Microtubules are nucleated from specific locations in the cell, and several of these microtubule nucleation pathways converge to form a particular cytoskeletal architecture (Kollman et al., 2011; Lin et al., 2015; Lüders and Stearns, 2007). Importantly, microtubules in cells are nucleated by the microtubule nucleator γ-TuRC (Kollman et al., 2011; Zheng et al., 1995) and its co-nucleation factor XMAP215 (Flor-Parra et al., 2018; Gunzelmann et al., 2018; Thawani et al., 2018). At the same time, each microtubule nucleation pathway requires a unique set of nucleation effectors to recruit and regulate γ-TuRC at distinct cellular locations (Lin et al., 2015). The identity of most of these effectors remains elusive, along with a mechanistic understanding of how they constitute the different microtubule nucleation pathways that generate the cytoskeleton.

Microtubules can nucleate from pre-existing microtubules, termed branching microtubule nucleation (Petry et al., 2013), which amplifies microtubule number while preserving their polarity, as is needed in the mitotic spindle and in axons (Cunha-Ferreira et al., 2018; David et al., 2019; Kamasaki et al., 2013; Petry et al., 2013; Sánchez-Huertas et al., 2016). The eight-subunit protein complex augmin is required for branching microtubule nucleation in plant, human and Drosophila cells, and meiotic Xenopus egg extract, where its depletion leads to reduced spindle microtubule density, less kinetochore fiber tension, metaphase arrest, and cytokinesis failure (David et al., 2019; Decker et al., 2018; Goshima et al., 2008; Hayward et al., 2014; Ho et al., 2011; Kamasaki et al., 2013; Lawo et al., 2009; Nakaoka et al., 2012; Petry et al., 2011; Uehara et al., 2009). Augmin is necessary to recruit γ-TuRC to spindle microtubules (Goshima et al., 2007), and following the recombinant expression of augmin (Hsia et al., 2014), this activity was confirmed using purified proteins (Song et al., 2018).

In meiotic Xenopus egg extract, the Ran-regulated protein TPX2 is released near chromatin (Gruss et al., 2001), where it stimulates branching microtubule nucleation (Petry et al., 2013), potentially by activating γ-TuRC via nucleation activator motifs (Alfaro-Aco et al., 2017). Recently, TPX2 was also observed to form a co-condensate with tubulin along the microtubule lattice, which enhances the kinetic efficiency of branching microtubule nucleation (King and Petry, 2019). In meiotic Xenopus egg extract, TPX2 needs to bind to microtubules before augmin/γ-TuRC to result in a successful nucleation event (Thawani et al., 2019). In contrast, in mitotic Drosophila cells TPX2 is not required, and augmin can bind to microtubules before γ-TuRC (Verma and Maresca, 2019). Despite these numerous studies to characterize each individual protein component, exactly how augmin, TPX2 and γ-TuRC together mediate branching microtubule nucleation, and whether they alone constitute a minimal system that nucleates branched microtubules, remains unclear. Here, we use biochemical reconstitution of its purified components to mechanistically dissect branching microtubule nucleation.

Branching microtubule nucleation has been studied in Xenopus egg extract, where it is elicited by the constitutively active version of Ran (RanQ69L) (Petry et al., 2013). In order to establish a controlled, minimal assay that furthers our mechanistic insight, we exposed a microtubule tethered to glass to sequential reaction mixtures of decreasing complexity and thereby regulated the availability of proteins necessary to stimulate branching microtubule nucleation. Using multicolor time-lapse total internal reflection (TIRF) microscopy, we first confirmed that an endogenous, pre-existing microtubule can serve as a template for branching microtubule nucleation when exposed to Ran-supplemented extract that releases branching factors (Figure 1A and Figure 1—video 1). This shows that a microtubule formed independent of Ran can serve as the site for binding of branching factors and subsequent nucleation events.

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To gain mechanistic insight, we hypothesized that all necessary Ran-regulated branching factors bind to the pre-existing microtubule prior to microtubule nucleation. To test this, Ran-regulated branching factors were allowed to bind to taxol-stabilized pre-existing microtubules in the presence of nocodazole, which inhibits new microtubule formation (Figure 1B and Figure 1—figure supplement 1). When another extract reaction was subsequently added, new microtubules nucleated almost exclusively from pre-existing microtubules, indicating that Ran-regulated branching factors bind to microtubules independent of their successful nucleation reactions (Figure 1B). Importantly, when RanQ69L was omitted and no branching factors were released in the second extract reaction, pre-existing microtubules simply elongated and branching microtubule nucleation was absent in the third reaction (Figure 1—figure supplement 2A).

