Structural analysis of the role of TPX2 in branching microtubule nucleation

Abstract

The mitotic spindle consists of microtubules (MTs), which are nucleated by the γ-tubulin ring complex (γ-TuRC). How the γ-TuRC gets activated at the right time and location remains elusive. Recently, it was uncovered that MTs nucleate from preexisting MTs within the mitotic spindle, which requires the protein TPX2, but the mechanism basis for TPX2 action is unknown. Here, we investigate the role of TPX2 in branching MT nucleation. We establish the domain organization of Xenopus laevis TPX2 and define the minimal TPX2 version that stimulates branching MT nucleation, which we find is unrelated to TPX2's ability to nucleate MTs in vitro. Several domains of TPX2 contribute to its MT-binding and bundling activities. However, the property necessary for TPX2 to induce branching MT nucleation is contained within newly identified γ-TuRC nucleation activator motifs. Separation-of-function mutations leave the binding of TPX2 to γ-TuRC intact, whereas branching MT nucleation is abolished, suggesting that TPX2 may activate γ-TuRC to promote branching MT nucleation.

Figure 1.

TPX2 α5–α7 is the minimal construct that stimulates branching MT nucleation. (A) Domain organization of the C-terminal half of X. laevis TPX2 based on secondary structure prediction using Jpred. (B) Graphical representation of the FKARP motif, which occurs after predicted α-helices in domains α3 through α6. (C) CD spectra of various TPX2 constructs in the region of 200–250 nm. All spectra suggest the presence of significant α-helical content, which is characterized by two minima near 208 nm and 220 nm. Estimates of secondary structure content using the K2D3 method agree with values obtained from the Jpred prediction. (D–M) Branching MT nucleation in Xenopus egg extracts in the presence of 2 µM of different truncated TPX2 constructs. The same results were obtained with a lower TPX2 concentration of 0.75 µM. EB1-GFP (green) and Alexa Fluor 568–labeled porcine brain tubulin (red) were added to the extract to follow MT plus ends and MTs, respectively. Vanadate was added to prevent dynein-mediated gliding of MTs. All images were taken after 5 min. Brightness and contrast were adjusted for each image individually to optimize visual comparison of MT structures. Bar, 10 µm. See Video 1. (N) The number of individual MTs was counted for each time frame and then plotted against time. The data are displayed for addition of TPX2 α3–α7 and all the inactive constructs. (O) Same as N, except the number of MTs is averaged over two experimental replicates for the addition of TPX2 α3–α7 and the other active constructs α4–α7 and α5–α7. Error bars represent absolute error. All extract experiments were performed at least three different times.

Figure 2.

Multiple domains of TPX2 contribute to its ability to bind and bundle MTs in vitro. (A) Co-sedimentation assay of 1 µM of various TPX2 constructs with Taxol-stabilized MTs in vitro. The ratio of protein in the supernatant and pellet after centrifugation was used to calculate the fraction of total protein bound to MTs. Values are the mean of two experiments, and error bars represent standard deviation. *, TPX2 α6 was only tested once. (B) MT bundling assay using Alexa Fluor 568–labeled GMPCPP-stabilized MTs in vitro with 1 µM of various TPX2 constructs. Bar, 10 µm. Assay was repeated three times.

Figure 3.

Identification of novel motifs within TPX2 α5–α7. (A) Domain organization of TPX2 α5–α7 showing newly found motifs. Amino acids 507–523 (cyan) share homology with the SPM, and amino acids 519–531 (orange) and 562–567 (purple) share homology with the γTuNA motif, both of which have a γ-TuRC activation function in the proteins where they were originally identified. Amino acids 620–626 (pink) have been suggested to negatively regulate MT nucleation in Xenopus egg extract, and their absence in X. tropicalis TPX2 leads to increased MT formation. Amino acids 689–716 (blue) are necessary for the interaction between TPX2 and the kinesin Eg5. (B) Graphical representation of the SPM of pericentrin/Spc110 across multiple species and the γTuNA motif of Mto1/centrosomin/CDK5RAP2 and the related myomegalin across multiple species. The motif sequence logos were made using WebLogo 3. Sequence alignment reveals homology between these motifs and sequences within TPX2 α5–α7 (red sequence), revealing the new motifs SPM (cyan box), γTuNAa (orange box), and γTuNAb (purple box). γTuNAa and γTuNAb are separated by a stretch of 29 amino acids.

