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EBs recognize a nucleotide-dependent structural cap at growing microtubule ends.

Maurer SP, Fourniol FJ, Bohner G, Moores CA, Surrey T - Cell (2012)

Bottom Line: By binding close to the exchangeable GTP-binding site, the CH domain is ideally positioned to sense the microtubule's nucleotide state.The same microtubule-end region is also a stabilizing structural cap protecting the microtubule from depolymerization.This insight supports a common structural link between two important biological phenomena, microtubule dynamic instability and end tracking.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.

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Cryo-EM Imaging and Reconstruction of the Mal3 Microtubule Complex, Related to Figure 1(A) Raw cryo-EM image (left) of a Mal3143-decorated GTPγS microtubule (bottom), grown from quantum dot-labeled GMPCPP microtubule seeds (top), prepared as described previously (Maurer et al., 2011) (scale bar, 50 nm). Only Mal3143-decorated GTPγS microtubules were included in our analysis. The 8 nm layerline characteristic of decorated microtubules is strong in power spectra of Mal3143-GTPγS-MTs (middle, bottom), and not visible for GMPCPP microtubules (middle, top). A Fourier-filtered Mal3143-decorated GTPγS microtubule shows extra densities every other monomer along the protofilaments of the microtubule (right, bottom), which is not seen on the undecorated GMPCPP seed (right, top).(B) Analysis of protofilament number and helix start for GTPγS microtubules grown from GMPCPP seeds in the absence and presence of Mal3143. Mal3143 promotes the assembly of 13 protofilament 3 start microtubules (13_3, 2% of total population without Mal3143, and 68% with Mal3143). A small fraction of 2 and 4 start microtubules are also assembled—these microtubules have no seam, reinforcing the preference of Mal3 for B lattice contacts.(C) Fourier shell correlation (FSC) curve of the asymmetric cryo-EM reconstruction of 13 protofilament Mal3143-decorated GTPγS microtubules (Figure 1), calculated between two independent half-data sets. The FSC is above 0.5 for resolutions up to 15 Å.(D) Plot of the seam orientations (PHI angles) of the 2D cryo-EM images that went into the reconstruction, showing an isotropic distribution. The orientation of the seam in the reference 3D model is indicated by an arrowhead.(E) Raw cryo-EM image (left) of a Mal3143-decorated GDP microtubule. Scale bar, 50 nm. The power spectrum of the Mal3143-GDP microtubule displays a strong 8 nm layerline characteristic of decorated microtubules (middle), and corresponding to extra densities every other monomer along the protofilaments of the microtubule seen in the Fourier-filtered image (right).(F) Asymmetric reconstruction of Mal3143-GDP microtubules. Mal3143 (green) binds the cleft between protofilaments (sky blue) making B lattice contacts and does not bind the A lattice seam. Weaker extra densities between protofilaments (light green) could be due to additional Mal3 CH domains weakly interacting with the interprotofilament valley and/or with other bound CH domains (green). The heterogeneity in binding is consistent with the more than 10-fold lower affinity of Mal3 for the GDP lattice compared with the growing end region (Maurer et al., 2011). Averaging B lattice contacts resulted in a relatively noisy map with lower resolution compared with the symmetrised Mal3143-GTPγS map (not shown).(G) FSC curve of the asymmetric cryo-EM reconstruction of 13 protofilament Mal3143-decorated GDP microtubules, calculated between two independent half-data sets. The FSC is above 0.5 for resolutions up to 15 Å.(H) Plot of the seam orientations (PHI angles) of the 2D cryo-EM images that went into the reconstruction, showing an isotropic distribution. The orientation of the seam in the reference 3D model is indicated by an arrowhead.
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figs1: Cryo-EM Imaging and Reconstruction of the Mal3 Microtubule Complex, Related to Figure 1(A) Raw cryo-EM image (left) of a Mal3143-decorated GTPγS microtubule (bottom), grown from quantum dot-labeled GMPCPP microtubule seeds (top), prepared as described previously (Maurer et al., 2011) (scale bar, 50 nm). Only Mal3143-decorated GTPγS microtubules were included in our analysis. The 8 nm layerline characteristic of decorated microtubules is strong in power spectra of Mal3143-GTPγS-MTs (middle, bottom), and not visible for GMPCPP microtubules (middle, top). A Fourier-filtered Mal3143-decorated GTPγS microtubule shows extra densities every other monomer along the protofilaments of the microtubule (right, bottom), which is not seen on the undecorated GMPCPP seed (right, top).(B) Analysis of protofilament number and helix start for GTPγS microtubules grown from GMPCPP seeds in the absence and presence of Mal3143. Mal3143 promotes the assembly of 13 protofilament 3 start microtubules (13_3, 2% of total population without Mal3143, and 68% with Mal3143). A small fraction of 2 and 4 start microtubules are also assembled—these microtubules have no seam, reinforcing the preference of Mal3 for B lattice contacts.(C) Fourier shell correlation (FSC) curve of the asymmetric cryo-EM reconstruction of 13 protofilament Mal3143-decorated GTPγS microtubules (Figure 1), calculated between two independent half-data sets. The FSC is above 0.5 for resolutions up to 15 Å.(D) Plot of the seam orientations (PHI angles) of the 2D cryo-EM images that went into the reconstruction, showing an isotropic distribution. The orientation of the seam in the reference 3D model is indicated by an arrowhead.(E) Raw cryo-EM image (left) of a Mal3143-decorated GDP microtubule. Scale bar, 50 nm. The power spectrum of the Mal3143-GDP microtubule displays a strong 8 nm layerline characteristic of decorated microtubules (middle), and corresponding to extra densities every other monomer along the protofilaments of the microtubule seen in the Fourier-filtered image (right).(F) Asymmetric reconstruction of Mal3143-GDP microtubules. Mal3143 (green) binds the cleft between protofilaments (sky blue) making B lattice contacts and does not bind the A lattice seam. Weaker extra densities between protofilaments (light green) could be due to additional Mal3 CH domains weakly interacting with the interprotofilament valley and/or with other bound CH domains (green). The heterogeneity in binding is consistent with the more than 10-fold lower affinity of Mal3 for the GDP lattice compared with the growing end region (Maurer et al., 2011). Averaging B lattice contacts resulted in a relatively noisy map with lower resolution compared with the symmetrised Mal3143-GTPγS map (not shown).(G) FSC curve of the asymmetric cryo-EM reconstruction of 13 protofilament Mal3143-decorated GDP microtubules, calculated between two independent half-data sets. The FSC is above 0.5 for resolutions up to 15 Å.(H) Plot of the seam orientations (PHI angles) of the 2D cryo-EM images that went into the reconstruction, showing an isotropic distribution. The orientation of the seam in the reference 3D model is indicated by an arrowhead.

