8 well chambered coverslip

U2OS cell lines (engineered from HTB-96; ATCC) stably coexpressing Halo-H2A and NES-mCherry-LINuS or NLS-mCherry-LEXY were maintained in DMEM (no. 10567022; Thermo Fisher) supplemented with 10% FBS (Thermo Fisher no. A31605 or GenClone no. 25-514) at 37°C in a humidified atmosphere with 5% CO₂. Cells were seeded at 10,000 to 15,000 cells per well in an 8-well chambered coverslip (no. 80826; ibidi) and grown in complete medium for 1 day before cotransfection of the mammalian expression vectors encoding GFP and the viral protein of interest using TransIT-2020 transfection reagent (no. MIR5404; Mirus). After 24 h, the growth medium was replaced with imaging medium: low-glucose (1 g/L) DMEM without phenol red (no. 11054020; Thermo Fisher), supplemented with 10% FBS, 50 IU mL⁻¹ penicillin, 50 μg mL⁻¹ streptomycin, and GlutaMAX (no. 35050061; Thermo Fisher). For nuclear staining, 500 nM JF646-HaloTag ligand (a gift from Luke Lavis) was added to the imaging medium. Image acquisition and kinetics measurements were performed as described previously (32 (link)).

FCS measurements were performed with an inverted Zeiss LSM 880 laser scanning confocal microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) equipped with a C-Apochromat 40×/1.2 NA water immersion objective. The LHCII surfactant solution was placed in an 8-well chambered coverslip (Ibidi GmbH, Gräfelfing Germany), and chlorophyll auto-fluorescence was excited by a HeNe 633 nm wavelength laser (intensity approx. 1.0 µW) and detected at 642–695 nm by photon-counting GaAsP detector. The pinhole was adjusted to 70 μm aperture. The raw signals of chlorophyll a fluorescence fluctuations were detected during 100 s in total (10 s × 10 times with almost continuous acquisition) for each measurement. The autocorrelation function was processed through a digital signal correlator card. For each condition under investigation (200 μM, 100 μM, or 25 μM of DDM, corresponding in these experiments to protein to detergent molar ratios of around 1:8400, 1:4200, and 1:1050, respectively; 10 mM HEPES (pH 7.3) or MES (pH 5.0)), we made 3 independent repetitions. All measurements were performed at room temperature.