was dependent on the known MT nucleator, γTuRC. To do this, we first attempted depleting γTuRC from extract using our previously published rabbit-derived, anti-gamma tubulin antibody (Thawani et al., 2020). This γTuRC-depleted extract would then be assayed in the presence of γTuNA in our TIRF assay. However, due to low antibody yields and batch-to-batch variability, we were unable to generate γTuRC-depleted extract at consistent levels via this method. As an alternative, we instead depleted extracts of γTuRC via pulldown of γTuNA-coupled beads. With a single round of depletion, we observed a loss of >75% of γ-tubulin signal indicating a depletion of γTuRC (Figure 1E). In the mock-treated extract where γTuRC was not depleted, the γTuNA domain’s ability to increase MT nucleation levels remained unchanged (Figure 1F). By contrast, exogenous γTuNA no longer activated MT nucleation in γTuRC-depleted extracts (Figure 1F). Hence, the γTuNA domain requires the universal MT template, γTuRC, to activate MT nucleation. The γTuNA domain can designate new artificial MTOCs by recruiting γTuRC As γTuNA co-depletes γTuRC, we wondered whether this interaction would be sufficient to generate artificial MT asters (Figure 2A). To that end, we coated micron-scale beads with wildtype or mutant γTuNA domains and added them to extract. After a pulldown step, we assayed these beads for MT aster formation in vitro in the presence of purified fluorescent tubulin and GTP under oblique TIRF (Figure 2B). We found that wildtype γTuNA-coated beads formed large MT asters mimicking the potent MT nucleation of the centrosome (Figure 2B). In contrast, the F75A mutant beads formed severely impaired asters (Figure 2B). Mock-treated beads did not form asters. To confirm the stable presence of γTuRC, we repeated the bead pulldown from extract and attached the beads via an antibody against Mzt1, a γTuRC subunit, to surface-treated coverslips (Figure 2A). We then added fluorescent tubulin and GTP before live imaging via TIRF microscopy (Figure 2C). Critically, we observed that wildtype γTuNA beads attached and formed large MT asters in vitro, indicating that these beads had retained γTuRC and any other necessary MT nucleation factors (Figure 2C, Video 1). Figure 2 with 3 supplements see all Download asset Open asset The γTuNA domain strongly recruits MT nucleation factors (including γTuRC) from Xenopus egg extract. (A) Schematic of experiments for B and C. (B) Oblique TIRF images of MT asters from beads in vitro after 10 min. HisPur magnetic beads coated with either bovine serum albumin (mock), Strep-His-Xenopus γTuNA wildtype (WT), or ‘F75A’ mutant were incubated with extract, pulled-down, and washed. These were then diluted 1/1000 with polymerization mix containing 15 µM tubulin and 1 mM GTP, before imaging with TIRF. 5% Cy5-tubulin was used to label MTs. Bar = 5 µm. (C) Time-lapse imaging of MT aster growth from wildtype γTuNA beads in vitro. As in part B, wildtype γTuNA beads were pulled-down from extract and washed. These were then incubated on DDS-surface treated coverslips coated in anti-Mzt1 antibody to attach beads containing γTuRC. After a wash step, polymerization mix was added prior to time-lapse TIRF imaging. Frames are shown over the course of 15 min (900 s). Bar = 5 µm. (D) Diagram showing purification of endogenous Xenopus γTuRC using magnetic beads coupled to Strep-His-HaloTag-3C-human γTuNA. Made partly with Biorender. (E) Representative image of purified γTuRCs via negative-stain electron microscopy. Magnification is 64,000 x, taken at 80 kV with a Philips CM100 transmission electron microscope. Bar = 50 nm. (F) 2D class averages of 546 γTuRC particles picked from negative-stain EM images like in E. Each image represents one of four top classes. See “Figure 2—source data 1” for uncropped images in B and E. Figure 2—source data 1 Uncropped images for Figure 2. https://cdn.elifesciences.org/articles/80053/elife-80053-fig2-data1-v2.zip Download elife-80053-fig2-data1-v2.zip Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Post-pulldown wildtype γTuNA beads nucleate asters in vitro. From this we conclude that γTuNA domains are sufficient to specify new sites of γTuRC-mediated MT nucleation. Critically, this finding allowed us to develop a new γTuRC purification scheme based on scaled-up pulldowns with Halo-human γTuNA (outlined in Figure 2D–F), which we discuss in more detail later. Finally, the ability of F75A beads to weakly nucleate