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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

Fast and complete microfluidics-controlled solution exchange.(A) Scaled layout of microfluidic channels used in this study. The channel width was 300 µm and the height was 95 ± 5 µm. (B) Representative kymograph (left) and a plot of the time course (right) of 20 µM Alexa568-tubulin (12.5% labelled; 2.5 µM in total) fluorescence intensity as measured by TIRF microscopy, showing fast and complete solution exchange (imaged at 7.7 Hz): from 9 experiments, the average time for 90% buffer exchange (5% - 95%) was 199 (± 32 s.e.m) ms. (C) Left: Time series of TIRF microscopy images showing microtubules (red) growing from a surface-immobilised seed during repeated sudden microfluidics-controlled exchanges between solutions containing 75 nM Mal3-GFP (green) and not containing any Mal3-GFP, in the constant presence of 15 µM Alexa568-tubulin. Right: a corresponding kymograph (top: GFP channel only, bottom: merge). Horizontal and vertical scale bars are 3 µm and 1 min, respectively. Persistent microtubule growth is not affected by solution exchanges. Time in seconds, recording frequency was 0.5 Hz. (D) Kymographs showing a Alexa568-microtubule growing from an immobilised seed; the growth speed changes abruptly in response to a sudden microfluidics-controlled change of the Alexa568-tubulin concentration from 12 μM to 25 μM (left) or from 25 μM to 12 μM (right), again demonstrating that solution exchange does not cause catastrophesDOI:http://dx.doi.org/10.7554/eLife.13470.004
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fig1s1: Fast and complete microfluidics-controlled solution exchange.(A) Scaled layout of microfluidic channels used in this study. The channel width was 300 µm and the height was 95 ± 5 µm. (B) Representative kymograph (left) and a plot of the time course (right) of 20 µM Alexa568-tubulin (12.5% labelled; 2.5 µM in total) fluorescence intensity as measured by TIRF microscopy, showing fast and complete solution exchange (imaged at 7.7 Hz): from 9 experiments, the average time for 90% buffer exchange (5% - 95%) was 199 (± 32 s.e.m) ms. (C) Left: Time series of TIRF microscopy images showing microtubules (red) growing from a surface-immobilised seed during repeated sudden microfluidics-controlled exchanges between solutions containing 75 nM Mal3-GFP (green) and not containing any Mal3-GFP, in the constant presence of 15 µM Alexa568-tubulin. Right: a corresponding kymograph (top: GFP channel only, bottom: merge). Horizontal and vertical scale bars are 3 µm and 1 min, respectively. Persistent microtubule growth is not affected by solution exchanges. Time in seconds, recording frequency was 0.5 Hz. (D) Kymographs showing a Alexa568-microtubule growing from an immobilised seed; the growth speed changes abruptly in response to a sudden microfluidics-controlled change of the Alexa568-tubulin concentration from 12 μM to 25 μM (left) or from 25 μM to 12 μM (right), again demonstrating that solution exchange does not cause catastrophesDOI:http://dx.doi.org/10.7554/eLife.13470.004

Mentions: In a microfluidic device, we immobilised Alexa568-labelled stabilised microtubule seeds on a functionalised glass surface. We then observed their growth in the presence of 20 µM Alexa568-labelled tubulin and GTP by time-lapse total internal reflection fluorescence (TIRF) microscopy (Figure 1A, Figure 1—figure supplement 1A). Solutions were exchanged within ~200 ms (Figure 1—figure supplement 1B); the exchange itself did not affect microtubule growth (Figure 1—figure supplement 1C & D). Sudden and complete removal of tubulin stopped growth, and induced a catastrophe after a delay of typically several seconds (Figure 1B, Video 1), similar to earlier observations after tubulin dilution (Walker et al., 1991). This delay is thought to be caused by the temporary survival of the stabilising cap.10.7554/eLife.13470.003Figure 1.Momentary microtubule stability assayed by fast tubulin washout and nm precision plus end tracking.


