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. 2013 Feb 14;152(4):768-77.
doi: 10.1016/j.cell.2012.12.044.

Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2

Sabine Petry  1 , Aaron C Groen, Keisuke Ishihara, Timothy J Mitchison, Ronald D Vale
Affiliations

Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2

Sabine Petry et al. Cell. .

Abstract

The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence has suggested that microtubules also might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires γ-tubulin and augmin and is stimulated by factors previously implicated in chromatin-stimulated nucleation, guanosine triphosphate(GTP)-bound Ran and its effector, TPX2. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance.

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Figures

Figure 1
Figure 1. Branching microtubule nucleation is stimulated by Ran and TPX2 in meiotic Xenopus egg extracts
(A) Branching microtubule nucleation in Xenopus egg meiotic extracts without added Ran or TPX2. EB1-GFP (green) was added to the extract to follow the microtubule plus ends and thus identify locations of new microtubule growth. Alexa568-labelled bovine brain tubulin (red) was added to visualize microtubules. Sodium orthovanadate was added to prevent dynein-mediated sliding of microtubules along the glass and allow better observation of branching microtubules. Large arrows indicate nucleated microtubules that emerge a clear angled branch from the mother microtubule. Short arrows indicate nucleated microtubules that grow along the length of the mother microtubule. The time of this sequence is shown in seconds. Scale bar, 5 μm. See Movie S1. (B) Branching nucleation is stimulated in the presence of a constitutively active Ran mutant (Ran Q69L). The asterisk indicate a region that is enlarged in the bottom panels. Scale bar, 5 μm. See Movie S2. (C) Microtubule nucleation in an extract containing both RanQ69L and TPX2, leading to branched-fan like structures in the absence of microtubule gliding. The asterisk indicates a region that is enlarged in the bottom panels. Scale bar, 5 μm. See Movie S3. (D) Branching occurs from an exogenously added template microtubule. GMPCPP-stabilized, Cy5-labeled pig brain microtubules were attached to a passivated glass coverslip (to prevent binding of endogenous microtubules), and a reaction mixture of Xenopus extract, RanQ69L, TPX2, EB1-GFP and alexa568-labelled bovine brain tubulin was added. New microtubule growth (EB1-GFP spots) can be seen emerging from the template microtubule. Scale bar, 10 μm. See also Figure S1.
Figure 2
Figure 2. Quantifying the effect of RanQ69L and TPX2 on microtubule nucleation
The number of EB1 spots is counted for each time frame and experimental condition, and plotted against time. Because each microtubule, even when branched, is marked by a single EB1 spot at its growing plus tip, counting the number of EB1 spots directly corresponds to the number of microtubules per frame and field of view (82.2 × 82.2 μm). Data from a control extract in which only vanadate was added or experiments in which further additions of RanQ69L, TPX2, or RanQ69L plus TPX2 were made.
Figure 3
Figure 3. Properties of branching microtubule nucleation in a RanQ69L-treated extract
(A) Quantification of branch angles between the mother microtubule and the newly growing daughter microtubule. A 0 degree angle corresponds to growth of a new microtubule parallel to the mother microtubule, whereas a 180 degree angle signifies antiparallel growth. The polarity of the mother microtubule is preserved if the branch angle is smaller than 90°. 89% of the branches are <30°. (B) Quantification of branch position along the template microtubule. The majority of branches occur along the middle of the microtubule, about a third originate at the microtubule minus end and more rarely the EB1-enriched microtubule plus tip branches.
Figure 4
Figure 4. Molecular factors required for branching microtubule nucleation in the presence of RanQ69L and TPX2
(A) A control immunodepletion of the extract with total IgG fraction antibodies still produced fan-shaped structures composed of branching microtubule and did not significantly affect levels of augmin (Dgt4 subunit), γ-TB and TPX2 levels as quantified in a Western blot (bottom, error bars represent s.d. of three independent experiments with different extracts). (B) Immunodepletion of γ-TB (bottom) almost extinguished microtubule nucleation; most fields had no or a few microtubules; after longer times (7 min), rare examples of fan-shaped structures with few, but long microtubules could be found on the coverslip (see insert). (C) Immunodepletion of the augmin Dgt4 subunit, which depletes the entire augmin complex and one third of γ-TB (bottom diagram), abolished branching microtubule nucleation, but still allowed for microtubule growth. (D) Similarly, immunodepletion of TPX2 abolished branching microtubule nucleation, but still allowed for microtubule growth. Scale bars, 10 μm. See The time shown indicates the approximate time of recording after adding the extracts to the flow cell. Movie S6 for sequences of these immunodepletions. See also Figure S2. (E) Quantification of microtubule number over time for different immunodepletions. Reactions were made in the presence of RanQ69L plus TPX2 (except for the TPX2 depletion, in which case only RanQ69L was added).
Figure 5
Figure 5. Rescue of branching microtubule nucleation by addition of recombinant TPX2 to TPX2-depleted Xenopus extracts
(A) Addition of purified TPX2 or CT-TPX2 to extract depleted of TPX2 (see Fig. 3D) could rescue branching microtubule nucleation and the formation of branched fan-like structures. In contrast, add-back of NT-TPX2 displayed the same phenotype as TPX2 immunodepletion (see Fig. 3D). For each experimental condition a montage of four adjacent fields is displayed, which was collected with the slide explorer function of the MicroManager software (Edelstein et al., 2010). Scale bar, 10 μm. See Movie S7. (B) Localization of full-length GFP-TPX2 (C) Localization of the N-terminus of GFP-TPX2 (GFP-NT-TPX2) (D) Localization of the C-terminus of GFP-TPX2 (GFP-CT-TPX2). The full-length and GFP-CT-TPX2 constructs binds along microtubules of the fan-like structures, whereas the N-terminus of GFP-TPX2 can hardly be detected. Scale bar, 10 μm. E) Immunoprecipitations using antibodies specific against TPX2, augmin, and γ-TB, and control antibodies (total IgG fraction antibodies). The same antibodies were used in the immunoblot as detection reagents. TPX2, augmin, and γ-TB interact with each other. The two controls reflect separate immunoprecipitation experiments.
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