Glucose oxidase

The kinetics of PI(4)P phosphorylation was measured on SLBs formed in IBIDI chambers and visualized using TIRF microscopy. Reaction buffer contained 20 mM HEPES (pH 7.0), 150 mM NaCl, 1 mM ATP, 5 mM MgCl₂, 0.5 mM EGTA, 20 mM glucose, 200 µg/mL beta-casein (Thermo Scientific, Cat# 37528), 20 mM BME, 320 µg/mL glucose oxidase (Serva, #22780.01 Aspergillus niger), 50 µg/mL catalase (Sigma, #C40-100MG Bovine Liver), and 2 mM Trolox (UV treated, see methods below). Perishable reagents (i.e., glucose oxidase, catalase, and Trolox) were added 5–10 min before image acquisition. For all experiments, we monitored the change in PI(4)P or PI(4,5)P₂ membrane density using a solution concentration of 20 nM Alexa647-DrrA(544-647) or 20 nM Alexa488-PLCδ, respectively. We calculated the density of PIP lipids (lipids/µm²) assuming a footprint of 0.72 nm² for DOPC lipids (Galush et al., 2008 (link); Vacklin et al., 2005 (link)).

For motility assays with stabilised microtubules, long GMPCPP microtubules were prepared as described (Roostalu et al, 2011) and immobilised in the same manner as described for dynamic microtubule assays. A protein mix consisting of 5 nM GFP‐dynein, 10 nM pig dynactin, 200 nM BicD2‐N and 50 nM Lis1 was incubated on ice for 10 min and was then added to the appropriate volume of cold assay buffer (AB) or, for control purposes, HEPES‐based buffer (60 mM HEPES pH 7.4, 50 mM K‐acetate, 2 mM MgCl₂, 10% glycerol (vol/vol), 0.1 mg/ml BSA, 0.5% Pluronic F‐127, 10 μM taxol, 0.2 mM κ‐casein, 20 mM glucose, 2 mM ATP, 5 mM ME), supplemented with 1.3 mg/ml glucose oxidase (Serva) and 0.66 mg/ml catalase (Sigma). HEPES‐based buffer is similar to the buffer used in Gutierrez et al (2017). The final volume of the assay mix was 30 μl. This final assay mix was warmed to room temperature and introduced into the flow chamber with immobilised biotin‐labelled Alexa647‐labelled GMPCPP microtubules.

DOPC and 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (Ni²⁺-NTA-DOGS; nickel salt) were purchased from Avanti Polar Lipids. Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 647 maleimide dyes were purchased from Thermo Fisher. Bovine serum albumin (BSA), (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, catalase, 2-mercaptoethanol (BME), NiCl₂, H₂SO₄, and ATP were purchased from Sigma-Aldrich. Glucose oxidase was purchased from Serva. Tris(2-carboxyethyl)phosphine (TCEP), glucose and H₂O₂ were purchased from Thermo Fisher Scientific. MgCl₂ was purchased from EMD Chemicals. Tris-buffered saline (TBS) was purchased from Corning.

Freshly prepared, ice-cold BRB80 buffer (80 mM K-PIPES, 1 mM MgCl₂, and 1 mM EGTA, pH 6.8; prepared as a 5× stock and diluted to 1× before use) was used to resuspend β-casein (to 25 mg/mL, Sigma-Aldrich), glucose oxidase (to 40 mg/mL, Serva), and catalase (to 20 mg/mL, Sigma-Aldrich). Resuspended proteins were centrifuged at 80,000 rpm in a TLA 100 rotor (Beckman Coulter) at 4 °C for 15 min, aliquoted, snap-frozen in liquid nitrogen and stored at −80 °C. ATP and guanosine triphospate (GTP) were dissolved in MilliQ water to 100 mM each, adjusted to pH 7 using KOH, and filtered (0.22-μm pore size, Millipore) and aliquots were stored at −80 °C. Docetaxel (Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO) to 1 mM and filtered (0.22-μm pore size), and aliquots were stored at −80 °C. Glucose was dissolved in MilliQ water to 1 M, filtered (0.22-μm pore size), and stored at 4 °C.

Cell lysates were diluted to yield desired EGFP-SOS concentrations (typically 1 nM) in reaction buffer supplemented with 320 μg ml⁻¹ glucose oxidase (Serva Electrophoresis GmbH), 50 μg ml⁻¹ catalase (Sigma), 2 mM trolox, 20 mM beta-mercaptoethanol (BME), 20 mM glucose and 0.2 mg ml⁻¹ casein. SOS concentration was adjusted based on fluorescence intensity calibration standards of recombinant EGFPs. Lysates with 120 μM unlabelled GDP or GTP were introduced over SLBs functionalized with fluorescent nucleotide-loaded Ras. Lysates were maintained in the reaction chamber throughout the measurements. Time-lapse epifluorescence images were recorded. Intensity traces for individual corrals were extracted using ImageJ and Matlab. Each kinetic trace was normalized to its own initial maximum intensity value. Intensity changes due to enzymatic nucleotide exchange reactions were calculated by dividing with the average trace of inactive corrals obtained with the same concentration of untransfected lysate (Supplementary Fig. 9). For this, total protein concentrations in transfected and untransfected cell lysates were determined by Bradford assay (Bio-Rad) and diluted accordingly.