Figure 4.

The newly identified motifs in TPX2 are necessary to stimulate branching MT nucleation. (A–H) Branching MT nucleation in Xenopus egg extracts and in the presence of 2 µM TPX2 α3–α7 or α5–α7, with and without various motif deletions. The same results were obtained with a lower TPX2 concentration of 0.75 µM. EB1-GFP (green) and Alexa Fluor 568–labeled porcine brain tubulin (red) were added to follow MT plus ends and MTs, respectively. Vanadate was added to prevent dynein-mediated gliding of MTs. All images were taken after 10 min. Brightness and contrast were adjusted for each image individually to optimize visual comparison of MT structures. Bar, 10 µm. See Video 2. (I and J) For each reaction, the number of individual MTs was counted for each time frame and then plotted against time. The data are displayed for TPX2 α3–α7 and TPX2 α5–α7 and their inactive deletion mutants. (K) Same as I and J, but here the number of MTs is averaged over two experimental replicates for TPX2 α5–α7 and the only active deletion mutant, TPX2 α5–α7 Δtrop. Error bars represent absolute error. All extract experiments were performed at least three different times.

Figure 5.

Branching MT nucleation activity of multiple TPX2 single-site mutants. (A) Domain organization of TPX2 α5–α7 showing the location of single-site mutations. (B–L) Branching MT nucleation in Xenopus egg meiotic extracts and in the presence of 2 µM TPX2 α5–α7 with and without various single-site mutations. The same results were obtained with a lower TPX2 concentration of 1 µM. Images for F492AzF, F629AzF, and F714AzF mutants, which show a high level of activity, as well as the image for the positive control TPX2 α5–α7, were collected using one extract. Images for the rest of the mutants and the negative control were collected with a different extract. This was necessary because the lifetime of one extract was incompatible with the high number of samples; however, both controls are representative and consistent among all extracts tested. EB1-mCherry (green) and Cy5-labeled porcine brain tubulin (red) were added to the extract to follow MT plus ends and MTs, respectively. Vanadate was added to prevent dynein-mediated MT gliding. All images were acquired after 15 min. Brightness and contrast were adjusted for each image individually to optimize visual comparison of MT structures. Bar, 10 µm. See Video 3. (M) The number of EB1 particles was counted for three different fields of view and the mean was plotted against time. The data are displayed for the addition of TPX2 α5–α7 and all the inactive mutants. Data collection for the positive control, TPX2 α5–α7, was interrupted in the region denoted by the dashed line. Error bars represent standard deviation. (N) Same as M, but the data are depicted for the addition of TPX2 α5–α7 and mutants that have full or intermediate activity. Measurements also represent the mean of three different fields of view, and the error bars denote standard deviation. All extract experiments were performed at least three different times.

Figure 6.

TPX2 α5–α7 does not induce MT assembly in vitro. (A) Representative images of MTs formed after incubation of 200 nM fluorescent full-length TPX2, TPX2 α3–α7, and TPX2 α5–α7 with 30 µM tubulin. (B) Same as A, but using 800 nM of each TPX2 construct. At this concentration, full-length TPX2 and TPX2 α3–α7 also form tubulin aggregates. Assay was repeated two times. Bars, 10 µm.

Figure 7.

Single-site mutations within TPX2 α5–α7 do not interfere with the interaction of γ-TuRC. Immunoprecipitation of 1 µM GFP-tagged TPX2 constructs from Xenopus egg extracts using an antibody specific against GFP. The same GFP antibody was used for detection in the immunoblot, along with commercial antibodies against GCP5 and γ-tubulin. Assay was repeated three times. WT, wild type.