Mentions: We used cryo-EM to determine how the CH domain of Mal3, the fission yeast EB, binds to GTPγS microtubules, which were previously shown to be static mimics of growing microtubule ends (Maurer et al., 2011). We found that Mal3 favors the assembly of GTPγS-tubulin into microtubules with mostly 13 protofilaments (Figure S1B available online), consistent with a previous study of EB1 in presence of GTP-tubulin (Vitre et al., 2008). Three-dimensional (3D) reconstruction from segments of 13 protofilament GTPγS microtubules decorated with an N-terminal fragment of Mal3 containing its CH domain (Mal3143) generated an asymmetric structure with 15 Å resolution (Figures 1, S1C, and S1D). Mal3143 binds regularly between neighboring protofilaments (B lattice, 12 such contacts per 13 protofilament microtubule) except along the seam (the single A lattice contact between protofilaments) of GTPγS microtubules (Figure 1B). This selectivity suggests a highly specific binding site. The longitudinal distance between bound CH domains is 8 nm (corresponding to one tubulin heterodimer), resulting in a stoichiometry of 12 CH domains per 13 tubulin dimers. This is in good agreement with the recently reported approximately stoichiometric binding measured by fluorescence microscopy (Maurer et al., 2011). A similar pattern of binding—including absence of interaction at the seam—was seen for Mal3143 on GDP microtubules (Figures S1E–S1H). This is in contrast to previous reports (des Georges et al., 2008; Sandblad et al., 2006) (see Discussion). Our data suggest that the affinity difference of Mal3 binding to growing microtubule ends compared to older parts of the microtubule lattice (Maurer et al., 2011) is not a result of different binding positions nor of dramatic A-to-B lattice transitions within the microtubule but rather a consequence of conformational rearrangements within its binding site.