asters points to residual, but persistent, binding of γTuRC. We believe this is due to the low stringency wash particular to this experiment, as we do not detect γTuRC on F75A beads after higher stringency washes in subsequent experiments (western blots in Figure 2—figure supplement 1C, Figure 3D–E). Figure 3 with 1 supplement see all Download asset Open asset γTuNA requires both dimerization and the F75 residue to bind γTuRC in extract. (A) Model of dimerized, coiled-coil γTuNA with labeled side-chains for residues F63, I67, L70, F75, and L77. Made using PyMOL (RRID:SCR_000305) and chains C/G (red color) and D/H (blue color) from PDB: 6X0 V (Wieczorek et al., 2020a). (B) Size-exclusion chromatograms for human (aa 53–98) and Xenopus (aa 56–101) Halo-γTuNA wildtype and mutant constructs. Proteins were run at 50 µM (monomer) on a Superdex 200 increase 10/300 GL column (Cytiva) on an Äkta Pure system. Absorbance traces (A280 nm) were normalized by their peaks and plotted stacked as shown. (C) Diagram of peak retention volumes for each construct tested. (D) Western blots for γTuRC components, GCP5 and γ-tubulin, pulled down by beads coupled to human and Xenopus Halo-γTuNAs incubated in egg extract. The Strep-tag blot is shown as a bead loading control. (E) Western blots as in D, except comparing pulldowns done with Halo-Xenopus γTuNA alanine point mutants, with wildtype and F75A mutants as positive and negative controls. (F) Quantification of γTuRC pulldowns shown in D, normalized to the band intensity for human wildtype Halo-γTuNA beads. N=3. Error bars are SEM. (G) Same quantification of γTuRC pulldowns as in F, except for pulldowns as done in E. Normalized to the band intensity of wildtype Xenopus γTuNA. N=2. Error bars are SEM. See “Figure 3—source data 1” for numerical data and “Figure 3—source data 2” for raw blots. Figure 3—source data 1 Numerical data for Figure 3, includes normalized size-exclusion chromatography traces for Figure 3B, quantified pulldowns in Figure 3F, and quantified pulldowns in Figure 3G. https://cdn.elifesciences.org/articles/80053/elife-80053-fig3-data1-v2.zip Download elife-80053-fig3-data1-v2.zip Figure 3—source data 2 Labeled and raw blots used in Figure 3D and E. https://cdn.elifesciences.org/articles/80053/elife-80053-fig3-data2-v2.zip Download elife-80053-fig3-data2-v2.zip γTuNA is an obligate dimer Having confirmed that the γTuNA domain strongly recruits γTuRC from extract, we next investigated the γTuNA-γTuRC interaction. In a recent structural study, the authors generated a model of a parallel coiled-coil that directly interacts with γTuRC (Wieczorek et al., 2020a). The authors suggested that this coiled-coil is in fact a γTuNA dimer, although biochemical validation of this dimer state and its effect on γTuRC activity were not provided (Wieczorek et al., 2020a). To that end, we selectively mutated hydrophobic residues found within a heptad-repeat region of γTuNA. Specifically, we mutated the hydrophobic residues F63, I67, L70, and L77 to either alanine or aspartate (Figure 3A). To validate the well-conserved nature of this domain, we generated both human and Xenopus versions, referred to here by the residue position in the human sequence (Figure 1B). We initially focused on the double, triple, and quadruple mutants for both human and Xenopus γTuNAs. We performed size-exclusion chromatography (SEC) and compared the peak retention volumes of wildtype and mutated γTuNAs. Our SEC data revealed that wildtype γTuNA is a dimer (Figure 3B–C). By comparing the SEC traces for the double, triple, or quadruple mutants from both Xenopus and human γTuNAs, we found that γTuNA dimerization was dependent on residues I67, L70, and L77 (Figure 3B). The double hydrophilic mutants (I67D/L70D) from both human and Xenopus versions were entirely monomeric. This was also true for the human double-alanine mutant, I67A/L70A (Figure 3B). To resolve each residue’s individual contribution to γTuNA dimerization, we generated alanine point mutants for F63, I67, L70, and L77 in Xenopus γTuNA. We also tested the F75A mutant of Xenopus γTuNA, as we wanted to know whether its loss-of-function coincided with loss of dimerization. We compared the SEC traces for these point mutants and found that mutating residues F63 or F75 to alanine had no deleterious effect on γTuNA dimerization (Figure 3B). By contrast, individually mutating residues I67, L70, or L77 increasingly interfered with dimerization, resulting in