Taxol

For the fixed drug treatment experiments, U2OS cells were seeded on 18-mm coverslips. The next day, cells were incubated with 0.1% DMSO (3 h), 10 µM Taxol (#T7402; Sigma-Aldrich; 2 h), 10 µM tubacin (BML-GR362; Enzo Life Sciences; 2 h), 10 µM nocodazole (Cat#M1404; Sigma-Aldrich; 1 h), 10 µM Taxol (2 h) followed by 10 µM Taxol + 10 µM nocodazole (1 h), or 10 µM tubacin (2 h) followed by 10 µM tubacin + 10 µM nocodazole (1 h) in full medium and subsequently fixed. For the detyrosination assay, U2OS cells were seeded on 18-mm coverslips and transfected with VSH1-GFP and SBVP-FLAG the next day. 24 h after transfection, cells were treated with 0.1% DMSO (1 h) or 10 µM nocodazole (1 h) and subsequently fixed. For the drug treatments of StableMARK-expressing cells, U2OS cells were seeded on 18-mm coverslips and transfected with StableMARK the next day. 24 h after transfection, cells were treated with 0.1% DMSO (2 h), 10 µM Taxol (2 h), or 10 µM tubacin (2 h) and subsequently fixed. For the nocodazole treatment of StableMARK-expressing cells during live-cell imaging, U2OS cells were plated on 25-mm coverslips and transfected the next day with StableMARK and mCherry-tubulin. The following day, nocodazole was added with a final concentration of 10 µM to the live cells on stage during a time-lapse acquisition. For live-cell imaging of lysosomes, cells were incubated with 1 µM SiR-lysosome (Spirochrome) and 10 µM Verapamil for 1 h and subsequently imaged in a medium without SiR-lysosome and Verapamil. For cold treatment, cells were incubated on ice for 10 min and subsequently extracted and fixed with precooled reagents.

Double-cycled MT seeds were prepared by combining TRITC-labeled (49%), biotinylated (18%), and unlabeled tubulin (33%; Cytoskeleton) reconstituted in MRB80 (80 mM K-Pipes, 1 mM EGTA, 4 mM MgCl₂; pH 6.80 with KOH) to a final concentration of 20 µM with 1 mM GMPCPP (Jena Bioscience) on ice. The mixture was incubated at 35°C for 30 min to allow MTs to polymerize. Seeds were pelleted by centrifugation in an airfuge (Beckman coulter) at 20 psi for 5 min, resuspended in MRB80, and depolymerized on ice for 25 min. The tubulin was then repolymerized upon the addition of fresh GMPCPP by incubating at 35°C for 30 min. These seeds were pelleted by centrifugation in an airfuge at 20 psi for 5 min, resuspended and diluted sixfold in MRB80 supplemented with 10% [vol/vol] glycerol, aliquoted, flash-frozen in liquid nitrogen, and stored at −80°C until use.
To prepare the chambers, a clean glass coverslip was plasma-treated and fixed to a clean glass slide using strips of double-sided tape to create two parallel chambers of ∼10 µl. The surface was blocked and functionalized by incubating with a mix of 95% PLL-g-PEG and 5% PLL-g-PEG-biotin (0.1 mg/ml in 10 mM Hepes, pH 7.40; SuSoS) for 10 min. After washing with MRB80 supplemented with 40% [vol/vol] glycerol (MRB80-gly40), NeutrAvidin was introduced and incubated for 10 min. After washing, 50-fold diluted GMPCPP seeds were introduced and incubated for 5 min before washing once more and then incubating with Κ-casein for >3 min.
All reaction mixtures (MT mix, expansion mix, rigor mix, washout mix) were prepared at double the volume for the paired compacted/expanded lattice samples and split into two equal parts prior to the addition of DMSO (compacted control) or 20 µM Taxol (expanded). Reagents were added to MRB80-gly40 such that the effective glycerol concentration in the MT mix was 20% and in the other mixes was ∼27%. All mixes contained 0.1% [wt/vol] methylcellulose, 0.5 mg/ml K-casein, 50 mM glucose, 0.2 mg/ml catalase, 0.5 mg/ml glucose oxidase, and 10 mM DTT. The MT mix additionally contained 1 mM GTP, 10.8 µM porcine tubulin (Cytoskeleton), and 0.6 µM TRITC-labeled porcine tubulin (Cytoskeleton). The expansion mix additionally contained 50 mM KCl and 20 µM Taxol (or the equivalent dilution of DMSO). The rigor mix additionally contained 50 mM KCl, 20 µM Taxol (or the equivalent dilution of DMSO), 2 mM ATP, and 15.2 pM StableMARK. The washout mixture additionally contained 50 mM KCl, 20 µM Taxol (or the equivalent dilution of DMSO), and 2 mM ATP. After preparation, these mixtures were spun in an airfuge at 20 psi for 5 min, transferred to clean tubes, and kept on ice until use.
Samples were then moved to the TIRF microscope equipped with a stage-top incubator to maintain them at a constant temperature of 30°C. MTs were grown by flowing in two chamber volumes (ChV) of the MT mix and letting it incubate for 15 min. Subsequently, the chambers were flushed with five ChV MRB80-gly40. Next, the lattices were (mock) expanded by adding two ChV expansion mix (or DMSO equivalent) and incubating for 10 min. Next, two ChV rigor mix was added and incubated for 90 s. Finally, four ChV washout mix was added before imaging. For imaging, the following sequence was used: 2 × Taxol, 4 × DMSO, 4 × Taxol, 4 × DMSO, and either 2 × Taxol or 4 × Taxol and 2 × DMSO (8 or 10 images/condition/assay), and images were taken at similar heights within the channels.