|
FIGURE 1:. TOG domains with unique and universally conserved determinants comprise the XMAP215-family TOG array. (A) Domain architecture of XMAP215 family members ch-TOG, Msps, and Stu2 (CC, coiled coil; CTD, C-terminal domain). (B) Sequence alignment of TOG domains from Msps, ch-TOG, and Stu2. Class-specific TOG conservation has been mapped based on a multispecies alignment: residues with 80% identity (highlighted green) and 80% similarity (highlighted yellow) are indicated for each individual TOG class (TOG1, TOG2,â¦, TOG5). Residue numbers are for Msps TOG4. Msps TOG4 solvent-accessible surface area (SASA) is plotted. Stu2 TOG1 residues that interact with α- and β-tubulin are boxed in blue and purple, respectively. (C) Identity matrix, comparing sequence identity between Msps and ch-TOG TOG domains.
|
|
FIGURE 2:. Structure of Msps TOG4. (A) Cartoon representation of the Msps TOG4 domain. TOG4 consists of six HRs, AâF. (B) Msps TOG4 in surface representation (oriented as in A, bottom) mapping TOG4-specific conservation (top) as in Figure 1B and electrostatics (bottom). (CâE) The 2Fo â Fc electron density map (2Ï) illustrating the HR A-loop W874 residue and the R877-D911 salt bridge (C), the HR C-loop D952âK954 interaction (D), and the buried HR C-D interaction network (E).
|
|
FIGURE 3:. Structure of ch-TOG TOG4. (A) Cartoon representation of ch-TOG TOG4. (B, C) The 2Fo â Fc electron density map (2Ï) of the ch-TOG TOG4 structure showing conserved determinants in the HR A (B) and HR E (C) loops. (D) Pairwise alignment of ch-TOG TOG4 (color) and Msps TOG4 (gray) showing structural conservation with a 1.4-Ã
rmsd over 226 Cα atoms.
|
|
FIGURE 4:. Msps TOG4 is structurally distinct from Msps TOG2. (A) Pairwise alignment of Msps TOG4 (color) and Msps TOG2 (gray) across the full domain yields a 4.5-Ã
rmsd. (B, C) Pairwise alignment of Msps TOG4 and Msps TOG2 across the first HR triad (B; 2.0 Ã
rmsd) and the second HR triad (C; 2.8 Ã
rmsd). (D) Superpositioning of Msps TOG4 and Msps TOG2 based on the first HR triad alignment shown in B, highlighting the 45° difference in the orientation of each domain's second HR triad and the 19-Ã
differential placement of HR F. (E) Comparative views of Msps TOG4 and Msps TOG2 structures oriented as in D; models shown below. (F) The Msps TOG4 α4Bâ²-α4Câ² interface contains bulky, conserved residues, whereas the Msps TOG2 α2Bâ²-α2Câ² interface has conserved residues with no or small side chains. (G) Msps TOG4 contains the α4D1 helical insert that forms an extensive interaction network at the base of HR C and HR D, absent in Msps TOG2.
|
|
FIGURE 5:. TOG4 is predicted to form TOG4-specific contacts with tubulin. (A) Msps TOG4 (color) and Stu2 TOG1 (gray) aligned pairwise over the first HR triad (HR AâC) with a 2.5-Ã
rmsd over HR AâC, highlighting the differential arrangement of each domain's second HR triad and the 17-Ã
shift in HR F (red arrow). Lower images show identical orientations in surface representation. (B) Stu2 TOG1 bound to curved αβ-tubulin (left; Ayaz et al., 2012) vs. straight tubulin (modeled at right). (C) Msps TOG4 was docked onto the Stu2 TOG1âαβ-tubulin structure and model presented in B by aligning the first HR triads as in A. (D) Msps TOG4 conserved HR A residues W874 and K875 can interact with β-tubulin residues T107 and S400, respectively, as observed in the Stu2 TOG1âαβ-tubulin structure. (E) Msps TOG4 conserved HR E residue R1038 is within 5 Ã
of α-tubulin E415 and is likely to reposition and form a salt bridge as observed with Stu2 TOG1 R200. (F) Superposition of the Stu2 TOG1âαβ-tubulin structure and the Msps TOG4âαβ-tubulin model reveals major differences in HR F's interaction with α-tubulin. (G, H) Zoom view of the region boxed in F (G) and shown after a 90° rotation about the x-axis (H).