EBs recognize a nucleotide-dependent structural cap at growing microtubule ends.

Maurer SP, Fourniol FJ, Bohner G, Moores CA, Surrey T - Cell (2012)

Cryo-EM Imaging and Reconstruction of the Mal3 Microtubule Complex, Related to Figure 1(A) Raw cryo-EM image (left) of a Mal3143-decorated GTPγS microtubule (bottom), grown from quantum dot-labeled GMPCPP microtubule seeds (top), prepared as described previously (Maurer et al., 2011) (scale bar, 50 nm). Only Mal3143-decorated GTPγS microtubules were included in our analysis. The 8 nm layerline characteristic of decorated microtubules is strong in power spectra of Mal3143-GTPγS-MTs (middle, bottom), and not visible for GMPCPP microtubules (middle, top). A Fourier-filtered Mal3143-decorated GTPγS microtubule shows extra densities every other monomer along the protofilaments of the microtubule (right, bottom), which is not seen on the undecorated GMPCPP seed (right, top).(B) Analysis of protofilament number and helix start for GTPγS microtubules grown from GMPCPP seeds in the absence and presence of Mal3143. Mal3143 promotes the assembly of 13 protofilament 3 start microtubules (13_3, 2% of total population without Mal3143, and 68% with Mal3143). A small fraction of 2 and 4 start microtubules are also assembled—these microtubules have no seam, reinforcing the preference of Mal3 for B lattice contacts.(C) Fourier shell correlation (FSC) curve of the asymmetric cryo-EM reconstruction of 13 protofilament Mal3143-decorated GTPγS microtubules (Figure 1), calculated between two independent half-data sets. The FSC is above 0.5 for resolutions up to 15 Å.(D) Plot of the seam orientations (PHI angles) of the 2D cryo-EM images that went into the reconstruction, showing an isotropic distribution. The orientation of the seam in the reference 3D model is indicated by an arrowhead.(E) Raw cryo-EM image (left) of a Mal3143-decorated GDP microtubule. Scale bar, 50 nm. The power spectrum of the Mal3143-GDP microtubule displays a strong 8 nm layerline characteristic of decorated microtubules (middle), and corresponding to extra densities every other monomer along the protofilaments of the microtubule seen in the Fourier-filtered image (right).(F) Asymmetric reconstruction of Mal3143-GDP microtubules. Mal3143 (green) binds the cleft between protofilaments (sky blue) making B lattice contacts and does not bind the A lattice seam. Weaker extra densities between protofilaments (light green) could be due to additional Mal3 CH domains weakly interacting with the interprotofilament valley and/or with other bound CH domains (green). The heterogeneity in binding is consistent with the more than 10-fold lower affinity of Mal3 for the GDP lattice compared with the growing end region (Maurer et al., 2011). Averaging B lattice contacts resulted in a relatively noisy map with lower resolution compared with the symmetrised Mal3143-GTPγS map (not shown).(G) FSC curve of the asymmetric cryo-EM reconstruction of 13 protofilament Mal3143-decorated GDP microtubules, calculated between two independent half-data sets. The FSC is above 0.5 for resolutions up to 15 Å.(H) Plot of the seam orientations (PHI angles) of the 2D cryo-EM images that went into the reconstruction, showing an isotropic distribution. The orientation of the seam in the reference 3D model is indicated by an arrowhead.
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figs1: Cryo-EM Imaging and Reconstruction of the Mal3 Microtubule Complex, Related to Figure 1(A) Raw cryo-EM image (left) of a Mal3143-decorated GTPγS microtubule (bottom), grown from quantum dot-labeled GMPCPP microtubule seeds (top), prepared as described previously (Maurer et al., 2011) (scale bar, 50 nm). Only Mal3143-decorated GTPγS microtubules were included in our analysis. The 8 nm layerline characteristic of decorated microtubules is strong in power spectra of Mal3143-GTPγS-MTs (middle, bottom), and not visible for GMPCPP microtubules (middle, top). A Fourier-filtered Mal3143-decorated GTPγS microtubule shows extra densities every other monomer along the protofilaments of the microtubule (right, bottom), which is not seen on the undecorated GMPCPP seed (right, top).(B) Analysis of protofilament number and helix start for GTPγS microtubules grown from GMPCPP seeds in the absence and presence of Mal3143. Mal3143 promotes the assembly of 13 protofilament 3 start microtubules (13_3, 2% of total population without Mal3143, and 68% with Mal3143). A small fraction of 2 and 4 start microtubules are also assembled—these microtubules have no seam, reinforcing the preference of Mal3 for B lattice contacts.