Metamorph

ATP levels in control and FUS‐transfected cells were determined using a FRET‐based plasmid reporter (Adenosine 5′‐Triphosphate indicator based on Epsilon subunit for Analytical Measurements; ATeam reporter) 46. To do so, NSC34 cells were co‐transfected with AT1.03 cytosolic ATeam reporter and either control vector, FUS, FUSR521C or FUSR518K. Cells were imaged in Hanks’ balanced salt solution (HBSS) without phenol red at 37°C by time‐lapse microscopy (12 s intervals) on a Zeiss Axiovert S100 microscope equipped with a 40×/1.3NA Plan‐Neofluar objective and a Photometrics Cascade‐II 512B EMCCD driven by MetaMorph (Molecular Dynamics). FRET filtersets (ECFP excitation filter ET 430/24×; ECFP emission filter 470/24 m; EYFP emission filter ET545/40 m) were from Chroma Technology. KCN 1 mM in HBSS was applied using a peristaltic pump (0.5 ml/min). YFP/CFP ratios prior to and after KCN treatment were measured as described and used to calculate relative ATP levels in the different samples, which were displayed as bar charts 46.

Aspergillus nidulans conidiospores were inoculated in uncoated glass-bottom µ-dishes (Ibidi GmbH) containing 2.5 mL watch minimal medium (WMM^{57 (link)}) supplemented with 25 mM NaH₂PO₄, 5 mM ammonium (+)-tartrate, and 0.5% glucose. After 16 h at 25 °C medium was replaced with freshly prepared WMM supplemented with 100 mM Na₂HPO_4. Samples were cultured for an additional 1.5 h at 37 °C prior to observation of fluorescence (see text for details of enaA and enaB induction).
Differential Interference Contrast (Normarski optics) and fluorescence images were acquired from in vivo cultures with a Leica DMI-6000b inverted microscope coupled to an ORCA-ER digital camera (Hamamatsu Photonics) and equipped with a 63 Plan Apochromat 1.4 N.A. oil immersion objective (Leica) and a GFP filter (excitation 470 nm; emission 525 nm). Images were acquired using Metamorph (Molecular Dynamics) software and processed using ImageJ free-software (https://imagej.nih.gov/ij).

ATP levels in cultured cells were measured using a ViaLight ATP kit (Lonza) according to the manufacturer’s instructions; luminescence signals were obtained with a FluoSTAR luminometer (BMG Labtech). To determine ATP levels generated by oxidative phosphorylation in mitochondria, cells were first treated with 100 μM iodoacetate (Sigma) for 2 h to inhibit ATP produced by glycolysis. ATP levels were also determined using a FRET based plasmid reporter (Adenosine 5′-Triphosphate indicator based on Epsilon subunit for Analytical Measurements; ATeam reporter) [42 (link)]. To do so, cells were transfected with AT1.03 cytosolic ATeam reporter and then imaged in Hanks Balanced Salt Solution (HBSS) without phenol red at 37 °C by timelapse microscopy (12 s intervals) on a Zeiss Axiovert S100 microscope equipped with a 40×/1.3NA Plan-Neofluar objective and a Photometrics Cascade-II 512B EMCCD driven by MetaMorph (Molecular Dynamics). FRET filtersets (ECFP excitation filter ET 430/24×; ECFP emission filter 470/24 m; EYFP emission filter ET545/40m) were from Chroma Technology. 1 mM KCN in HBSS was applied using a peristaltic pump (0.5 ml/min). YFP/CFP ratios prior to and after KCN treatment were measured as described and used to calculate relative ATP levels in the different samples which were displayed as bar charts [42 (link)].