Limits...
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

Delay times and microtubule orientations.(A) Top: distribution of individual microtubule orientations, each averaged over 10 s growth before tubulin washout, for the conditions without Mal3 (Figures 1, 2) and with 200 nM Mal3-GFP (Figures 3, 4). The microchannel axis is 0° and corresponds to the flow direction. Bottom: Delay times after washout versus orientation before washout, for the individual microtubules shown in the top panel. (B) As (A) for the tubulin concentration variation datasets (Figure 6A). For each dataset, the Pearson’s r correlation coefficients were calculated using the magnitude of the orientations relative to the distribution mean.DOI:http://dx.doi.org/10.7554/eLife.13470.005
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4829430&req=5

fig1s2: Delay times and microtubule orientations.(A) Top: distribution of individual microtubule orientations, each averaged over 10 s growth before tubulin washout, for the conditions without Mal3 (Figures 1, 2) and with 200 nM Mal3-GFP (Figures 3, 4). The microchannel axis is 0° and corresponds to the flow direction. Bottom: Delay times after washout versus orientation before washout, for the individual microtubules shown in the top panel. (B) As (A) for the tubulin concentration variation datasets (Figure 6A). For each dataset, the Pearson’s r correlation coefficients were calculated using the magnitude of the orientations relative to the distribution mean.DOI:http://dx.doi.org/10.7554/eLife.13470.005

Mentions: We imaged 101 microtubules in the presence of 20 μM tubulin at 4 Hz and tracked their plus ends with a precision of ~30 nm (Figure 1C). Fitting traces of the end position and fluorescence background (Materials and methods) allowed us to determine the washout and catastrophe times with sub-sampling time precision, as well as the instantaneous growth speeds measured over a 10 s time period just before washout (Figure 1D, Figure 1—figure supplement 1B). Growth speeds varied considerably around a mean of 28 nm/s (Figure 1E, left). The delay times between washout and catastrophe also showed a broad and non-exponential distribution, with a mean of 7.3 s (Figure 1E, right), similar to previous dilution experiments (Walker et al., 1991). As bending can affect the material properties of microtubules (Schaedel et al., 2015), we tested whether the measured delay times were influenced by mechanical stress, potentially induced by microtubule bending in our assay. We determined the orientation of the growing microtubule end regions relative to the flow direction before tubulin washout, and found that the variation of orientations was relatively small (mean orientation 3.1° with a standard deviation of 7.3°) indicative of good microtubule alignment. No correlation between the delay time and the magnitude of the orientation was observed (Figure 1—figure supplement 2A, blue data), indicating that mechanical stress is not responsible for the observed variations of the momentary microtubule stabilities in our assay.


The size of the EB cap determines instantaneous microtubule stability.

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

Delay times and microtubule orientations.(A) Top: distribution of individual microtubule orientations, each averaged over 10 s growth before tubulin washout, for the conditions without Mal3 (Figures 1, 2) and with 200 nM Mal3-GFP (Figures 3, 4). The microchannel axis is 0° and corresponds to the flow direction. Bottom: Delay times after washout versus orientation before washout, for the individual microtubules shown in the top panel. (B) As (A) for the tubulin concentration variation datasets (Figure 6A). For each dataset, the Pearson’s r correlation coefficients were calculated using the magnitude of the orientations relative to the distribution mean.DOI:http://dx.doi.org/10.7554/eLife.13470.005
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829430&req=5

fig1s2: Delay times and microtubule orientations.(A) Top: distribution of individual microtubule orientations, each averaged over 10 s growth before tubulin washout, for the conditions without Mal3 (Figures 1, 2) and with 200 nM Mal3-GFP (Figures 3, 4). The microchannel axis is 0° and corresponds to the flow direction. Bottom: Delay times after washout versus orientation before washout, for the individual microtubules shown in the top panel. (B) As (A) for the tubulin concentration variation datasets (Figure 6A). For each dataset, the Pearson’s r correlation coefficients were calculated using the magnitude of the orientations relative to the distribution mean.DOI:http://dx.doi.org/10.7554/eLife.13470.005
Mentions: We imaged 101 microtubules in the presence of 20 μM tubulin at 4 Hz and tracked their plus ends with a precision of ~30 nm (Figure 1C). Fitting traces of the end position and fluorescence background (Materials and methods) allowed us to determine the washout and catastrophe times with sub-sampling time precision, as well as the instantaneous growth speeds measured over a 10 s time period just before washout (Figure 1D, Figure 1—figure supplement 1B). Growth speeds varied considerably around a mean of 28 nm/s (Figure 1E, left). The delay times between washout and catastrophe also showed a broad and non-exponential distribution, with a mean of 7.3 s (Figure 1E, right), similar to previous dilution experiments (Walker et al., 1991). As bending can affect the material properties of microtubules (Schaedel et al., 2015), we tested whether the measured delay times were influenced by mechanical stress, potentially induced by microtubule bending in our assay. We determined the orientation of the growing microtubule end regions relative to the flow direction before tubulin washout, and found that the variation of orientations was relatively small (mean orientation 3.1° with a standard deviation of 7.3°) indicative of good microtubule alignment. No correlation between the delay time and the magnitude of the orientation was observed (Figure 1—figure supplement 2A, blue data), indicating that mechanical stress is not responsible for the observed variations of the momentary microtubule stabilities in our assay.

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