To further test whether these microtubule-bound branching factors are sufficient for generating branches, we reduced the complexity of our assay by introducing only purified tubulin and GTP in the third reaction step. Surprisingly, short branched microtubules nucleated from pre-existing microtubules (Figure 1—figure supplement 2B), showing that upon binding of branching factors and γ-TuRC, tubulin is the only protein required to form new branched microtubules from the localized factors. Further addition of XMAP215 to the final tubulin reaction made the short branches grow longer (Figure 1C). This revealed that branched microtubules retained the polarity of the pre-existing microtubule, and new microtubules do not appear to nucleate from other branched microtubules, suggesting that the branching factors do not relocate between microtubules (Figure 1C and Figure 1—video 2). Thus, solely the deposition of branching factors and γ-TuRC to the pre-existing microtubule determines branching architecture.

Because the key for branching microtubule nucleation is to target γ-TuRC along the length of a microtubule, we verified as a proof of concept that purified γ-TuRC, when tethered along the microtubule lattice via artificial linkers, can still nucleate microtubules, which indeed was the case (Figure 2—figure supplement 1A–B). Therefore, if all branching factors are known, it should be possible to reconstitute branching microtubule nucleation from a template microtubule using only purified components. To test this, we purified the essential proteins for branching microtubule nucleation in Xenopus egg extract (Petry et al., 2013). The GFP-labeled eight-subunit X. laevis augmin holocomplex was co-expressed in insect cells and co-purified (Song et al., 2018), the native 2.2 MDa γ-TuRC was purified from Xenopus egg extract (Thawani et al., 2018), and GFP-TPX2 was expressed from E. coli and purified (King and Petry, 2019) (Figure 2—figure supplement 2A–C).

First, we assessed how the nucleator γ-TuRC gets targeted along the microtubule lattice. Purified TPX2, augmin and γ-TuRC were added in various combinations to surface-bound, GMPCPP-stabilized microtubules and imaged via TIRF microscopy (Figure 2A). γ-TuRC, visualized by a fluorescently-labeled antibody, bound along the length of microtubules in the presence of augmin (Figure 2B–C and Figure 2—source data 1) consistent with previous studies (Song et al., 2018). Unexpectedly, TPX2 alone marginally increased the binding of γ-TuRC to microtubules compared to non-specific γ-TuRC binding. A higher amount of γ-TuRC was recruited all along the microtubule lattice in the presence of both TPX2 and augmin (Figure 2B–C and Figure 2—source data 1). Surprisingly, augmin and TPX2 formed distinct puncta on microtubules, where γ-TuRC was recruited. Using negative stain electron microscopy, we visualized this binding at higher resolution and indeed observed that γ-TuRC is recruited to regularly spaced patches, where it accumulates (Figure 2D). Next, we tested whether the microtubule binding proteins augmin and TPX2 need to bind in a certain sequence. Surprisingly, microtubule-bound TPX2 increased the amount of augmin bound to the microtubule, whereas the presence of augmin did not change the level of bound TPX2 (Figure 2E–F and Figure 2—source data 2). In sum, TPX2 has the unexpected capacity to directly recruit γ-TuRC as well as augmin, which in turn targets more γ-TuRC along the microtubule lattice.

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Figure 2—source data 1

Binding of γ-TuRC to a template microtubule.

Numerical data obtained from the experiment in Figure 2B, and represented graphically in Figure 2C.

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Figure 2—source data 2

Binding of TPX2 and augmin to a template microtubule.

Numerical data obtained from the experiment in Figure 2E, and represented graphically in Figure 2F.

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Having established that purified TPX2 and augmin recruit γ-TuRC to template microtubules, can they indeed cause branching microtubule nucleation? All three factors were bound to a stabilized microtubule as above, followed by addition of tubulin and GTP in polymerization buffer (Figure 3A). Remarkably, branching microtubule nucleation from a template microtubule occurred using only purified proteins (Figure 3B and Figure 3—video 1). Live microscopy allowed us to accurately distinguish branching microtubule nucleation from microtubules that were spontaneously nucleated before contacting the microtubule template (Figure 3—figure supplement 1). Thus, TPX2, augmin and γ-TuRC are sufficient to specifically nucleate new branched microtubules, which remain attached at the nucleation site on the template microtubule (Figure 3B). Occasionally, enough microtubules branched from a single microtubule template (Figure 3B, bottom) to create structures reminiscent of those created after TPX2, augmin and γ-TuRC were deposited from Xenopus egg extract (Figure 1—figure supplement 2B).

