P7127

MTs are mixed with the motor clusters that act as cross-linkers, and with ATP (Sigma; A2383) that drives the activity of the gel. The aqueous dispersion contains a non-adsorbing polymer (PEG, 20 kDa) (Sigma; 95172) that promotes the formation of filament bundles through depletion interaction (Supplementary Fig. 2). To maintain a constant concentration of ATP during the experiments, an enzymatic ATP-regenerator system is used, consisting on phosphoenolpyruvate (Sigma; P7127) that fuels pyruvate kinase/lactate dehydrogenase (PK/LDH) (Invitrogen; 434301) to convert ADP back into ATP. Several antioxidant components are also included in the solution to avoid protein denaturation, and to minimize photobleaching during characterization by means of fluorescence microscopy. The PEG-based triblock copolymer surfactant Pluronic F-127 (Sigma; P-2443) is added at 1% (wt/wt) (final concentration) to procure a biocompatible water/oil interface in subsequent steps.

The activity of acyl‐CoA synthetase (ACS) was measured by a continuous coupled enzymatic assay as previously described.^[47] The reaction mixture contained 100 mM Tris‐HCl buffer (pH 8.0), 10 mM ATP (10519979001, Sigma‐Aldrich), 15 mM MgCl₂, 5 mM dithiothreitol, 150 mM KCl, 1 mM potassium phosphoenolpyruvate (P7127, Sigma‐Aldrich), 0.3 mM nicotinamide adenine dinucleotide (NADH, N8129, Sigma‐Aldrich) in 100 mM triethanolamine (pH 8.2), and 500 µM sodium palmitate (P9767, Sigma‐Aldrich). 4.5 µg adenylate kinase (M3003, Sigma‐Aldrich), 3 µg pyruvate kinase (P1506, Sigma‐Aldrich), 3 µg lactate dehydrogenase (L8080, Solarbio), and 3 µg mitochondrial protein sample were added in a total reaction volume of 100 µL. The reaction system was incubated at 37 °C for 1 min, and the reaction was initiated by the addition of CoA (final concentration 600 µM) (C4282, Sigma‐Aldrich). Changes in absorbance of the reaction system at 334 nm were measured every 10 s for 15 min with a recording spectrophotometer (Thermo Fisher Scientific). The reaction rate was calculated using the slope and intercept created from a NADH standard curve.
Total cellular and mitochondrial PKA activities were measured by the nonradioactive PKA Kinase Activity Assay Kit (ab139435, Abcam) following manufacturers' instructions.