Effects of Confinement on the Self-Organization of Microtubules and Motors

Abstract

The regulation of the cytoskeleton is essential for the proper organization and function of eukaryotic cells. For instance, radial arrays of microtubules (MTs), called asters, determine the intracellular localization of organelles. Asters can be generated through either MT organizing center (MTOC)-dependent regulation or self-organization processes. In vivo, this occurs within the cell boundaries. How the properties of these boundaries affect MT organization is unknown. To approach this question, we studied the organization of microtubules inside droplets of eukaryotic cellular extracts with varying sizes and elastic properties. Our results show that the size of the droplet determined the final steady-state MT organization, which changed from symmetric asters to asymmetric semi-asters and, finally, to cortical bundles. A simple physical model recapitulated these results, identifying the main physical parameters of the transitions. The use of vesicles with more elastic boundaries resulted in very different morphologies of microtubule structures, such as asymmetrical semi-asters, "Y-branching" organizations, cortical-like bundles, "rackets," and bundled organizations. Our results highlight the importance of taking into account the physical characteristics of the cellular confinement to understand the formation of cytoskeleton structures in vivo.

Spatiotemporal control of microtubule nucleation and assembly using magnetic nanoparticles

“…The mineral oil contained a biocompatible block copolymer to stabilize the emulsion and facilitate observations. This method has been described previously and allows the formation of microtubule asters in droplets 30,31 . Arlacel P135 block copolymer was first dissolved in mineral oil (0.05 mg ml 21 ).…”

“…First, to reproduce the physical confinement conditions existing in cells, we encapsulated Xenopus extracts within spherical droplets with typical diameters ranging from 40 to 80 mm by forming an emulsion of the extract within oil (see Methods) 30,31 . In these confined extracts, when Ran-NPs were at a concentration above the microtubule nucleation threshold ( 6 mM for instance), we observed microtubule asters after 30 min of incubation.…”

Motor-mediated Cortical versus Astral Microtubule Organization in Lipid-monolayered Droplets

“…Aster formation in encapsulated extract has been shown to depend on confinement size. Spatial constraints led to microtubule bundling and bending in small droplets (17). A systematic investigation, however, of how microtubules arrange in droplets of different sizes in the absence of any other proteins, and how the presence of a purified and well characterized microtubule sliding motor changes these arrangements, is currently lacking.…”

“…Comparable experiments for encapsulated active motor/microtubule assemblies produced with purified proteins are lacking. The droplets-inoil method has been used to investigate self-organized microtubule assemblies in encapsulated Xenopus egg extract, an experimental system of a highly complex composition (17). When the microtubule stabilizer paclitaxel is added to such Xenopus egg extracts in the absence of confinement, microtubule asters are generated by a set of microtubule cross-linking and sliding activities, including the motor protein dynein (18,19).…”

Experiments inside a box lead to out-of-the-box ideas on cellular organization

“…In the second research direction the complexity is increased much more dramatically. Pinot et al (2009) for example, also grew microtubule asters in droplets, using cellular extracts instead of purified components. Nevertheless, the positioning of those asters is in agreement with predictions Fig.…”