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Figure 3—source data 1

Position of microtubule branches along the template microtubule.

Numerical data obtained from the experiment in Figure 3A–B, and represented graphically in Figure 3C.

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Figure 3—source data 2

Quantification of branched microtubules.

Numerical data obtained from the experiment in Figure 3A–B and Figure 3—figure supplement 2, and represented graphically in Figure 3E.

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Figure 3—source data 3

Angles of branching microtubule nucleation.

Numerical data obtained from the experiment in Figure 3A–B and Figure 3—figure supplement 2, and represented graphically in Figure 3F.

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Figure 3—source data 4

Quantification of non-branched microtubules.

Numerical data obtained from the experiment in Figure 3A–B and Figure 3—figure supplement 2, and represented graphically in Figure 3—figure supplement 3A.

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Figure 3—source data 5

Quantification of branched microtubules.

Numerical data represented graphically in Figure 3G.

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How are the nucleation sites spatially organized along the template microtubule? New microtubules nucleate all along the template microtubule without any preference for the template’s plus- or minus-ends (Figure 3C and Figure 3—source data 1), likely because the template microtubule was fully available for simultaneous binding of branching factors in our assay set-up. Thus, there is no signature on the stabilized microtubule lattice that determines where a branch occurs, only that microtubule nucleation events occur from distinct TPX2/augmin puncta distributed along the microtubule lattice (Figure 3D). Multiple microtubules can be generated from the same puncta as resolved by light microscopy (Figure 3D), which presumably nucleate from neighboring γ-TuRCs (Figure 2D). Because both TPX2 and augmin were concurrently tagged with GFP, an unlabeled version of TPX2 was used to confirm that augmin was indeed present in the observed puncta on template microtubules (Figure 3—figure supplement 2A).

What does each protein contribute to branching microtubule nucleation? To test this, each purified factor was assessed alone for its nucleation potential from a template microtubule, combined in pairs and ultimately altogether. Notably, γ-TuRC is essential for branching microtubule nucleation (Figure 3E and Figure 3—source data 2). Despite the fact that TPX2 can recruit tubulin (King and Petry, 2019), it alone or together with augmin cannot nucleate branched microtubules. γ-TuRC can infrequently bind to the microtubule lattice on its own (Figure 2D), leading to rare nucleation events without TPX2 and augmin (Figure 3E and Figure 3—source data 2). Not surprisingly, augmin and γ-TuRC can cause branching microtubule nucleation to a limited extent (Figure 3E, Figure 3—figure supplement 2B–C and Figure 3—source data 2), as augmin can directly recruit γ-TuRC to a pre-existing microtubule in vitro (Song et al., 2018). Surprisingly, TPX2 and γ-TuRC can also cause branching microtubule nucleation to a similar extent as augmin and γ-TuRC (Figure 3E, Figure 3—figure supplement 2D–E and Figure 3—source data 2). Although TPX2 only marginally recruits γ-TuRC to the microtubule lattice, its ability to form a co-condensate with tubulin (King and Petry, 2019), and to possibly activate γ-TuRC (Alfaro-Aco et al., 2017), may facilitate γ-TuRC-dependent microtubule nucleation from a pre-existing microtubule.

Similar to the pattern observed when both TPX2 and augmin are present, augmin or TPX2 alone with γ-TuRC also form distinct puncta along the microtubule lattice, implying that augmin may have a similar mode of binding to the microtubule lattice as TPX2. However, fewer of these puncta actually nucleate branched microtubules (Figure 3E, Figure 3—figure supplement 2C, Figure 3—figure supplement 2E and Figure 3—source data 2). Importantly, only when augmin, TPX2 and γ-TuRC are present, branching microtubule nucleation occurs most often (Figure 3E and Figure 3—source data 2). Branched microtubules are preferentially formed in angles < 90 degrees, with 0–15 degrees being the most common (Figure 3F and Figure 3—source data 3). This way, most branched microtubules maintain the same polarity as the mother microtubule, a hallmark of branching microtubule nucleation. The angle of microtubule branches did not drastically change when augmin or TPX2 were combined with γ-TuRC, only that augmin/γ-TuRC alone caused a higher proportion of shallow branch angles (Figure 3F and Figure 3—source data 3).

