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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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Validation of EB-Tubulin Contact Sites using Reconstituted Dynamic Microtubule-End Tracking(A) Localization of single mutated residues on the Mal3 microtubule-binding surface: residues chosen for mutations (red) in the context of all Mal3 residues within 5 Å of tubulin (rendered as a blue mesh).(B) Extract of a sequence alignment of different EBs: Mal3 residues contacting the microtubule (blue boxes) and residues that were mutated in this study (red asterisks).(C) TIRF microscopy images of Alexa 568-labeled microtubules (red) grown in the presence of GTP and wild-type (WT) or mutant Mal3-GFP (green) as indicated.(D) TIRF microscopy images illustrating the differences between the Mal3-GFP fluorescence signal (green) for WT and the different mutants on single microtubules. Imaging conditions and display settings are identical for all six experiments. The regions used to measure Mal3-GFP intensity at the microtubule end and on the lattice are indicated by red lines.(E and F) Quantification of Mal3-GFP intensities at the microtubule ends and on the lattice. Error bars represent standard error of the mean (SEM). (E) Mean intensities relative to the mean WT intensity at growing microtubule ends are shown. Concentrations were 30 nM Mal3-GFP, 22 μM tubulin, and 1 mM GTP. (F) Same as (E) with 300 nM Mal3-GFP.See also Figure S4 and Movie S2.
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fig5: Validation of EB-Tubulin Contact Sites using Reconstituted Dynamic Microtubule-End Tracking(A) Localization of single mutated residues on the Mal3 microtubule-binding surface: residues chosen for mutations (red) in the context of all Mal3 residues within 5 Å of tubulin (rendered as a blue mesh).(B) Extract of a sequence alignment of different EBs: Mal3 residues contacting the microtubule (blue boxes) and residues that were mutated in this study (red asterisks).(C) TIRF microscopy images of Alexa 568-labeled microtubules (red) grown in the presence of GTP and wild-type (WT) or mutant Mal3-GFP (green) as indicated.(D) TIRF microscopy images illustrating the differences between the Mal3-GFP fluorescence signal (green) for WT and the different mutants on single microtubules. Imaging conditions and display settings are identical for all six experiments. The regions used to measure Mal3-GFP intensity at the microtubule end and on the lattice are indicated by red lines.(E and F) Quantification of Mal3-GFP intensities at the microtubule ends and on the lattice. Error bars represent standard error of the mean (SEM). (E) Mean intensities relative to the mean WT intensity at growing microtubule ends are shown. Concentrations were 30 nM Mal3-GFP, 22 μM tubulin, and 1 mM GTP. (F) Same as (E) with 300 nM Mal3-GFP.See also Figure S4 and Movie S2.

Mentions: In order to test whether our model derived from monomeric Mal3 on GTPγS microtubules reflects the behavior of full-length EB end tracking on dynamic microtubules, we selected conserved Mal3 residues from the tubulin contact sites (Figures 5A and 5B), produced the corresponding single-point mutants in full-length dimeric Mal3-GFP (Figure S4), and tested their ability to track the ends of microtubules grown with GTP in vitro. Using total internal reflection fluorescence (TIRF) microscopy, we found that most selected Mal3 mutants either abolished or severely weakened end tracking (Figure 5; Movie S2), validating our model. Inverting charges at either of the contacts with α-tubulin (Mal3K63D-GFP and Mal3K76D-GFP) resulted in weaker binding of Mal3 (Figures 5D–5F), whereas the Mal3Y56A-GFP mutant (β4-tubulin contact) also strongly reduced Mal3 binding (Figures 5D–5F), indicating that the exact charge and geometry of these interfaces are important for interaction of Mal3 with the microtubule. However, when we investigated the importance of the β3-tubulin H3 contact, we found that the Mal3Q89E-GFP mutant had strongly impaired microtubule-end tracking, whereas Mal3Q89A-GFP bound with higher affinity to the entire microtubule than the wild-type (WT) (Figures 5C–5F; Movie S2). A recent in vivo study reported a similar enhancement of lattice binding and loss of end tracking for a Mal3 mutation at the same position (Mal3Q89R; Iimori et al., 2012). This finding together with our in vitro mutational analysis of the EB-H3 helix interface underlines the importance of this contact for EB microtubule-end tracking.