|
|
FIGURE 6:. Paired Msps TOG domains show differential tubulin-binding and MT polymerization activities in vitro. (A) Msps TOG1-2 (40 μM) binds and shifts tubulin (20 μM) to an earlier elution peak over gel filtration. (B) Msps TOG3-4 (40 μM) fails to shift tubulin (20 μM) over gel filtration. (C) Light scattering curves of tubulin (15 μM) polymerized at 37°C alone or in the presence of Msps TOG1-2 or TOG3-4 constructs (1 μM). TOG constructs alone showed no scattering activity. (D) Images of in vitro MT polymerization in the absence (left) and presence (right) of 1 nM Msps TOG34. Tubulin (20 μM) was doped with 10% rhodamine-labeled tubulin. Polymerization reactions were terminated after 180 s by diluting the samples into buffer containing 20 μM Taxol at 25°C. Scale bar, 50 μm. Gel filtration elution profiles, light scattering curves, and tubulin microscopy images shown are from individual experiments and are representative of experiments done in triplicate.
|
|
FIGURE 7:. TOG3 and TOG4 contribute to Msps MT lattice localization in cell culture. (A) Msps tRFP constructs analyzed; vertical line represents an HR A-loop W â E mutation. (B) Distribution of Msps 498â1079 tRFP lattice localization in Drosophila S2 cells cotransfected with α-tubulin GFP. Msps localization was binned into three categories: strong MT lattice localization (black), weak MT lattice localization (dark gray), and cytoplasmic localization (light gray). Error bars represent SD of the mean. Numbers at right list the total number of cells examined and the number of independent experiments performed. Representative images from each category are displayed (CâF). Mutating individual (DâE) or paired (F) conserved HR A-loop tryptophans resulted in an increase in cytoplasmic localization. Scale bars, 10 μm (images), 5 μm (insets). Statistical significance was determined using an unpaired t test to calculate two-tailed p values for the cytoplasmic bin.
|
|
FIGURE 8:. Msps requires a functional TOG array to rescue MT polymerization in cells. (A) Msps GFP constructs analyzed; vertical lines indicate an HR A-loop W â E mutation. (B) Western blot showing RNAi-mediated Msps depletion in Drosophila S2 cells (SK dsRNA control; antibodies: anti-Msps, anti-actin loading control). (C) Distribution of EB1 comet velocities from Msps rescue experiments. (D) Projection image (30 s) of an S2 cell expressing EB1-tRFP treated with control dsRNA (top) and a kymograph of a representative EB1 comet from this cell (bottom). (EâG) EB1 comet velocity is reduced when Msps is depleted (E) but can be largely rescued with Msps TOG1â4 (F) or Msps TOG1-5 (G). (H) Distribution of EB1 comet velocities in Msps dsRNA-treated cells transfected with Msps TOG1â4 mutant rescue constructs. Mutating the conserved HR A-loop tryptophan individually (IâL), in pairs (MâN), or across all four TOG domains (O) fails to rescue EB1 comet velocities. Scale bar, 10 μm (projection images), 30 s and 1 μm (kymographs). Box-and-whisker plots in C and H confer the following information: whiskers, 10th and 90th percentiles; boxes, 25th and 75th percentiles; line, median; cross, mean. Numbers in parentheses are the number of cells analyzed and the total number of EB1 comets tracked. Two-tailed p values were calculated using an unpaired t test.
|
