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The size of the EB cap determines instantaneous microtubule stability.

Duellberg C, Cade NI, Holmes D, Surrey T - Elife (2016)

Bottom Line: Using a microfluidics-assisted multi-colour TIRF microscopy assay with close-to-nm and sub-second precision, we measured the sizes of the stabilizing cap of individual microtubules.Nevertheless, the trigger of instability lies in a short region at the end of the cap, as a quantitative model of cap stability demonstrates.Our study establishes the spatial and kinetic characteristics of the protective cap and provides an insight into the molecular mechanism by which its loss leads to the switch from microtubule growth to shrinkage.

View Article: PubMed Central - PubMed

Affiliation: Lincoln's Inn Fields Laboratory, The Francis Crick Institute, London, United Kingdom.

ABSTRACT
The function of microtubules relies on their ability to switch between phases of growth and shrinkage. A nucleotide-dependent stabilising cap at microtubule ends is thought to be lost before this switch can occur; however, the nature and size of this protective cap are unknown. Using a microfluidics-assisted multi-colour TIRF microscopy assay with close-to-nm and sub-second precision, we measured the sizes of the stabilizing cap of individual microtubules. We find that the protective caps are formed by the extended binding regions of EB proteins. Cap lengths vary considerably and longer caps are more stable. Nevertheless, the trigger of instability lies in a short region at the end of the cap, as a quantitative model of cap stability demonstrates. Our study establishes the spatial and kinetic characteristics of the protective cap and provides an insight into the molecular mechanism by which its loss leads to the switch from microtubule growth to shrinkage.

No MeSH data available.


Related in: MedlinePlus

Growth and shrinkage speeds and delay times of the data sets with varied tubulin concentrations.(A) Illustration of speed-sorting: Histogram showing all growth speeds from the 4 data sets with different tubulin concentrations presented in Figure 6A. The data was then sorted into 7 groups each containing 31 microtubules, according to their measured speed before washout, irrespective of the original tubulin concentration. (B) Delay times as a function of tubulin concentration: box plots depicting the delay times of microtubules grown at different tubulin concentrations. Same data set as in Figure 6A. (C) Scatter plot of the slow shrinkage speed vs after washout against the growth speed vg before washout. A mild correlation was observed; the explicit relationship between vs and vg was necessary for the model fits to the experimental data in Figure 6A and Figure 6—figure supplement 2. This observation potentially supports earlier proposals that predict higher koff rates for faster and more tapered microtubule ends (Coombes et al., 2013; Gardner et al., 2011a).DOI:http://dx.doi.org/10.7554/eLife.13470.016
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fig6s1: Growth and shrinkage speeds and delay times of the data sets with varied tubulin concentrations.(A) Illustration of speed-sorting: Histogram showing all growth speeds from the 4 data sets with different tubulin concentrations presented in Figure 6A. The data was then sorted into 7 groups each containing 31 microtubules, according to their measured speed before washout, irrespective of the original tubulin concentration. (B) Delay times as a function of tubulin concentration: box plots depicting the delay times of microtubules grown at different tubulin concentrations. Same data set as in Figure 6A. (C) Scatter plot of the slow shrinkage speed vs after washout against the growth speed vg before washout. A mild correlation was observed; the explicit relationship between vs and vg was necessary for the model fits to the experimental data in Figure 6A and Figure 6—figure supplement 2. This observation potentially supports earlier proposals that predict higher koff rates for faster and more tapered microtubule ends (Coombes et al., 2013; Gardner et al., 2011a).DOI:http://dx.doi.org/10.7554/eLife.13470.016

Mentions: We observed that a large part of the EB binding region is lost at catastrophe, however part of it is still present when catastrophe occurs. This suggests that a threshold may have to be reached to induce catastrophe, raising the question of what exactly constitutes this threshold. Two extreme possibilities can be envisaged: A minimal total number of cap sites anywhere in the entire cap might be required for stability. Alternatively a minimal density of cap sites only at its very end where the cap site density is highest might be needed for stability of the cap. In other words, either the entire cap or only its highest density region could be critical for stability. These two scenarios predict different dependencies of the delay times on the growth speed and hence cap length (Materials and methods). To explore the momentary microtubule stabilities over a larger range of cap sizes, we performed tubulin washout experiments at a range of different tubulin concentrations from 10 μM to 35 μM, and at an increased magnesium ion concentration to further increase the velocity range (O'Brien et al., 1990). In total 210 microtubules were analysed. As the growth speed distributions at different tubulin concentrations overlapped strongly (Figure 6A, top), we speed-sorted the data when calculating averages as a function of speed (Figure 6—figure supplement 1A) (Maurer et al., 2014). Average delay times extracted for seven speed groups from over 200 individual microtubule tracks displayed the expected positive correlation between delay times and growth speeds, however showing a weaker dependence especially for the higher speed range (Figure 6A bottom). This correlation was masked when displaying the delay times simply as a function of tubulin concentration (Figure 6—figure supplement 1B), again emphasizing the importance of correlating delay times with momentary speeds in the presence of growth fluctuations.10.7554/eLife.13470.015Figure 6.An end density threshold explains the dependence of the mean delay times on growth speed.


