Limits...
Active contraction of microtubule networks.

Foster PJ, Fürthauer S, Shelley MJ, Needleman DJ - Elife (2015)

Bottom Line: 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.These results demonstrate that the motor-driven clustering of filament ends is a generic mechanism leading to contraction.

View Article: PubMed Central - PubMed

Affiliation: John A. Paulson School of Engineering and Applied Sciences, FAS Center for Systems Biology, Harvard University, Cambridge, United States.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Inhibition of Kinesin-5 has little effect on the contraction process.(A) Comparison of (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
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4764591&req=5

fig6s1: Inhibition of Kinesin-5 has little effect on the contraction process.(A) Comparison of (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

Mentions: Finally, we sought to determine the molecular basis of the contraction process, and check prediction (iii), that the number of motors driving the contraction affects the rate of contraction, but not the final density the network contracts to. Aster assembly is dynein-dependent in Xenopus egg extracts (Gaglio et al.,1995; Verde et al., 1991), and dynein (Heald et al., 1996) and Kinesin-5 (Sawin et al., 1992) are two of the most dominant motors in spindle assembly in this system. We inhibited these motors to test their involvement in the contraction process. Extracts supplemented with STLC for Kinesin-5 inhibition or p150-CC1 for dynein inhibition were loaded into channels with a width, , of 0.9 mm and imaged at low magnification. Inhibiting Kinesin-5 had little effect on the contraction process (Figure 6—figure supplement 1). In contrast, inhibiting dynein caused a dose-dependent slowdown of the contraction (Figure 6A). In spindle assembly, inhibiting Kinesin-5 suppresses the morphological changes caused by dynein inhibition (Mitchison et al., 2005). We, therefore, tested how simultaneously inhibiting both motors influences the contraction process, but found that the effects of dynein inhibition were not rescued by the simultaneous inhibition of Kinesin-5 (Figure 6—figure supplement 1), suggesting that in this context, Kinesin-5 is not generating a counteracting extensile stress. This further suggests the possibility that in the spindle, the role of Kinesin-5 may be in orienting, polarity sorting, and sliding microtubules as opposed to active stress generation. Curves of (t) were fit using Equation (2) to extract the final fraction contracted, , and the characteristic time of contraction, . By varying the concentration of p150-CC1, the characteristic time, , could be tuned over a wide range from 3 min to 75 min (Figure 6B). Fitting a sigmoid function to the vs. p150-CC1 concentration curve yields an EC50 value of 0.22 . 02 M (mean standard error), similar to the value of 0.3 M reported for the effect of p150-CC1 on spindle length in Xenopus extracts (Gaetz and Kapoor, 2004), which is consistent with active stress generated by dynein being required for pole focusing. Despite this large change in the contraction timescale, we found no apparent differences in (Figure 6C). Thus, the microtubule networks contract to approximately the same final density irrespective of the concentration of p150-CC1. The observation that inhibiting dynein affects the timescale of contraction but not the final density to which the network contracts is consistent with the predictions of our model. We note that even at the highest p150-CC1 concentrations used, the network still undergoes a bulk contraction. This could possibly be due to incomplete inhibition of dynein by p150-CC1, or by another motor protein present in the extract that also contributes to the contraction process. As the characteristic time, , by comparing the characteristic times in the uninhibited and 2 M p150-CC1 cases, we can estimate that the strength of the active stress, , in the 2 M p150-CC1 condition is only 4% of the strength of the active stress in the uninhibited case, arguing that even if another motor is involved in the contraction, dynein contributes 96% of the active stress.10.7554/eLife.10837.015Figure 6.Network contraction is a dynein-dependent process.


Active contraction of microtubule networks.

Foster PJ, Fürthauer S, Shelley MJ, Needleman DJ - Elife (2015)

Inhibition of Kinesin-5 has little effect on the contraction process.(A) Comparison of (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
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4764591&req=5

fig6s1: Inhibition of Kinesin-5 has little effect on the contraction process.(A) Comparison of (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
Mentions: Finally, we sought to determine the molecular basis of the contraction process, and check prediction (iii), that the number of motors driving the contraction affects the rate of contraction, but not the final density the network contracts to. Aster assembly is dynein-dependent in Xenopus egg extracts (Gaglio et al.,1995; Verde et al., 1991), and dynein (Heald et al., 1996) and Kinesin-5 (Sawin et al., 1992) are two of the most dominant motors in spindle assembly in this system. We inhibited these motors to test their involvement in the contraction process. Extracts supplemented with STLC for Kinesin-5 inhibition or p150-CC1 for dynein inhibition were loaded into channels with a width, , of 0.9 mm and imaged at low magnification. Inhibiting Kinesin-5 had little effect on the contraction process (Figure 6—figure supplement 1). In contrast, inhibiting dynein caused a dose-dependent slowdown of the contraction (Figure 6A). In spindle assembly, inhibiting Kinesin-5 suppresses the morphological changes caused by dynein inhibition (Mitchison et al., 2005). We, therefore, tested how simultaneously inhibiting both motors influences the contraction process, but found that the effects of dynein inhibition were not rescued by the simultaneous inhibition of Kinesin-5 (Figure 6—figure supplement 1), suggesting that in this context, Kinesin-5 is not generating a counteracting extensile stress. This further suggests the possibility that in the spindle, the role of Kinesin-5 may be in orienting, polarity sorting, and sliding microtubules as opposed to active stress generation. Curves of (t) were fit using Equation (2) to extract the final fraction contracted, , and the characteristic time of contraction, . By varying the concentration of p150-CC1, the characteristic time, , could be tuned over a wide range from 3 min to 75 min (Figure 6B). Fitting a sigmoid function to the vs. p150-CC1 concentration curve yields an EC50 value of 0.22 . 02 M (mean standard error), similar to the value of 0.3 M reported for the effect of p150-CC1 on spindle length in Xenopus extracts (Gaetz and Kapoor, 2004), which is consistent with active stress generated by dynein being required for pole focusing. Despite this large change in the contraction timescale, we found no apparent differences in (Figure 6C). Thus, the microtubule networks contract to approximately the same final density irrespective of the concentration of p150-CC1. The observation that inhibiting dynein affects the timescale of contraction but not the final density to which the network contracts is consistent with the predictions of our model. We note that even at the highest p150-CC1 concentrations used, the network still undergoes a bulk contraction. This could possibly be due to incomplete inhibition of dynein by p150-CC1, or by another motor protein present in the extract that also contributes to the contraction process. As the characteristic time, , by comparing the characteristic times in the uninhibited and 2 M p150-CC1 cases, we can estimate that the strength of the active stress, , in the 2 M p150-CC1 condition is only 4% of the strength of the active stress in the uninhibited case, arguing that even if another motor is involved in the contraction, dynein contributes 96% of the active stress.10.7554/eLife.10837.015Figure 6.Network contraction is a dynein-dependent process.

Bottom Line: 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.These results demonstrate that the motor-driven clustering of filament ends is a generic mechanism leading to contraction.

View Article: PubMed Central - PubMed

Affiliation: John A. Paulson School of Engineering and Applied Sciences, FAS Center for Systems Biology, Harvard University, Cambridge, United States.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus