Spatiotemporal organization of branched microtubule networks
- Princeton University, United States
Figures
Figure 1 with 3 supplements
Branched microtubule networks and stochastic models for their assembly.
(A) Branched microtubule (MT) networks were generated in Xenopus egg extracts with 10 μM RanQ69L, and time-lapse is displayed for one representative branched network. MTs were labeled with …
Figure 1—figure supplement 1
Hybrid tracking of branched microtubule networks.
(A) Microtubules (MTs) in branched networks were tracked in the following four steps. (1) EB1 comets were pre-processed using temporal median filter, which isolates only moving particles in an image …
Figure 1—figure supplement 2
Characterization of branched microtubule networks.
(A) Schematic representation of a branched network depicting parameters measured in (B–E). (B) Growth speed of plus-ends was obtained from EB1 tracks as described in Materials and methods. Histogram …
Figure 1—figure supplement 3
Stochastic models of branched microtubule networks.
(A) Distribution of mother MT lengths at the first branching event, used for parameter estimation of the single-step model and the sequential model, is displayed. The fit parameters are tabulated …
Figure 2 with 3 supplements
Nucleation profile of first branching event and spatial organization of microtubules in dense branched networks.
Spatial location of first branching event on a naked mother MT was recorded during the formation of individual branched MT networks, and the following distributions were measured. See Figure …
Figure 2—figure supplement 1
Measurement of nucleation profile and testing robustness of sequential model.
(A) Position of first branching event on a naked mother MT was recorded during the formation of individual branched networks. Two branched networks were selected in the field of view and time-lapse …
Figure 2—figure supplement 2
Spatial organization of microtubules in dense branched networks.
(A) Distribution of MT plus-ends was measured in dense branched networks as follows. EB1 comets were pre-processed (left panel) and detected (red circles, right panel) as described in Materials and m…
Figure 2—figure supplement 3
Self-similarity in the architecture of branched networks.
(A–B) Positions of the minus-ends were recorded in simulated branched networks. The distances of all minus-ends were calculated from the origin, normalized by seed MT’s length, and the resulting …
Figure 3 with 1 supplement
Network architecture changes with varying protein concentrations as predicted by the sequential model.
(A) Branched MT networks were generated in Xenopus egg extracts with 10 μM RanQ69L for four conditions: buffer addition or control depletion, addition of 260 nM TPX2 (9.6x), partial depletion of …
Figure 3—figure supplement 1
Controls for varying TPX2 and augmin concentration in Xenopus egg extracts.
(A) Endogenous TPX2 concentration was measured using quantitative, fluorescence western blot. A series of known concentrations of recombinant GFP-TPX2 were compared with endogenous TPX2 and probed …
Figure 4 with 2 supplements
Sequential mechanism of branching microtubule nucleation and binding rate of TPX2 to microtubules.
Sequential mechanism of branching MT nucleation (A-C). (A) De novo MTs (blue) were generated by performing branching reaction in augmin-depleted Xenopus egg extracts where TPX2 is present. Non …
Figure 4—figure supplement 1
Control immunodepletion reactions and measurement of length of initial branched microtubules.
(A) Augmin, TPX2 or γ-TuRC were immunodepleted from Xenopus egg extracts, and western blot analysis against TPX2, γ-tubulin or HAUS1 (augmin subunit) was performed to verify depletion. Branching MT …
Figure 4—figure supplement 2
Controls for TPX2’s binding rate measurement, and visualization of augmin/γ-TuRC on branched networks.
(A) Distribution of branching effector1 in sequential model was measured along the length of mother MT at 100 s. The number of molecules is plotted after rescaling the length of mother MT to 1 (n = 2…
Videos
Video 1
Assembly of branched microtubule networks in Xenopus egg extracts.
Branched microtubule (MT) networks were generated in Xenopus egg extracts with 10 μM RanQ69L, and time-lapse is displayed for one representative branched network. MTs are labeled with Cy5-tubulin …
Video 2
Tracking of microtubules in branched networks.
MTs in branched networks were tracked. Branched networks (‘raw data’) were imaged in Xenopus egg extracts with fluorescently-labeled plus-tip protein EB1-mCherry (green, displayed as ‘EB1 channel’) …
Video 3
Simulation of single-step reaction model.
Three stochastic simulations of branched networks using the single-step model are displayed with parameters specified in Figure 1—figure supplement 3A. Individual MTs are labelled with a rotating …
Video 4
Simulation of the sequential reaction model.
Three stochastic simulations of branched networks using sequential model are displayed with parameters specified in Figure 1—figure supplement 3A. Individual MTs are labelled with a rotating color …
Video 5
Branched microtubule networks generated with varying augmin and TPX2 concentrations.
Branched MT networks were generated in Xenopus egg extracts with 10 μM RanQ69L for four conditions: buffer addition or control depletion, addition of 260 nM TPX2, partial depletion of TPX2 (20% of …
Video 6
Sequence of TPX2 and augmin in branching microtubule nucleation by two-color solution exchange experiments.
Left movie: De novo MTs (blue) were generated in Xenopus egg extracts containing TPX2 but no augmin first, followed by exchange to Xenopus egg extracts containing augmin but no TPX2. Branched MTs …
Video 7
Sequence of γ-TuRC, TPX2 and augmin in branching microtubule nucleation by two-color solution exchange experiments.
De novo MTs (blue) were generated in Xenopus egg extracts containing TPX2 and γ-TuRC but no augmin first, followed by exchange to Xenopus egg extracts containing TPX2 and augmin but no γ-TuRC. No …
Video 8
Binding of TPX2 to branched microtubule networks in Xenopus egg extracts.
Endogenous TPX2 was immunodepleted from Xenopus egg extracts and replaced with 20–30 nM recombinant GFP-TPX2. Branched MT networks were generated 10 μM RanQ69L, and TPX2’s association with MTs was …
Additional files
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Supplementary file 1
MATLAB-based image analysis and simulation software.
- https://doi.org/10.7554/eLife.43890.023
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Transparent reporting form
- https://doi.org/10.7554/eLife.43890.024
