Structural insight into TPX2-stimulated microtubule assembly
- Lawrence Berkeley National Laboratory, United States
- The Francis Crick Institute, United Kingdom
- University of California, Berkeley, United States
- Howard Hughes Medical Institute, University of California, Berkeley, United States
Figures
Figure 1 with 5 supplements
High-resolution cryo-EM structure of TPX2 bound to GMPCPP-MTs.
(A) Schematic of domain structure for full-length TPX2 and TPX2mini. (B) Cryo-EM reconstruction of mGFP-TPX2mini decorated GMPCPP-MT, with pseudo-helical symmetry applied. α-tubulin, β-tubulin and …
Figure 1—figure supplement 1
The sequence of human TPX2.
Predicted secondary structure for TPX2 obtained using PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/). α-helices are colored in magenta and beta sheets in yellow. The starting and ending residues for …
Figure 1—figure supplement 2
Coomassie Blue stained SDS page gels of purified TPX2 constructs used in this study.
Equal amounts of intact (A) wild-type and mutant versions of mGFP-TPX2micro, (B) wild-type and triple mutant of mGFP-TPX2mini, (C) wild-type and triple mutant of biotinylated TPX2mini were loaded on …
Figure 1—figure supplement 3
Cryo-EM images of GMPCPP-MTs decorated with TPX2mini or TPX2micro constructs.
Cryo-EM images of GMPCPP-MTs in the presence of saturating amounts of either mGFP-TPX2mini (A) or mGFP-TPX2micro (B) clearly show extra densities on the MT surface corresponding to the TPX2 molecules.
Figure 1—figure supplement 4
Resolution estimation of the cryo-EM structures of GMPCPP-MTs decorated with TPX2.
(A) Fourier Shell Correlation (FSC) curves for GMPCPP-MTs decorated with mGFP-TPX2mini and mGFP-TPX2micro. The final resolution for each reconstruction was estimated by calculating the Fourier Shell …
Figure 1—figure supplement 5
Cryo-EM reconstructions of GMPCPP-MTs decorated with TPX2 molecules.
(A–B) Zoom-in views of the symmetric reconstructions of 14-PF (A) and 13-PF (B) GMPCPP-MTs decorated with TPX2mini molecules show similar density levels for the TPX2. (C–D) Zoom-in view of the …
Figure 2
TPX2micro retains the MT lattice specificity for GMPCPP-MTs.
(A) Schematic of the TPX2mini and TPX2micro constructs. (B–C) TIRF microscopy images (B) and representative kymographs (C) showing mGFP-TPX2micro (green in merge) binding preferentially to the …
Figure 3 with 1 supplement
Residues important for TPX2 interaction with MTs.
(A) Zoom-in view of the atomic model of the TPX2 ridge (left) and wedge (right). (B) Schematic of TPX2micro indicating the ‘ridge’ and the ‘wedge’ regions as well as the residues that were mutated …
Figure 3—figure supplement 1
Alignment of TPX2 amino acid sequences from different species.
The alignment shows the region around the wedge, ridge and NLS. The figure was prepared using ESPript (Notredame et al., 2000) based on alignment results from T-coffee (Robert and Gouet, 2014). The …
Figure 4
Perturbing critical residues for MT interaction disrupts GMPCPP ‘seed’ and growing MT end binding of TPX2mini.
(A) Schematic of the TPX2mini indicating the three mutated residues (top), and representative TIRF microscopy images (bottom) comparing wild-type mGFP-TPX2mini and the F307E F334E H335E triple …
Figure 5 with 2 supplements
TPX2 binds at inter-dimer interfaces that change during the MT lattice compaction linked to GTP hydrolysis.
(A) Comparison of atomic models between the kinesin-bound GMPCPP-MT and GDP-MT states (both in the absence of TPX2 binding). The two models are aligned on the β1-tubulin subunit. Both α- and β-tubuli…
Figure 5—figure supplement 1
Plot of MT lattice parameters for different functional states.
Dimer twist versus dimer rise for MT cryo-EM reconstructions in different states, displaying values for the most abundant 13- (A) or 14- (B) start helix of the tubulin dimer. Note 14-PF MTs …
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Figure 5—figure supplement 1—source data 1
Lattice parameters for different MT states.
- https://doi.org/10.7554/eLife.30959.014
Figure 5—figure supplement 2
Effect of TPX2 binding on tubulin conformational transitions with nucleotide state.
Comparing cryo-EM structures of MTs in different nucleotide states reveals a pair of tubulin side chains at the inter-dimer interface, α:K326 and β:F214, that adopt different rotamer conformations …
Figure 6 with 1 supplement
Model for the RanGTP-regulated interaction of TPX2 with tubulin assemblies during MT nucleation and MT growth.
Binding of importins by RanGTP releases the sequestration of structural elements in TPX2 that are involved in interaction with tubulin assemblies at polymerization interfaces. TPX2 then functions to …
Figure 6—figure supplement 1
Comparison of the binding sites of TPX2, EB3 and doublecortin (DCX) on MTs.
α-tubulin, β-tubulin, TPX2, EB3 (PDB ID: 3JAK) and DCX (PDB ID: 2XRP) are colored in green, blue, magenta, orange and gold, respectively. Tubulin residues within 4 Å of the MAPs (TPX2, EB3 and DCX) …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.30959.018