A key question that we next addressed with this system is whether TPX2 and augmin merely act as localizing factors, or whether they indeed activate γ-TuRC at the branching nucleation sites. To test whether branching was specifically stimulated, we counted the number of non-branched microtubules nucleated in the reactions shown in Figure 3E. Reactions where γ-TuRC was present have approximately twice as many non-branched microtubules per field of view compared to the reactions where γ-TuRC was absent. Notably, the addition of TPX2 or augmin does not change the number of non-branched microtubules (Figure 3—figure supplement 3A and Figure 3—source data 4). This suggests that the increase in branching microtubule nucleation by TPX2 and augmin is not just a result of increased microtubule nucleation overall, but specifically occurs from a pre-existing microtubule.

To more directly assess whether augmin or TPX2 increase the nucleation activity of γ-TuRC, we performed a microtubule nucleation assay in solution. Augmin did not enhance the nucleation activity of purified γ-TuRC in vitro (Figure 3—figure supplement 3B), consistent with previous studies (Song et al., 2018). A low concentration of TPX2 (10 nM) did not enhance the nucleation activity of γ-TuRC either (Figure 3—figure supplement 3C). A higher concentration of TPX2 (50 nM) was also tested, but at this concentration and in the absence of pre-formed microtubules, TPX2 forms co-condensates with tubulin in solution that nucleate microtubules in vitro (Figure 3—figure supplement 3D) (King and Petry, 2019), which precludes the accurate assessment of γ-TuRC-mediated microtubule nucleation at higher concentrations. These nucleation experiments suggest that TPX2 and augmin do not activate γ-TuRC in solution. There remains the possibility that they increase the nucleation activity of γ-TuRC specifically when bound to a microtubule. To evaluate whether the increase in branching microtubule nucleation activity in Figure 3E is larger than the increase in γ-TuRC binding in Figure 2C we calculated the ratio of branching to binding, and normalized it to the γ-TuRC only sample. Although the increase in branching when both TPX2 and augmin are present with γ-TuRC is marginally larger than what is expected from the increase in γ-TuRC localization alone, the difference is not statistically significant based on the current data.

Last, we tested whether branching microtubule nucleation is further enhanced by having XMAP215 present. Indeed, XMAP215 co-localizes to the template microtubule and appears to increase both microtubule nucleation rate and length (Figure 3—figure supplement 4). Exact quantification of this effect was not possible because branched microtubules were already formed before imaging was possible and microtubules quickly grew into each other, preventing the accurate identification of branching microtubule nucleation. Lastly, knowing that the binding sequence of TPX2 and augmin matters for maximum factor recruitment, does this have an effect on nucleation? Indeed, only when TPX2 was bound first and augmin/γ-TuRC second, a higher level of branching microtubule nucleation was measured (Figure 3G and Figure 3—source data 5).

Via an in vitro reconstitution, we demonstrate that the three factors TPX2, augmin and γ-TuRC are sufficient to cause branching microtubule nucleation and defined the roles of each protein. Although microtubule nucleation effectors alone, both as monomers or as phase-separated entities, can generate microtubules in vitro (Roostalu et al., 2015; Woodruff et al., 2017; King and Petry, 2019), γ-TuRC is required for physiological microtubule nucleation (Kollman et al., 2011; Thawani et al., 2018), and this reconstitution highlights the importance of including it when studying microtubule nucleation. Considered a poor nucleator, γ-TuRC that has been hypothesized to require activation (Lüders and Stearns, 2007). In our in vitro system, γ-TuRC’s activity could be tuned via the tubulin concentration and the presence of XMAP215. Assay conditions in which not all nucleation events occur at once were necessary in order to clearly identify and observe branching microtubule nucleation events and to differentiate them from microtubules growing over one another. Based on this, we could investigate the effect of TPX2 and augmin on the activity level of γ-TuRC. The microtubule nucleation capacity of γ-TuRC was not enhanced by TPX2 or augmin in solution, but these factors specifically enable γ-TuRC-dependent microtubule nucleation from a pre-existing microtubule. This can be explained if TPX2 and augmin act as localization factors, but there is also the possibility that they increase the nucleation activity of γ-TuRC specifically when bound to a microtubule. Our results do not conclusively support or rule our either of these models. In the future, single molecule and kinetic measurements along with structural studies are required to determine how exactly TPX2 and augmin interact with γ-TuRC, and whether they increase its activity to stimulate microtubule nucleation from a template microtubule.

