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

Abstract

Background: How cytoskeleton architecture is established within the confining boundary of the cell membrane is not understood. Results: Together, spatial constraints and cross-linking motor activities determine distinct motor/microtubule organizations in a biomimetic system. Conclusion: Characteristic microtubule architectures result from basic design principles. Significance: Spatial confinement plays an important role in cytoskeleton organization.

Physical Determinants of Bipolar Mitotic Spindle Assembly and Stability in Fission Yeast

“…Quantitative force-balance models were later applied to Drosophila spindle assembly (28)(29)(30)(31), and the same ideas have more recently been extended to human cells (32). In parallel with work on spindles, force-balance ideas have been studied in reconstituted MTmotor systems (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45).…”

Self-organization of motors and microtubules in lipid-monolayered droplets

“…Single microtubule asters can self-organize reliably in the presence of the purified minusend directed microtubule cross-linking and sliding kinesin, XCTK2 (added in the range of hundreds of nanomoles), provided the droplets have a diameter larger than 40 mm (Figure 7(A) top) (Baumann & Surrey, 2014). In smaller droplets, asters fail to form, because their size would exceed the droplet diameter forcing the microtubules to arrange into motor-bundled cortical arrays (Figure 7(A) bottom).…”

“…3. For confined motor-mediated microtubule self-organization: recombinant mCherry-XCTK2 (mCherry-tagged Xenopus leavis Carboxy-Terminal Kinesin 2, 0.4e1.0 mg/mL, snap frozen in aliquots of 2 mL), expressed and purified as described (Hentrich & Surrey, 2010) with some modifications (His-tag cleavage and phosphate buffer) (Baumann & Surrey, 2014). 4.…”

“…The compositions of the lipid monolayer and the buffer are optimized in this method to ensure good maintenance of droplet shape, tubulin solubility, and efficient microtubule nucleation (Baumann & Surrey, 2014;Keber et al, 2014). In the absence of lipids, droplets in close proximity to the glass tend to spread onto the glass and/or fuse, to a certain extent even if the surface is treated with a water repellent ( Figure 3 and Figure 4, panel 1).…”

“…Here we describe a method that allows the encapsulation of purified motors and microtubules in oil-surrounded droplets with diameters of several micrometers (Baumann & Surrey, 2014), intended to produce microcontainers in the size range of typical cell bodies. To minimize contact with the oil and to provide a more natural boundary, a lipid monolayer is generated at the interface between the aqueous solution and the oil (Pautot, Frisken, & Weitz, 2003;Pinot et al, 2009).…”