(C) Fourier shell correlation (FSC) curve of the asymmetric cryo-EM reconstruction of 13 protofilament Mal3143-decorated GTPγS microtubules (Figure 1), calculated between two independent half-data sets. The FSC is above 0.5 for resolutions up to 15 Å.(D) Plot of the seam orientations (PHI angles) of the 2D cryo-EM images that went into the reconstruction, showing an isotropic distribution. The orientation of the seam in the reference 3D model is indicated by an arrowhead.(E) Raw cryo-EM image (left) of a Mal3143-decorated GDP microtubule. Scale bar, 50 nm. The power spectrum of the Mal3143-GDP microtubule displays a strong 8 nm layerline characteristic of decorated microtubules (middle), and corresponding to extra densities every other monomer along the protofilaments of the microtubule seen in the Fourier-filtered image (right).(F) Asymmetric reconstruction of Mal3143-GDP microtubules. Mal3143 (green) binds the cleft between protofilaments (sky blue) making B lattice contacts and does not bind the A lattice seam. Weaker extra densities between protofilaments (light green) could be due to additional Mal3 CH domains weakly interacting with the interprotofilament valley and/or with other bound CH domains (green). The heterogeneity in binding is consistent with the more than 10-fold lower affinity of Mal3 for the GDP lattice compared with the growing end region (Maurer et al., 2011). Averaging B lattice contacts resulted in a relatively noisy map with lower resolution compared with the symmetrised Mal3143-GTPγS map (not shown).(G) FSC curve of the asymmetric cryo-EM reconstruction of 13 protofilament Mal3143-decorated GDP microtubules, calculated between two independent half-data sets. The FSC is above 0.5 for resolutions up to 15 Å.(H) Plot of the seam orientations (PHI angles) of the 2D cryo-EM images that went into the reconstruction, showing an isotropic distribution. The orientation of the seam in the reference 3D model is indicated by an arrowhead.
Mentions: We used cryo-EM to determine how the CH domain of Mal3, the fission yeast EB, binds to GTPγS microtubules, which were previously shown to be static mimics of growing microtubule ends (Maurer et al., 2011). We found that Mal3 favors the assembly of GTPγS-tubulin into microtubules with mostly 13 protofilaments (Figure S1B available online), consistent with a previous study of EB1 in presence of GTP-tubulin (Vitre et al., 2008). Three-dimensional (3D) reconstruction from segments of 13 protofilament GTPγS microtubules decorated with an N-terminal fragment of Mal3 containing its CH domain (Mal3143) generated an asymmetric structure with 15 Å resolution (Figures 1, S1C, and S1D). Mal3143 binds regularly between neighboring protofilaments (B lattice, 12 such contacts per 13 protofilament microtubule) except along the seam (the single A lattice contact between protofilaments) of GTPγS microtubules (Figure 1B). This selectivity suggests a highly specific binding site. The longitudinal distance between bound CH domains is 8 nm (corresponding to one tubulin heterodimer), resulting in a stoichiometry of 12 CH domains per 13 tubulin dimers. This is in good agreement with the recently reported approximately stoichiometric binding measured by fluorescence microscopy (Maurer et al., 2011). A similar pattern of binding—including absence of interaction at the seam—was seen for Mal3143 on GDP microtubules (Figures S1E–S1H). This is in contrast to previous reports (des Georges et al., 2008; Sandblad et al., 2006) (see Discussion). Our data suggest that the affinity difference of Mal3 binding to growing microtubule ends compared to older parts of the microtubule lattice (Maurer et al., 2011) is not a result of different binding positions nor of dramatic A-to-B lattice transitions within the microtubule but rather a consequence of conformational rearrangements within its binding site.

Bottom Line: By binding close to the exchangeable GTP-binding site, the CH domain is ideally positioned to sense the microtubule's nucleotide state.The same microtubule-end region is also a stabilizing structural cap protecting the microtubule from depolymerization.This insight supports a common structural link between two important biological phenomena, microtubule dynamic instability and end tracking.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.

Show MeSH
Related in: MedlinePlus