Augmin can recruit γ-TuRC to the side of a pre-existing microtubule (Song et al., 2018), which can subsequently nucleate branched microtubules. This may be reflective of cell types where TPX2 is not needed for branching microtubule nucleation, such as mitotic Drosophila cells (Verma and Maresca, 2019). Unexpectedly, TPX2 with γ-TuRC can also cause branching microtubule nucleation to a similar extent as augmin with γ-TuRC. This could be explained by our observation that TPX2 increases γ-TuRC localization to microtubules to a small extent. Additionally, it was recently shown that TPX2 can recruit tubulin to the lattice of a pre-existing microtubule by forming a co-condensate (King and Petry, 2019), potentially making lattice-bound γ-TuRC more likely to nucleate branched microtubules. Finally, TPX2 contains γ-TuRC activator motifs that have been proposed to activate γ-TuRC (Alfaro-Aco et al., 2017). All three of these functions may be the reason why TPX2, which recruited less γ-TuRC than augmin, subsequently caused γ-TuRC-dependent branching microtubule nucleation at a similar level as augmin. Besides operating via these three modes in the full reconstitution, TPX2 additionally recruits more augmin, which in turn can localize more γ-TuRC. Finally, TPX2 alone creates a slightly wider angle distribution of branches, than augmin alone and both factors together. Thus, by bridging the microtubule and γ-TuRC with a defined architecture based on its h-shaped structure (Hsia et al., 2014; Song et al., 2018), augmin may keep the angle variation in a more shallow range, which is critical for creating parallel structures that retain microtubule polarity as is necessary in the spindle and axons.

Although TPX2 + γ-TuRC or augmin + γ-TuRC can nucleate microtubules from a pre-existing microtubule in this purified system, both factors are required in Xenopus egg extract for branching microtubule nucleation (Petry et al., 2013). This indicates that microtubule nucleation in a physiological environment is more stringently regulated. Another important difference is that while it is beneficial that TPX2 binds to microtubules before augmin in our in vitro reconstitution, TPX2 binding to microtubules is absolutely necessary for augmin/γ-TuRC binding and subsequent branching microtubule nucleation in Xenopus egg extract (Thawani et al., 2019). This implies that TPX2 directly regulates augmin’s binding to microtubules in Xenopus egg extract, in a manner that still needs to be uncovered.

In summary, TPX2, augmin and γ-TuRC are required for efficient branching microtubule nucleation in vitro. Surprisingly, TPX2, despite being only a single protein amongst large multi-subunit complexes, lies at the very heart of controlling this reaction, as demonstrated in this purified in vitro system and in Xenopus egg extract (Thawani et al., 2019). Finally, even though TPX2, augmin and γ-TuRC are sufficient to reconstitute the nucleation of branched microtubules in vitro, we cannot eliminate the possibility that other proteins are involved in additional regulation, such as the microtubule and augmin binding protein EML3 (Luo et al., 2019).

Mechanistic insight obtained from this work helps explain how microtubules are made within spindles to orchestrate chromosome segregation. It is analogous to the in vitro reconstitution of actin branching (Mullins et al., 1998), which paved the way to explain how the actin cytoskeleton supports cell locomotion. In this biochemical reconstitution, γ-TuRC was localized to a specific location, from which it nucleates a microtubule in vitro as it would occur in the cell. As such, this work serves as a platform to study how microtubule nucleation creates different microtubule architectures that support cell function. This approach presents a pioneering example for the biochemical reconstitution of other microtubule nucleation pathways or microtubule organizing centers. It also serves as a stepping-stone to reconstitute larger structures based on this microtubule nucleation pathway, such as the mitotic spindle.

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2 and 3.

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Author details

1. Raymundo Alfaro-Aco

   Department of Molecular Biology, Princeton University, Princeton, United States

   Present address

   Department of Molecular Biology, Princeton University, Princeton, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4196-3797

2. Akanksha Thawani

   Department of Chemical and Biological Engineering, Princeton University, Princeton, United States

   Present address

   Department of Chemical and Biological Engineering, Princeton University, Princeton, United States

   Contribution

   Resources, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4168-128X

3. Sabine Petry

   Department of Molecular Biology, Princeton University, Princeton, United States

   Present address

   Department of Molecular Biology, Princeton University, Princeton, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   spetry@Princeton.EDU

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8537-9763

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