Foster PJ et al. (2015), Active contraction of microtubule networks.

XB-ART-51662

Elife 2015 Jan 06;4. doi: 10.7554/eLife.10837.

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Active contraction of microtubule networks.

Foster PJ , Fürthauer S , Shelley MJ , Needleman DJ .

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Many cellular processes are driven by cytoskeletal assemblies. It remains unclear how cytoskeletal filaments and motor proteins organize into cellular scale structures and how molecular properties of cytoskeletal components affect the large-scale behaviors of these systems. Here, we investigate the self-organization of stabilized microtubules in Xenopus oocyte extracts and find that they can form macroscopic networks that spontaneously contract. We propose that these contractions are driven by the clustering of microtubule minus ends by dynein. Based on this idea, we construct an active fluid theory of network contractions, which predicts a dependence of the timescale of contraction on initial network geometry, a development of density inhomogeneities during contraction, a constant final network density, and a strong influence of dynein inhibition on the rate of contraction, all in quantitative agreement with experiments. These results demonstrate that the motor-driven clustering of filament ends is a generic mechanism leading to contraction.

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Species referenced: Xenopus
Genes referenced: abl1 fubp1

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	Figure 1. Stabilized microtubules form asters in Xenopus egg extracts.(A) Experiments were performed in thin rectangular channels of width W0, height H0, and length L0. (B) In some regions of the channel, microtubules organize into asters, with minus ends localized in the aster core (Scale bar, 5 μm). (C) Isolated asters fuse together over minute timescales (Scale bar, 5 μm). (D) Aster-like structures form in other regions of the channel (Scale bar, 10 μm) (E) Aster-like structures show large scale movement on minute timescales. (Scale bar, 25 μm). (F) NUMA localizes to the network interior (Scale bar, 20 μm). (G) Closeup of aster-like structure showing NUMA localized on the interior (Scale bar, 10 μm).DOI: http://dx.doi.org/10.7554/eLife.10837.003
	Figure 2. Stabilized microtubules form a contractile network in Xenopus egg extracts.(A) Low magnification imaging shows that microtubules form a contractile network (Scale bar, 500 μm). (B) The width of the microtubule network decreases with time (n = 6 experiments). (Inset) Representative plot of ϵ(t) (Blue line) and fit from (Equation 2) (Pink line), with ϵ∞=0.81, τ=3.49 min, Tc=1.06 min.DOI: http://dx.doi.org/10.7554/eLife.10837.006Figure 2—figure supplement 1. Plots of ϵ(t) from data in Figure 1F (Blue lines) along with fits from (Equation 2) (Pink lines).DOI: http://dx.doi.org/10.7554/eLife.10837.007
	Figure 3. Contraction dynamics in channels of different width provide a means to test potential contraction mechanisms.(A) Microtubules form contractile networks in channels with various widths (Scale bar, 500 μm, t=10 min). (B) Width of the networks as a function of time in channels with various widths. (C) Fraction contracted as a function of time, ϵ(t), calculated from the data in B. The networks all contract to a similar final fraction, while the timescale of contraction differs. (D) The scaling of the characteristic time, τ, with channel width does not vary as W02, as would result for a poroelastic timescale, and is not a constant, independent of width, as would result from a viscoelastic timescale. The scaling is well described by an active fluid model (green line analytic scaling, fit to (Equation 6); green dots numerical solution). (E) The characteristic time, τ, is found to be independent of channel height. The dashed line is the mean value of τ. (F) ϵ∞ is constant for all channel widths and heights, indicating that the network contracts to a constant final density. The dashed line is the mean value of ϵ∞. All panels display mean ± s.e.m.DOI: http://dx.doi.org/10.7554/eLife.10837.010
	Figure 4. Cartoon of the microscopic model underlying the active fluid theory of network contractions by minus end clustering.(A) Microtubule sliding by dynein drives microtubule minus ends together. (B) Minus end clustering leads to the formation of aster-like structures. Due to steric interactions between microtubules, there is an upper limit to the local microtubule density. (C) The microtubule network is composed of interacting asters. Motor activity driving aster cores together leads to bulk contraction of the network.DOI: http://dx.doi.org/10.7554/eLife.10837.012
	Figure 5. Microtubule density increases at the network’s edges during contraction.(A) Time series of contraction showing intensity averaged along the length of the channel. The average intensity peaks at the network’s edges due to increased local microtubule density. (Scale bars, 500 μm) (B) Comparison of measured density profiles (solid lines) with density profiles from simulation (dashed lines). Data are plotted at 1 min intervals starting at t = 40 s. (C) Representative frame from PIV showing the network’s local velocity component along the network’s width. (D) Comparison between measured (solid red line) and simulated (dashed red line) velocity along the width of the channel at t = 80 s. The measured and simulated velocities increase superlinearly with distance from the center of the network, as can be seen by comparison to a linear velocity profile (dashed black line).DOI: http://dx.doi.org/10.7554/eLife.10837.013Figure 5—figure supplement 1. Comparison between measured (solid lines) and simulated (dashed lines) density profiles.Data are plotted at 2 min intervals starting at t = 40 s.DOI: http://dx.doi.org/10.7554/eLife.10837.014
	Figure 6. Network contraction is a dynein-dependent process.(A) Fraction contracted as a function of time, ϵ(t), when dynein is inhibited using p150-CC1. (B) The characteristic time of contraction, τ, increases with increasing p150-CC1 concentration. Solid green line indicates fit of sigmoid function. (C) ϵ∞ has no apparent variation with p150-CC1 concentration. Solid green line indicates the mean value of ϵ∞. All panels display mean ± s.e.m.DOI: http://dx.doi.org/10.7554/eLife.10837.015Figure 6—figure supplement 1. Inhibition of Kinesin-5 has little effect on the contraction process.(A) Comparison of ϵ(t) curves for samples where Kinesin-5 was inhibited using STLC and control where no STLC was added. (B) Simultaneous inhibition of dynein with p150-CC1 and Kinesin-5 with STLC does not rescue the effects of dynein inhibition alone. All panels display mean ± s.e.m.DOI: http://dx.doi.org/10.7554/eLife.10837.016
	Figure 6—figure supplement 2. Plots of ϵ(t) from experiments with 2 μM p150-CC1 (blue lines) along with fits from Equation (2) (pink lines).DOI: http://dx.doi.org/10.7554/eLife.10837.017

References [+] :

Belmonte, Large-scale microtubule networks contract quite well. 2016, Pubmed, Xenbase