The size of the EB cap determines instantaneous microtubule stability.

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

Fast and complete microfluidics-controlled solution exchange.(A) Scaled layout of microfluidic channels used in this study. The channel width was 300 µm and the height was 95 ± 5 µm. (B) Representative kymograph (left) and a plot of the time course (right) of 20 µM Alexa568-tubulin (12.5% labelled; 2.5 µM in total) fluorescence intensity as measured by TIRF microscopy, showing fast and complete solution exchange (imaged at 7.7 Hz): from 9 experiments, the average time for 90% buffer exchange (5% - 95%) was 199 (± 32 s.e.m) ms. (C) Left: Time series of TIRF microscopy images showing microtubules (red) growing from a surface-immobilised seed during repeated sudden microfluidics-controlled exchanges between solutions containing 75 nM Mal3-GFP (green) and not containing any Mal3-GFP, in the constant presence of 15 µM Alexa568-tubulin. Right: a corresponding kymograph (top: GFP channel only, bottom: merge). Horizontal and vertical scale bars are 3 µm and 1 min, respectively. Persistent microtubule growth is not affected by solution exchanges. Time in seconds, recording frequency was 0.5 Hz. (D) Kymographs showing a Alexa568-microtubule growing from an immobilised seed; the growth speed changes abruptly in response to a sudden microfluidics-controlled change of the Alexa568-tubulin concentration from 12 μM to 25 μM (left) or from 25 μM to 12 μM (right), again demonstrating that solution exchange does not cause catastrophesDOI:http://dx.doi.org/10.7554/eLife.13470.004
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fig1s1: Fast and complete microfluidics-controlled solution exchange.(A) Scaled layout of microfluidic channels used in this study. The channel width was 300 µm and the height was 95 ± 5 µm. (B) Representative kymograph (left) and a plot of the time course (right) of 20 µM Alexa568-tubulin (12.5% labelled; 2.5 µM in total) fluorescence intensity as measured by TIRF microscopy, showing fast and complete solution exchange (imaged at 7.7 Hz): from 9 experiments, the average time for 90% buffer exchange (5% - 95%) was 199 (± 32 s.e.m) ms. (C) Left: Time series of TIRF microscopy images showing microtubules (red) growing from a surface-immobilised seed during repeated sudden microfluidics-controlled exchanges between solutions containing 75 nM Mal3-GFP (green) and not containing any Mal3-GFP, in the constant presence of 15 µM Alexa568-tubulin. Right: a corresponding kymograph (top: GFP channel only, bottom: merge). Horizontal and vertical scale bars are 3 µm and 1 min, respectively. Persistent microtubule growth is not affected by solution exchanges. Time in seconds, recording frequency was 0.5 Hz. (D) Kymographs showing a Alexa568-microtubule growing from an immobilised seed; the growth speed changes abruptly in response to a sudden microfluidics-controlled change of the Alexa568-tubulin concentration from 12 μM to 25 μM (left) or from 25 μM to 12 μM (right), again demonstrating that solution exchange does not cause catastrophesDOI:http://dx.doi.org/10.7554/eLife.13470.004
Mentions: In a microfluidic device, we immobilised Alexa568-labelled stabilised microtubule seeds on a functionalised glass surface. We then observed their growth in the presence of 20 µM Alexa568-labelled tubulin and GTP by time-lapse total internal reflection fluorescence (TIRF) microscopy (Figure 1A, Figure 1—figure supplement 1A). Solutions were exchanged within ~200 ms (Figure 1—figure supplement 1B); the exchange itself did not affect microtubule growth (Figure 1—figure supplement 1C & D). Sudden and complete removal of tubulin stopped growth, and induced a catastrophe after a delay of typically several seconds (Figure 1B, Video 1), similar to earlier observations after tubulin dilution (Walker et al., 1991). This delay is thought to be caused by the temporary survival of the stabilising cap.10.7554/eLife.13470.003Figure 1.Momentary microtubule stability assayed by fast tubulin washout and nm precision plus end tracking.

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