Live cell imaging experiments

Cells were seeded onto 35-mm glass bottom culture dishes. The following day, for HEK293T cells, calcium phosphate transfection methods were used to transfect 500 ng of each specified FUS, Q72HTT, or CRY2olig plasmid DNA, and 1000 ng of each C-BLOCK protein. Unless specified otherwise, cells were moved to HBSS imaging buffer (Hanks Balanced Salt Solution, 1.26 mM CaCl₂, 0.41 mM MgSO₄, .49 mM MgCl₂, 5.33 mM KCL, 138 mM NaCl, 0.44 mM KH₂PO₄, 0.34 mM Na₂HPO₄, 4.17 mM NaHCO₃, 5.56 mM D-glucose, and 20 mM HEPES) for live cell imaging 18 to 22 h after transfection. Where specified, 333 nM rapamycin was added directly to the samples during live cell imaging.

To examine the effect of DisCo over arsenite-induced condensates of FUS, COS-7 cells were transfected with 250 ng of EGFP-FRB-FUS-FRB or EGFP-FRB-FUS and 500 ng of mCh-FKBP plasmid. 18–22 h after transfection, cells were imaged during treatment with 1 mM sodium arsenite. After arsenite-induced formation of FUS condensates, 333 nM rapamycin was added.

To examine recruitment of CRY2olig to mitochondria, COS-7 cells were transfected with 500 ng of each specified plasmid DNA. 18–22 h after transfection, cells were moved to imaging buffer containing 100 nM Far-Red MitoTracker (Invitrogen). For zapalog experiments, HEK293T cells were transfected with 500 ng EGFP-DHFR-FUS and 500 ng mCh-FKBP. 4 h after transfection, media was replaced and 500 nM zapalog was added. Cells were subsequently kept in the dark. 18–22 h after transfection, cells were moved to imaging buffer containing 500 nM zapalog. For experiments involving CaM/CBP cells were moved to PBS prior to live cell imaging. 2.5 mM CaCl₂ and 3 µM ionomycin were added directly to samples during imaging to increase intracellular calcium. To reverse the Ca²⁺ increase, 10 mM EGTA was added.

During imaging, samples were kept at 33.5˚C in a light-tight stage top incubator enclosure. Cells were imaged using either of three systems: (1) an Andor Dragonfly 301 spinning disc imaging system with Olympus IX73 base and four-line ILE laser merge module and controller. Images were taken using a 60x UplanSApo 1.35 NA oil objective and collected on a 1024 ×1024 pixel Andor iXon EM-CCD camera. Data was acquired using Fusion 2.0 or iQ3 (Andor). (2) A Zeiss AxioObserver Z1 inverted microscope equipped with a Yokogawa CSU-X1 spinning disk unit, a 63x/NA 1.4 oil immersion objective, a mSAC unit for correction of spherical ablation (3i), a Vector live specimen scanner (3i), a mSwitcher ms optical switching unit (3i) and a Photometrics 512×512 EM-CCD camera. Slidebook 6 was used for all collection of images on this system. 3) Olympus IX71 microscope equipped with a spinning disc scan head (Yokogawa) and a 60x/NA 1.4 oil immersion objective. Excitation illumination was delivered from an AOTF controlled laser launch (Andor Technology) and images collected on a 1024 ×1024 pixel EM-CCD camera (iXon; Andor Technology). After initial image collection, ImageJ 1.46r was used for all image analysis.