intermediate populations between full dimer and full monomer (Figure 3B–C). Mutation of the L70 or L77 residues resulted in the most drastic impairment, further confirming that this central region is crucial for γTuNA dimerization. Both dimerization of γTuNA and its F75 residue are critical for binding γTuRC With the insight that the γTuNA domain is an obligate dimer, we next asked whether dimerization was required to bind γTuRC. We performed pulldowns of N-terminally Halo-tagged γTuNA mutants from Xenopus egg extract. We determined the amount of γTuRC bound for each γTuNA construct by probing for the γTuRC components GCP5 and γ-tubulin (Figure 3D–G). We found that both human and Xenopus double aspartate mutants (I67D/L70D), as well as the human triple mutant (I67D/L70D/L77D) did not bind γTuRC, indicating that loss of dimerization results in loss of γTuRC binding (Figure 3D and F). Interestingly, we found that the intermediate dimer mutants (I67A, L70A, or L77A) had correspondingly intermediate levels of γTuRC binding ability (Figure 3E). The I67A mutant, for example, was only weakly impaired in terms of dimerization (Figure 3B) and subsequently retained its ability to bind γTuRC (Figure 3E and G). As dimerization was increasingly impaired in the L70A and L77A mutants, γTuRC binding became increasingly weaker (Figure 3G). In the most extreme example, the L77A mutant, which had the most substantial dimerization defect, had complete loss of γTuRC binding (Figure 3G). Critically, the known F75A mutant did not bind γTuRC, as expected (Figure 3D–G). As our SEC data shows that F75A does not affect γTuNA dimerization, we conclude that both γTuNA dimerization and the F75 residue are required for binding γTuRC (Figure 3B and D–G). Finally, we found that forcing γTuNA dimerization via the addition of a constitutively dimeric coiled-coil domain (GCN4) did not rescue the ability of the intermediate dimer mutants to bind γTuRC (Figure 3—figure supplement 1). This suggests that simply bringing intermediate dimer mutants within tight proximity is not enough to induce restoration of the proper γTuRC binding interface. Both γTuNA dimerization and the F75 residue are required for full γTuRC activation in extract Having identified specific mutations that impaired γTuNA’s ability to dimerize and bind γTuRC, we next asked what effect these mutants had on MT nucleation in extract. We added wildtype or mutant Xenopus γTuNA to freshly prepared extracts and again tracked MT plus-ends via fluorescent EB1 as a measure of MT number (Figure 4). As before, wildtype γTuNA triggered an increase in MT nucleation, when compared to the buffer control (Figure 4). The F75A mutant had little effect on extract MT levels (Figure 4). Similarly, the L77A mutant, which cannot bind γTuRC in extract (Figure 3G), did not increase MT nucleation (Figure 4). Intriguingly, when we examined the intermediate γTuRC-binding mutants I67A and L70A, we found that the I67A mutant activated MT nucleation to ~50% of wildtype levels, but L70A had no activity (Figure 4B). This was surprising as I67A’s activation effect was on the order of its γTuRC-binding ability (~50% vs~67%; compared to wildtype), suggesting binding ability was predictive of the activation effect in extract (Figure 3G). However, because the L70A mutant had little activity in extract (~6%, Figure 4B) but retained ~35% binding ability (Figure 3G), it appears that there is a threshold to γTuRC’s activation in extract. We further analyze the implications of this divergent behavior between γTuNA mutants in our Discussion. Figure 4 Download asset Open asset Complete γTuNA dimerization is required to maximally increase MT nucleation in extract. (A) TIRF assay of MT nucleation in extract after addition of 2 µM (1 µM dimer) wildtype or single alanine mutants of Strep-His-Xenopus γTuNA. EB1-mCherry was used to count MTs (MT nucleation) and is shown pseudo-colored green. Images were taken after 5 min at 18–20°C. Bar = 5 µm. (B) Quantification of MT nucleation (MT number) normalized by the wildtype condition across four independent experiments. Red bar denotes wildtype level, while the blue bar denotes the effect of the I67A mutant. Error bars are SEM. See “Figure 4—source data 1” for numerical data. Figure 4—source data 1 Numerical data used in Figure 4. https://cdn.elifesciences.org/articles/80053/elife-80053-fig4-data1-v2.zip Download elife-80053-fig4-data1-v2.zip The γTuNA domain directly activates MT nucleation by