Optima ultracentrifuge

To study the interaction between tau and microtubules we adapted a previously published protocol [25 (link)]. Briefly, HeLa cells were cultured in 60 mm plates and transfected at 90% confluence with 5 μg of the different MAPT constructs using Lipofectamine 2000 (Invitrogen), according to manufacturer’s instructions. After 24 hours, cells were washed twice with warm PBS and lysed in 500 μL of warm 80 mM PIPES buffer pH 6.8, 1mM guanosine-5’-triphosphate, 1 mM MgCl₂, 1 mM EGTA, 0.5% w/v Triton X-100 and 30% v/v glycerol, supplemented with 1 mM phenylmethylsulfonylfluoride, complete protease inhibitors (Roche), 0.5 μM okadaic acid (Calbiochem), and 10 μM taxol (Sigma). Cell lysates were spun at 5000×g for 5 minutes at room temperature (RT) to pellet the nuclei. Clarified lysates were then centrifuged at 100,000×g for 1 hour at RT using an Optima ultracentrifuge (Beckman). The supernatants were retained as the microtubule unbound fraction while the pellets were washed once with PIPES buffer and resuspended in 150 μL of 2X Laemmli buffer (microtubule bound fraction). Equal volumes of both bound and unbound fractions were probed by Western blot using an anti-FLAG antibody. The relative affinity of each Tau mutant for microtubules was then expressed as the ratio between the signal intensity of the bound over the unbound fraction.

Neurons were treated with or without 100 μM CESP in balanced salt solution (BSS) (140 mM NaCl, 5 mM KCl, 1.5 mM pH 7.4) for 60 min at 37°C. Cells MgCl₂, 5 mM glucose, 10 mM HEPES and 2.5 mM CaCl₂ were then washed twice with cold PBS (phosphate buffered saline) (Invitrogen Cat # 14190-250). Cold PBS /1 mM EGTA was added to the dish and cells transferred into tubes. The tubes were centrifuged at 1000 g for 1 min at 4°C. The pellets were suspended with K-EGTA buffer (125 mM KCl, 20 mM Hepes pH 7.4, 1 mM EGTA) and homogenized with a glass pestle B (40 strokes). The homogenates were centrifuged at 60,000 g for 30 min at 4°C (Beckman Optima Ultracentrifuge, rotor TLA100.1). The pellet contained mitochondria and the supernatant was defined to be the cytosolic fraction. The pellet (crude mitochondrial fraction was homogenized in K⁺⁻EGTA buffer with a Dounce homogenizer (B) and centrifuged 1,000 g for 10 min at 4°C. The supernatants were transferred into new tubes and centrifuged at 8,000 g at 4°C for 10 min. The pellet contained the mitochondria.