June 1, 2017
Two separate solutions containing 50-200 nM SPD-5 in Condensate buffer and 6%â24% PEG-3350 in Condensate buffer were dispensed into two rows of a 96 well plate. A pipetting robot (Tecan, Model Freedom EVO 200) equipped with a TeMo 96 pipetting head with disposable tips (50 ml) was used to mix the SPD-5 and PEG solutions 1:1 and then transfer each condition in quadruplicate to a 384 well plastic bottom imaging plate (15 mL per well) to synchronize condensate formation
Centrosomes are non-membrane-bound compartments that nucleate microtubule arrays. They consist of nanometer-scale centrioles surrounded by a micron-scale, dynamic assembly of protein called the pericentriolar material (PCM). To study how PCM forms a spherical compartment that nucleates microtubules, we reconstituted PCM-dependent microtubule nucleation in vitro using recombinant C. elegans proteins. We found that macromolecular crowding drives assembly of the key PCM scaffold protein SPD-5 into spherical condensates that morphologically and dynamically resemble in vivo PCM. These SPD-5 condensates recruited the microtubule polymerase ZYG-9 (XMAP215 homolog) and the microtubule-stabilizing protein TPXL-1 (TPX2 homolog). Together, these three proteins concentrated tubulin âź4-fold over background, which was sufficient to reconstitute nucleation of microtubule asters in vitro. Our results suggest that in vivo PCM is a selective phase that organizes microtubule arrays through localized concentration of tubulin by microtubule effector proteins. Copyright Š 2017 Elsevier Inc. All rights reserved.
Woodruff, JB; Ferreira Gomes, B; Widlund, PO; Mahamid, J; Honigmann, A; Hyman, AA;
Journal: Cell Pages: 1066-1077.e10