Light microscopy

2.4.1. Antibody coupling

2 mg of the respective antibody were mixed with 100 µl 1 M NaHCO3 and the respective fluorescent dye was dissolved in water free DMF to 10 µg/µl. 20 µl of dissolved dye were mixed with the antibody solution and incubated at RT for 1 h. Subsequently 100 µl 1 M Tris was added and the mixture stirred at RT for 5 min. Purification of dye-coupled antibodies was done using a PD-10 desalting column (GE Healthcare, Little Chalfort, UK). Peak fractions were pooled, aliquoted to 50 µl portions and flash frozen in liquid nitrogen for subsequent storage at -80°C.

33 2.4.2. Indirect immunofluorescence staining

Human cells were cultured on glass cover slips until they reached a confluence of about 70%

and fixed in 37°C prewarmed 4% (w/v) PFA (paraformaldehyde) in PBS at RT for 5 min. The cells were permeabilized using 0.5 % (v/v) Triton-X-100 in PBS for 5 min followed by subsequent incubation in blocking buffer (5% (w/v) BSA in PBS containing 100 mM glycin) for 5 min. Primary antibodies were diluted in blocking buffer and incubated with the coverslips at room temperature for 1 h. The following primary antibodies were used: rabbit anti-HMG-I (EPR7839; 1:400; Abcam), mouse anti-Vimentin (V9; 1:100; Santa Cruz Biotechnology), mouse anti-Zyxin (ZOL301, 1:400, Abcam); rabbit anti-PHB1 (EP2803Y, 1:200, Abcam), rabbit anti-PHB2 (EPR14523, 1:400, Abcam); mouse anti-ESR1 (D12, 1:500, Santa Cruz Biotechnology), chicken anti-GFP (1:1000, Abcam). After three washing steps in PBS, fluorophore-coupled secondary antibodies were diluted 1:1000 and added for incubation at room temperature for 1h. The following secondary antibodies were used: sheep anti-mouse, goat anti-rabbit or goat anti-chicken (all Dianova, Hamburg, Germany) coupled to KK114 (Kolmakov et al., 2010) or Atto 590 (Atto-Tec, Siegen, Germany). After three PBS washing steps, cells were embedded in Mowiol 4-88 mounting medium containing 1 µg/ml 4′,6 -Diamidin-2-phenylindol (DAPI) and 2.5 % (w/v) 1,4-diazabicyclo-[2,2,2]-octane (DABCO).

2.4.3. Widefield microscopy

Widefield fluorescence microscopy was done using an upright Leica DM6000 B epifluorescence microscope (Leica, Wetzlar, Germany). The microscope was equipped with a 100x oil immersion objective (HCX PL APO 100x/1.40-0.70 oil), a charge-coupled device (CCD) camera (DFC350FX) and various filter cubes: A4 (excitation: 360/40 nm; emission:

470/40 nm), L5 (excitation: 480/40 nm; emission: 527/30 nm), GFP (excitation: 470/40 nm;

emission: 525/50 nm), N3 (excitation: 546/12 nm; mission: 600/40 nm), BGR (excitation:

420/30 nm, 495/15 nm or 570/20 nm; emission: 465/20 nm, 530/30 nm or 640/40 nm) and SFR (excitation: 630/20 nm; emission: 667/30 nm). Light source was a metal-halide lamp (EL6000, Leica Microsystems).

2.4.4. Confocal microscopy

Confocal microscopy was done using the Leica TCS SP5 Confocal Microscope (Leica, Wetzlar, Germany). All recordings were done using a pinhole diameter of one Airy unit (1.22λ/NA), a scan speed of 400 Hz and a 63x oil immersion objective (HCX PL APO CS 63x/1.40-0.60 oil). The following laser lines were used for fluorescence excitation: a 405

2. Materials and Methods

Diode (405 nm), an argon laser (458 nm/ 476 nm/ 488 nm/ 496 nm/ 514 nm), a diode-pumped solid-state (DPSS) laser (561 nm) and a helium-neon (HeNe) laser (630 nm). Fluorescence detection was done using photomultipliers (PMTs) operated within the dynamic range.