γTuRC in vitro While we had explored the effect of wildtype γTuNA and its dimer mutants on MT nucleation in extract, we had yet to determine if γTuNA directly increased γTuRC’s activity in vitro. As we briefly mentioned (Figure 2D–F), we used beads coupled to a Halo-human γTuNA construct to purify endogenous Xenopus γTuRC from extract (Figure 2D–F), similar to previous work (Wieczorek et al., 2020b). Mass spectrometry confirmed that the dominant co-precipitant was indeed Xenopus γTuRC (Figure 2—figure supplement 1). We also confirmed the presence of fully assembled γTuRC rings via negative-stain electron microscopy (Figure 2E–F and Figure 2—figure supplement 3). Using this purified γTuRC, we investigated the effect of wildtype and mutant γTuNAs on γTuRC’s activity in vitro via in vitro TIRF assays (Figure 5). In these assays, biotinylated γTuRCs were attached to passivated coverslips before imaging with TIRF microscopy (schematized in Figure 5—figure supplement 1). This not only offers high signal-to-noise but also allows tracking of individual γTuRC-mediated MT nucleation events. Figure 5 with 3 supplements see all Download asset Open asset γTuNA dimers directly activate γTuRC MT nucleation ability in vitro. (A) Single molecule TIRF assays of γTuRC-mediated MT nucleation in vitro. Purified Xenopus γTuRCs were biotinylated and attached to passivated coverslips via surface-bound Neutravidin molecules. Polymerization mix containing 15 μM tubulin,1 mM GTP, and either control buffer or 3.3 μM (1.7 μM dimer) Strep-His-Xenopus γTuNA was then added. Wildtype, F75A, and L77A versions of γTuNA were tested. 5% Alexa 568-tubulin was used to visualize MTs. Images were taken every 2 s, for 5 min total, at 33.5 °C. Wildtype γTuNA (n=8), buffer control (n=6), γTuNA-F75A (n=5), and γTuNA-L77A (n=3). (B) Mean MT signal (MT mass) over time, normalized to the buffer condition at 300 s. (C) Mean MT number over time (measured for the first 150 s). The box shows the mean MT number ± SEM at 150 s for each condition. (B and C) Solid lines are the mean over time, with error clouds representing SEM. (D) Initial nucleation rates (Mts nucleated per sec) for each condition (± SEM). The curves shown in part C were fit to an exponential function to determine k (the nucleation rate). Each k was then averaged; see Materials and methods. The following are mean nucleation rate ± SEM. Buffer: 1.2±0.15 MTs/s, WT: 24.5±3.27 MTs/s, F75A: 2.3 ± 0.41 MTs/s, L77A: 2.4±0.55 MTs/s. (E and F) Violin plots of MT growth speeds (in E) or MT lengths (in F) for each condition. Means (μ) are shown alongside p-values. Wildtype γTuNA (n=2303 MTs), buffer control (n=355 MTs), γTuNA-F75A (n=368 MTs), and γTuNA-L77A (n=302 MTs). (C-F) Two-sample unpaired t-tests were used to compare the buffer control to the experimental values. Significance is p<0.05. See “Figure 5—source data 1” for all numerical data presented here. Figure 5—source data 1 Numerical data from Figure 5’s in vitro TIRF assays with purified γTuRC and γTuNA: including MT mass measurements, MT number, MT growth speed, and MT lengths. https://cdn.elifesciences.org/articles/80053/elife-80053-fig5-data1-v2.zip Download elife-80053-fig5-data1-v2.zip We started by first comparing total MT mass generated in our assay (Figure 5B). Strikingly, the addition of γTuNA triggered a 5-fold increase in MT mass as compared to the buffer control (Figure 5B, Video 2 and Video 3). To determine if this was a direct stimulation of γTuRC’s activity, we then quantified the number of γTuRC-nucleated MTs within the first 150 s (Figure 5C), the MT nucleation rate (Figure 5D), the mean MT growth speed (Figure 5E), and the mean maximum MT length (Figure 5F). These quantifications revealed that wildtype γTuNA sharply increased the γTuRC nucleation rate from 1.2 MTs/s to 24.5 MTs/s (~20-fold increase, Figure 5C–D). While there was a slight increase in mean MT growth speed (+0.2 µm/min), this did not translate into a significant effect on MT length (Figure 5E–F). We also" @default.
• W4320073612 created "2023-02-12" @default.
• W4320073612 creator A5063329457 @default.
• W4320073612 date "2022-06-13" @default.
• W4320073612 modified "2023-10-18" @default.
• W4320073612 title "Editor's evaluation: The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC)" @default.
• W4320073612 doi "https://doi.org/10.7554/elife.80053.sa0" @default.
• W4320073612 hasPublicationYear "2022" @default.
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