The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy
- Department of Chemical and Biological Engineering, Princeton University, United States
- Department of Molecular Biology, Princeton University, United States
- Department of Cell Biology, New York University School of Medicine, United States
- Department of Mechanical and Aerospace Engineering, Princeton University, United States
- Lewis-Sigler Institute for Integrative Genomics, United States
- Department of Physics, Princeton University, United States
Figures
Figure 1 with 1 supplement
Single molecule microscopy of microtubule nucleation from γ-TuRC.
(A) Schematic for microtubule nucleation from γ-TuRC. Biochemical features of γ-TuRC including the γ-/αβ-tubulin interaction affinity and conformation of γ-TuRC determine to MT nucleation activity …
Figure 1—figure supplement 1
Controls for γ-TuRC-mediated microtubule nucleation.
(A) Protein gel of purified γ-TuRC was stained with SYPRO Ruby stain (left). Biotinylated sites on γ-TuRC was visualized with alkaline phosphatase conjugated to avidin (middle), and …
Figure 2
Molecular composition of transition state in γ-TuRC-mediated nucleation.
(A) Titrating tubulin concentration with constant the density of γ-TuRC. MT nucleation from γ-TuRC begins at 7μM tubulin. (B) MT plus-end growth speed increases linearly with tubulin concentration. …
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Figure 2—source data 1
Source data for Figure 2B–D.
Each excel sheet is labelled with individual figure panel. For Figure 2C, all three experimental replicates are supplied, and dataset one is plotted.
- https://cdn.elifesciences.org/articles/54253/elife-54253-fig2-data1-v3.xlsx
Figure 3 with 1 supplement
Comparison of γ-TuRC-mediated, spontaneous and seed-templated nucleation.
(A) Spontaneous MT nucleation (schematized) was measured with increasing tubulin concentration and high concentrations. 14 μM tubulin is required. (B) Number of MTs (N(t=τ)) nucleated spontaneously …
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Figure 3—source data 1
Source data for Figure 3 panels B, D, E and Figure 3—figure supplement 1C–D.
Each excel sheet is labelled with individual figure panel. For Figure 3D, all three experimental replicates are supplied, and dataset one is plotted.
- https://cdn.elifesciences.org/articles/54253/elife-54253-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Controls for spontaneous and blunt seed-mediated microtubule nucleation.
(A) Representative kymographs of spontaneously nucleated MT is displayed, demonstrating that MTs grow from both the minus-end (dotted line) and the plus-end (solid line) and can be distinguished …
Figure 4 with 2 supplements
γ-tubulin binds to αβ-tubulin with a high affinity.
(A) Size-exclusion chromatography was performed with 150 nM of γ-tubulin alone (i) and with 35 μM and 10 μM αβ-tubulin in (ii) and (iii), respectively. Gel filtration fractions were analyzed via …
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Figure 4—source data 1
Source data for Figure 4C.
- https://cdn.elifesciences.org/articles/54253/elife-54253-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Purified γ-tubulin binds αβ-tubulin with high affinity in vitro.
(A) Size-exclusion chromatography was performed with 35μM and 10μM αβ-tubulin alone in (i) and (ii), respectively. Gel filtration fractions were analyzed via SDS–PAGE followed by immunoblot with …
Figure 4—figure supplement 2
Purified γ-tubulin nucleates microtubules and assembles laterally into filaments.
(A) MTs nucleate from high concentration of purified γ-tubulin efficiently and (B) minus-ends of γ-tubulin-nucleated MTs remain capped while the plus-ends polymerize. The experiments were repeated …
Figure 5 with 2 supplements
Monte Carlo simulations of microtubule nucleation from γ-TuRC.
(A) Kinetic Monte Carlo simulations of MT nucleation were performed. Helical MT lattice was simulated with 13 protofilaments and a pitch of 3 tubulin monomers across the seam. Native γ-TuRC was …
Figure 5—figure supplement 1
Parameter variation in Monte Carlo simulations of γ-TuRC-mediated nucleation.
(A) MT plus-end assembly was simulated. Parameter values used: , . 20 simulations each were performed at tubulin concentration 2-20 μM. MT growth speed was obtained from each MT length versus …
Figure 5—figure supplement 2
Simulations of microtubule nucleation from γ-TuRC and from blunt seeds.
(A) Simulations recapitulated experimental data for γ-TuRC-mediated MT nucleation. Parameter values used: , , , , and . 500 simulations each were performed for a range of tubulin …
Figure 6 with 1 supplement
Regulation of γ-TuRC-mediated nucleation by putative activation factors.
(A) A constant density of γ-TuRC molecules were attached without (left) and with (right) 6μM CDK5RAP2’s γ-TuNA motif and 10.5μM tubulin ± 3μM additional γ-TuNA was added. Scale bar, 10 μm. (B) MTs …
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Figure 6—source data 1
Source data for Figure 6 panels B, D.
- https://cdn.elifesciences.org/articles/54253/elife-54253-fig6-data1-v3.xlsx
Figure 6—figure supplement 1
Effect of putative activation factor NME7 on γ-TuRC-mediated nucleation.