The size of the EB cap determines instantaneous microtubule stability.

Duellberg C, Cade NI, Holmes D, Surrey T - Elife (2016)

Growth and shrinkage speeds and delay times of the data sets with varied tubulin concentrations.(A) Illustration of speed-sorting: Histogram showing all growth speeds from the 4 data sets with different tubulin concentrations presented in Figure 6A. The data was then sorted into 7 groups each containing 31 microtubules, according to their measured speed before washout, irrespective of the original tubulin concentration. (B) Delay times as a function of tubulin concentration: box plots depicting the delay times of microtubules grown at different tubulin concentrations. Same data set as in Figure 6A. (C) Scatter plot of the slow shrinkage speed vs after washout against the growth speed vg before washout. A mild correlation was observed; the explicit relationship between vs and vg was necessary for the model fits to the experimental data in Figure 6A and Figure 6—figure supplement 2. This observation potentially supports earlier proposals that predict higher koff rates for faster and more tapered microtubule ends (Coombes et al., 2013; Gardner et al., 2011a).DOI:http://dx.doi.org/10.7554/eLife.13470.016
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Related In: Results  -  Collection

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fig6s1: Growth and shrinkage speeds and delay times of the data sets with varied tubulin concentrations.(A) Illustration of speed-sorting: Histogram showing all growth speeds from the 4 data sets with different tubulin concentrations presented in Figure 6A. The data was then sorted into 7 groups each containing 31 microtubules, according to their measured speed before washout, irrespective of the original tubulin concentration. (B) Delay times as a function of tubulin concentration: box plots depicting the delay times of microtubules grown at different tubulin concentrations. Same data set as in Figure 6A. (C) Scatter plot of the slow shrinkage speed vs after washout against the growth speed vg before washout. A mild correlation was observed; the explicit relationship between vs and vg was necessary for the model fits to the experimental data in Figure 6A and Figure 6—figure supplement 2. This observation potentially supports earlier proposals that predict higher koff rates for faster and more tapered microtubule ends (Coombes et al., 2013; Gardner et al., 2011a).DOI:http://dx.doi.org/10.7554/eLife.13470.016
Mentions: We observed that a large part of the EB binding region is lost at catastrophe, however part of it is still present when catastrophe occurs. This suggests that a threshold may have to be reached to induce catastrophe, raising the question of what exactly constitutes this threshold. Two extreme possibilities can be envisaged: A minimal total number of cap sites anywhere in the entire cap might be required for stability. Alternatively a minimal density of cap sites only at its very end where the cap site density is highest might be needed for stability of the cap. In other words, either the entire cap or only its highest density region could be critical for stability. These two scenarios predict different dependencies of the delay times on the growth speed and hence cap length (Materials and methods). To explore the momentary microtubule stabilities over a larger range of cap sizes, we performed tubulin washout experiments at a range of different tubulin concentrations from 10 μM to 35 μM, and at an increased magnesium ion concentration to further increase the velocity range (O'Brien et al., 1990). In total 210 microtubules were analysed. As the growth speed distributions at different tubulin concentrations overlapped strongly (Figure 6A, top), we speed-sorted the data when calculating averages as a function of speed (Figure 6—figure supplement 1A) (Maurer et al., 2014). Average delay times extracted for seven speed groups from over 200 individual microtubule tracks displayed the expected positive correlation between delay times and growth speeds, however showing a weaker dependence especially for the higher speed range (Figure 6A bottom). This correlation was masked when displaying the delay times simply as a function of tubulin concentration (Figure 6—figure supplement 1B), again emphasizing the importance of correlating delay times with momentary speeds in the presence of growth fluctuations.10.7554/eLife.13470.015Figure 6.An end density threshold explains the dependence of the mean delay times on growth speed.

Bottom Line: Using a microfluidics-assisted multi-colour TIRF microscopy assay with close-to-nm and sub-second precision, we measured the sizes of the stabilizing cap of individual microtubules.Nevertheless, the trigger of instability lies in a short region at the end of the cap, as a quantitative model of cap stability demonstrates.Our study establishes the spatial and kinetic characteristics of the protective cap and provides an insight into the molecular mechanism by which its loss leads to the switch from microtubule growth to shrinkage.

View Article: PubMed Central - PubMed

Affiliation: Lincoln's Inn Fields Laboratory, The Francis Crick Institute, London, United Kingdom.

ABSTRACT
The function of microtubules relies on their ability to switch between phases of growth and shrinkage. A nucleotide-dependent stabilising cap at microtubule ends is thought to be lost before this switch can occur; however, the nature and size of this protective cap are unknown. Using a microfluidics-assisted multi-colour TIRF microscopy assay with close-to-nm and sub-second precision, we measured the sizes of the stabilizing cap of individual microtubules. We find that the protective caps are formed by the extended binding regions of EB proteins. Cap lengths vary considerably and longer caps are more stable. Nevertheless, the trigger of instability lies in a short region at the end of the cap, as a quantitative model of cap stability demonstrates. Our study establishes the spatial and kinetic characteristics of the protective cap and provides an insight into the molecular mechanism by which its loss leads to the switch from microtubule growth to shrinkage.

No MeSH data available.


Related in: MedlinePlus