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

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

Validation of EB-Tubulin Contact Sites using Reconstituted Dynamic Microtubule-End Tracking(A) Localization of single mutated residues on the Mal3 microtubule-binding surface: residues chosen for mutations (red) in the context of all Mal3 residues within 5 Å of tubulin (rendered as a blue mesh).(B) Extract of a sequence alignment of different EBs: Mal3 residues contacting the microtubule (blue boxes) and residues that were mutated in this study (red asterisks).(C) TIRF microscopy images of Alexa 568-labeled microtubules (red) grown in the presence of GTP and wild-type (WT) or mutant Mal3-GFP (green) as indicated.(D) TIRF microscopy images illustrating the differences between the Mal3-GFP fluorescence signal (green) for WT and the different mutants on single microtubules. Imaging conditions and display settings are identical for all six experiments. The regions used to measure Mal3-GFP intensity at the microtubule end and on the lattice are indicated by red lines.(E and F) Quantification of Mal3-GFP intensities at the microtubule ends and on the lattice. Error bars represent standard error of the mean (SEM). (E) Mean intensities relative to the mean WT intensity at growing microtubule ends are shown. Concentrations were 30 nM Mal3-GFP, 22 μM tubulin, and 1 mM GTP. (F) Same as (E) with 300 nM Mal3-GFP.See also Figure S4 and Movie S2.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3368265&req=5

fig5: Validation of EB-Tubulin Contact Sites using Reconstituted Dynamic Microtubule-End Tracking(A) Localization of single mutated residues on the Mal3 microtubule-binding surface: residues chosen for mutations (red) in the context of all Mal3 residues within 5 Å of tubulin (rendered as a blue mesh).(B) Extract of a sequence alignment of different EBs: Mal3 residues contacting the microtubule (blue boxes) and residues that were mutated in this study (red asterisks).(C) TIRF microscopy images of Alexa 568-labeled microtubules (red) grown in the presence of GTP and wild-type (WT) or mutant Mal3-GFP (green) as indicated.(D) TIRF microscopy images illustrating the differences between the Mal3-GFP fluorescence signal (green) for WT and the different mutants on single microtubules. Imaging conditions and display settings are identical for all six experiments. The regions used to measure Mal3-GFP intensity at the microtubule end and on the lattice are indicated by red lines.(E and F) Quantification of Mal3-GFP intensities at the microtubule ends and on the lattice. Error bars represent standard error of the mean (SEM). (E) Mean intensities relative to the mean WT intensity at growing microtubule ends are shown. Concentrations were 30 nM Mal3-GFP, 22 μM tubulin, and 1 mM GTP. (F) Same as (E) with 300 nM Mal3-GFP.See also Figure S4 and Movie S2.
Mentions: In order to test whether our model derived from monomeric Mal3 on GTPγS microtubules reflects the behavior of full-length EB end tracking on dynamic microtubules, we selected conserved Mal3 residues from the tubulin contact sites (Figures 5A and 5B), produced the corresponding single-point mutants in full-length dimeric Mal3-GFP (Figure S4), and tested their ability to track the ends of microtubules grown with GTP in vitro. Using total internal reflection fluorescence (TIRF) microscopy, we found that most selected Mal3 mutants either abolished or severely weakened end tracking (Figure 5; Movie S2), validating our model. Inverting charges at either of the contacts with α-tubulin (Mal3K63D-GFP and Mal3K76D-GFP) resulted in weaker binding of Mal3 (Figures 5D–5F), whereas the Mal3Y56A-GFP mutant (β4-tubulin contact) also strongly reduced Mal3 binding (Figures 5D–5F), indicating that the exact charge and geometry of these interfaces are important for interaction of Mal3 with the microtubule. However, when we investigated the importance of the β3-tubulin H3 contact, we found that the Mal3Q89E-GFP mutant had strongly impaired microtubule-end tracking, whereas Mal3Q89A-GFP bound with higher affinity to the entire microtubule than the wild-type (WT) (Figures 5C–5F; Movie S2). A recent in vivo study reported a similar enhancement of lattice binding and loss of end tracking for a Mal3 mutation at the same position (Mal3Q89R; Iimori et al., 2012). This finding together with our in vitro mutational analysis of the EB-H3 helix interface underlines the importance of this contact for EB microtubule-end tracking.

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