(A) A constant density of γ-TuRC molecules were attached without (left) and with (right) 6 μM NME7 and 10.5 μM tubulin ±1 μM additional NME7 was added. Experiments were repeated twice on different …
Figure 7 with 2 supplements
Role of XMAP215 and microtubule-associated proteins in microtubule nucleation with γ-TuRC.
(A) γ-TuRCs were attached and 7μM tubulin (pseudo-colored in red) ± 20nM XMAP215-GFP (pseudo-colored in green) was added. Scale bar, 10 μm. Experiments and analyses in (A–B) were repeated thrice …
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Figure 7—source data 1
Source data for Figure 7 panels B, D, E, F and Figure 7—figure supplement 1 panels B, E.
Each excel sheet is labelled with individual figure panel. For Figure 7D, all five experimental replicates are supplied, and dataset one is plotted. Source Code. MATLAB code for Monte Carlo simulations used to model the dynamics of γ-TuRC-mediated nucleation. Materials and methods section details how the simulation was set up and performed. Figure 5—figure supplement 2 provide the parameters used to model our experimental data.
- https://cdn.elifesciences.org/articles/54253/elife-54253-fig7-data1-v3.xlsx
Figure 7—figure supplement 1
Role of XMAP215 on γ-TuRC-mediated microtubule nucleation.
(A) γ-TuRCs were attached and 3.5-7μM tubulin ± 20nM XMAP215-GFP was added. Experiment was repeated identically two times with independent γ-TuRC purifications. One additional replicated was …
Figure 7—figure supplement 2
MCAK and Stathmin inhibit γ-TuRC-mediated nucleation.
(A) γ-TuRC molecules were attached to coverslips and either 10.5 μM tubulin alone (left panels), 10 μM tubulin + 10 nM MCAK (top-right) or 10 μM tubulin + 5 μM Stathmin (bottom-right) was added to …
Author response image 1
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Videos
Video 1
Microtubule nucleation from γ-TuRC complexes.
γ-TuRC was attached to functionalized coverslips and MT nucleation was observed upon introducing fluorescent αβ-tubulin (gray). MTs nucleated from individual γ-TuRC molecules from zero length at 14 …
Video 2
Microtubule nucleation from fluorescent, single γ-TuRC molecules.
Dual Alexa-568 and biotin-labeled γ-TuRC (green) was attached to functionalized coverslips and MT nucleation was observed upon introducing fluorescent αβ-tubulin (red). MTs nucleated from single …
Video 3
γ-TuRC molecules nucleate microtubules efficiently.
Constant density of γ-TuRC was attached while concentration of fluorescent αβ-tubulin was titrated (3.5–21 μM) and MT nucleation was observed. γ-TuRC molecules nucleated MTs starting from 7 μM …
Video 4
Spontaneous microtubule nucleation occurs at high tubulin concentration.
Concentration of fluorescent αβ-tubulin was titrated (7–21 μM) and spontaneous MT nucleation was assayed. MTs nucleated spontaneously starting from high concentration of 14 μM tubulin and MT …
Video 5
Microtubule assembly from blunt plus-ends resembles polymerization.
MTs with blunt ends (seeds, cyan) were generated and attached to functionalized coverslips. Varying concentration of fluorescent αβ-tubulin was added (1.4–8.7 μM, pseudo-colored as magenta) and MT …
Video 6
γ-TuNA motif from CDK5RAP2, NME7 and TPX2 do not significantly increase γ-TuRC-mediated microtubule nucleation.
Top panels: γ-TuRC was immobilized on coverslips with control buffer (left) or with 6 μM γ-TuNA motif from CDK5RAP2 (right) and MT nucleation was observed upon introducing fluorescent 10.5 μM …
Video 7
XMAP215 increases microtubule nucleation activity of γ-TuRC.
γ-TuRC was immobilized on coverslips and MT nucleation was assayed with low concentration of fluorescent αβ-tubulin (3.5 μM and 7 μM) without (top panels) or with 20 nM XMAP215-GFP (bottom panels). …
Video 8
Synergistic microtubule nucleation by γ-TuRC and XMAP215.
Triple-color fluorescence microscopy was performed to observe the molecular sequence of events during MT nucleation from γ-TuRC and XMAP215. γ-TuRC (blue) and XMAP215 (red) formed a complex before …
Video 9
MCAK and Stathmin inhibit γ-TuRC-mediated microtubule nucleation.
Top panels: γ-TuRC was immobilized on coverslips and MT nucleation was observed upon introducing fluorescent 10.5 μM αβ-tubulin without (left) or with 10 nM MCAK (right). Bottom panels: γ-TuRC was …
Additional files
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Source code 1
MATLAB code for simulations of γ-TuRC-mediated microtubule nucleation.
- https://cdn.elifesciences.org/articles/54253/elife-54253-code1-v3.zip
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Transparent reporting form
- https://cdn.elifesciences.org/articles/54253/elife-54253-transrepform-v3.pdf
