The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability
- The Francis Crick Institute, United Kingdom
- Barcelona Institute of Science and Technology, Spain
- ICREA, Spain
Figures
Figure 1 with 2 supplements
Recombinant human tubulin lacking GTPase activity strongly nucleates microtubules.
(a) The evolutionarily conserved catalytic glutamic acid of α-tubulin (E, pink). (b) Coomassie Blue-stained SDS gel of purified wildtype (WT) and E254A mutant recombinant human tubulin. (c) HPLC …
Figure 1—figure supplement 1
Expression and purification of recombinant human α/β tubulin.
(a) Schematic of the human TUBA1B α-tubulin and TUBB3 β-tubulin expression constructs. (b) Flow chart of the purification steps to obtain recombinant human α/β-tubulin from insect cells. The hash …
Figure 1—figure supplement 2
iSCAT microscopy of unlabeled porcine brain microtubules.
(a) Schematic of a dynamic microtubule assay. (b) iSCAT microscopy images of unlabeled porcine brain microtubules growing from GMPCPP-seeds at 12 µM porcine brain tubulin. (c) Kymographs showing the …
Figure 2 with 1 supplement
GTPase deficient microtubules are stable and bind EB3 with high affinity.
(a) iSCAT/TIRF microscopy image of unlabeled wildtype microtubules (WT, magenta) growing from GMPCPP-seeds at 12 µM wildtype human tubulin in the presence of 20 nM human mGFP-EB3 (green). (b) …
Figure 2—figure supplement 1
Characterization of EB3 binding to wildtype and GTPase deficient human microtubules.
(a) Coomassie Blue-stained SDS gel of purified recombinant mGFP-EB3 and Alexa647-labeled SNAP-EB3 (Alexa647-EB3). (b, c) Comparative iSCAT/TIRF microscopy images of 20 nM mGFP-EB3 binding to (b) …
Figure 3
Slowing down GTP hydrolysis extends the GTP cap and stabilizes growing microtubules.
(a) Coomassie Blue-stained SDS gel with purified E254D mutant recombinant human tubulin. (b, c) iSCAT/TIRF microscopy images of unlabeled E254D mutant microtubules growing from GMPCPP-seeds and also …
Figure 4 with 3 supplements
Conformational gradient in the GTP cap revealed by spatially resolved single EB3 dwell time distributions.
(a) TIRF microscopy kymograph of single mGFP-EB3 molecules (green) at 25 pM in the plus end region of an E254D microtubule (E254D MT) growing at 10 µM E254D tubulin (vg = 58.5 nm/s) in the …
Figure 4—figure supplement 1
Flow chart of single molecule localization and spatially-resolved dwell time analysis.
For single molecule analysis of mGFP-EB3 binding to microtubules, TIRF microscopy movies were recorded in the presence of 25 pM mGFP-EB3 and additional 1 nM Alexa647-EB3 to visualize the growing …
Figure 4—figure supplement 2
Spatially-resolved EB3 dwell time distributions at the ends of E254D and wildtype microtubules.
(a) Local dwell time survival functions at specific distance bins from the microtubule end for E254D microtubules. Dashed black lines are mono-exponential fits. The global reduced χ2 value for all …
Figure 4—figure supplement 3
Wildtype and E254D microtubules grow with similar speeds under single molecule experiment conditions.
Quantification of microtubule growth speeds at 19 µM wildtype (WT) and 10 µM E254D tubulin concentrations in the presence of 1 nM Alexa647-EB3 and 25 pM mGFP-EB3. The boxes extend from 25th to 75th …
Videos
Video 1
Pure recombinant human wildtype tubulin polymerizes into dynamic microtubules.
iSCAT microscopy time-lapse movie of unlabeled microtubules growing in the presence of 12 µM human wildtype tubulin from immobilized stable microtubule seeds at 30°C. Scale bar is 5 µm. Time stamp …
Video 2
Human wildtype tubulin does not nucleate microtubules at low tubulin concentration.
iSCAT microscopy time-lapse movie showing lack of microtubule nucleation at 5 µM human wildtype tubulin on a surface with an immobilized rigor kinesin (Kin1rigor) at 30°C. Scale bar is 5 µm. Time …
Video 3
Human E254A tubulin mutant lacking GTPase activity strongly nucleates microtubules.
iSCAT microscopy time-lapse movie showing strong microtubule nucleation at 4 µM human E254A tubulin on a surface with an immobilized rigor kinesin (Kin1rigor) at 30°C. Scale bar is 5 µm. Time stamp …
Video 4
EB3 binds to the growing ends of human wildtype microtubules.
iSCAT/TIRF microscopy time-lapse movie of unlabeled wildtype microtubules (magenta) growing from immobilized stable microtubule seeds in the presence of 12 µM human wildtype tubulin and 20 nM human …
Video 5
Slowing down GTP hydrolysis extends the GTP cap and stabilizes growing microtubules.
iSCAT/TIRF microscopy time-lapse movie of unlabeled E254D mutant microtubules (magenta) growing from immobilized microtubule seeds and also spontaneously nucleating in the presence of 12 µM human …
Video 6
Wildtype microtubules are very dynamic at low tubulin concentrations.
iSCAT/TIRF microscopy time-lapse movie of human wildtype microtubules (magenta) undergoing frequent catastrophes when growing from immobilized microtubule seeds in the presence of 5 µM human …
Video 7
Microtubules with reduced GTPase activity are more stable than wildtype microtubules.
iSCAT/TIRF microscopy time-lapse movie of human E254D microtubules (magenta) show persistent growth when growing from immobilized microtubule seeds in the presence of 5 µM E254D tubulin and 20 nM …