Separation of excitation and emission light was accomplished using an AOTF (acousto-optic tunable filter). Multicolor imaging was done using sequential acquisition between frames. For image digitization a sampling rate according to the Nyquist criterion was chosen. Each image was recorded at least twice for averaging.

2.4.5. STED super-resolution microscopy

STED (stimulated emission depletion) super-resolution microscopy was done using an Abberior STED 775 QUAD scan nanoscope (Abberior Instruments, Göttingen, Germany). The nanoscope was equipped with a 100x oil immersion objective (Olympus UPlanSApo 100x/1.4). Fluorescence excitation was done using two pulsed laser sources at 594 nm 594 nm (Abberior Instruments, Göttingen, Germany) and 640 nm (Picoquant, Berlin, Germany).

Fluorescence depletion was achieved using a donut shaped pulsed laser at 775 nm leading to a lateral resolution of about 30 nm. Image acquisition was done in the sequential line-scanning mode. Image acquisition and processing was performed using the software ImSpector (Andreas Schönle, MPIbpc, Göttingen). Besides smoothing with a Gaussian filter and contrast stretching, no image processing was performed.

2.4.6. RESOLFT super-resolution microscopy

The home-built RESOLFT microscope utilized three separate beam paths for generating co-aligned focal spots: two at a wavelength of 491 nm for excitation and OFF-switching, and one at 405 nm for ON-switching. The two focal spots at 491 nm comprised: (i) a normally focused pulsed beam for reading out the fluorescence signal; (ii) a ‘doughnut-shaped’ focal intensity distribution with a central minimum (‘zero’) for OFF-switching at the focal periphery in the xy-plane, obtained by passing a continuous wave beam through a vortex phase mask (463nm mask, vortex plate VPP-A, RPC Photonics, Rochester, NY). The two focal intensity spots were generated by two different lasers diodes: one for OFF-switching (50 mW, continuous wave, Calypso 50, Cobolt, Stockholm, Sweden) and the second (10mW, 80-100ps pulse width PicoQuant, Berlin, Germany) for fluorescence readout. The third focal spot, again with a regularly focused profile, was generated by a laser diode at 405nm wavelength (30mW, BCL-030-405-S, CrystaLaser, Reno, NV, USA) and used for the ON-switching of the fluorescent protein. An oil-immersion objective lens (HCX PC APO, 100×, 1.4NA, oil; Leica Microsystems,

35 Wetzlar, Germany) was used to image the different cell lines. A piezo actuator ENV40/20 (Piezosystem Jena, Jena, Germany) was used to move the objective lens along the optical axis in a range of 120 µm. A separate piezo stage NV40 (Piezosystem Jena) was implemented to translate the sample with nanometer precision in the xy-plane. The fluorescence signal was filtered by a bandpass filter (532/70 nm) and detected by an epitaxial silicon single photon avalanche diode SPAD (MPD, Bolzano, Italy); fluorescence photons were counted only when the 491 nm pulse read-out beam was switched on. The individual laser beam paths were triggered either by an acousto-optic modulator MTS 130A3 (Pegasus Optik GmbH, Wallenhorst, Germany) or by an acousto-optic tunable filter AOTF.nC/TN (Pegasus Optik GmbH). The pulse sequence and duration were defined by a pulse generator Model 9514 (QUANTUM COMPOSERS, Bozeman, MT, USA) and triggered by a time-correlated single photon counting module (Becker & Hickl, Berlin, Germany) pixel by pixel.

Each image was recorded by applying a specific pulse scheme, pixel by pixel. For details on all shown images, see Table 12. All intensity values are referring to the light intensities in the focal plane. Image acquisition and processing was performed using the software ImSpector (Andreas Schönle, MPIbpc, Göttingen).

The RESOLFT super-resolution microscope was built and operated by Dr. Ilaria Testa (Department of NanoBiophotonics, MPI-BPC, Göttingen, Germany).