Cells and expression vectors

A variant of the human Burkitt lymphoma B-cell line Ramos that lacks expression of endogenous immunoglobulin heavy and light chains (RHLKO) was provided by Dr. Michael Reth (Freiburg, Germany). All cells were maintained in RPMI 1640 + GlutaMAX (Biochrom, Berlin, Germany) supplemented with 10% heat-inactivated FCS and antibiotics. All cDNAs encoding immunoglobulin heavy and light chains were synthesized by Eurofins Genomics (Ebersberg, Germany) and ligated into the retroviral expression vector pMSCVpuro (Clontech, Takara Bio Inc., Kyoto, Japan). Retroviral particles were produced using the packaging cell line Plat-E. The vesicular stomatitis virus (VSV) glycoprotein was used to pseudotype MMLV particles for human cell transduction. Transduced cells were selected in the presence of 2 µg/ml puromycin for 5‒7 days, followed by expression analysis via flow cytometry and Western blotting. When necessary, the transduced cells were sorted for purity using a BD FACSAria II.

Flow cytometry

The cell surface expression of the immunoglobulin chains was analyzed via flow cytometry using the following reagents: FluoTag®-X2 anti-ALFA AlexaFluor 647 (Nanotag Biotechnologies, Göttingen, Germany), Brilliant Violet 421™ anti-HA.11 Epitope tag antibody (BioLegend, San Diego, CA, USA) and NP-PE (Biosearch Technologies, Petaluma, CA, USA). For each sample, 1 × 106 cells were stained for 30 min on ice in the dark, followed by three wash steps. For the staining of intracellular proteins, the cells were fixed in Cytofix buffer (BD Biosciences) for 20 min on ice, followed by incubation in Perm/Wash Buffer I (BD) for another 20 min at 22 °C. Staining was performed in Perm/Wash buffer I using anti-phospho-CD79A-AF647 (Y182, Cell Signaling Technology, Danvers, MA, USA) and anti-phospho-Syk-APC (Y348, Invitrogen) antibodies. Finally, the cells were washed three times for 10 min each in Perm/Wash buffer I. All flow cytometry data were acquired on FACSCelesta or LSRII flow cytometers (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed via FlowJo 10.8 software. Imaging flow cytometry of TFEB was performed as described previously [18]. In brief, the cells were fixed with CytoFix buffer for 20 min at 4 °C, washed with PBS and resuspended in 200 μl of PBS containing 0.1% Triton X-100, 2% FCS and a primary antibody against human TFEB (clone D2O7D, CST, Danvers, MA, USA). After incubation at 4 °C for 30 min and washing with PBS, the cells were stained with an AlexaFluor-647-conjugated secondary anti-rabbit antibody (Abcam) and incubated at 4 °C for 30 min. After washing with PBS, the nuclei were stained with 50 µl of PBS containing 1 µg/ml DAPI (Thermo Fisher). For each sample, at least 2 × 104 single, focused cells (as determined via the area-to-aspect ratio and GradientRMS, respectively) were recorded via an ImageStreamX MkII imaging flow cytometer (Luminex). Apoptotic cells were excluded by means of their nuclear morphology (‘Apoptosis Wizard’ algorithm, DAPI staining). The nuclear localization of TFEB was assessed via the ‘Nuclear Translocation Wizard’ algorithm, which is based on channels 11 (TFEB/AF647) and 7 (nucleus/DAPI). The nucleus was defined via the mask ‘Dilate(Object(M05,Ch05,Tight),1)’. Events with a ‘SimilarityDilate’ colocalization score >1.0 were considered cells with nuclear localization of TFEB.

Measurement of intracellular free Ca2+

The day before the measurement, ~1 × 106 cells were seeded in a 10 cm culture dish. The next day, the cells were gently mixed for 30 min at 30 °C with 1 µM Indo-1-AM (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) in RPMI containing 10% fetal bovine serum and 0.015% Pluronic-F-127 (Invitrogen). The cells were subsequently washed twice and resuspended in Krebs-Ringer solution composed of 10 mM HEPES (pH 7.0), 140 mM NaCl, 10 mM glucose, 4 mM KCl, 1 mM MgCl2 and 1 mM CaCl2. After 30 min of rest prior to measurement, the fluorescence ratio of Ca2+-bound Indo-1 (405 nm) to Ca2+-unbound Indo-1 (530 nm) was monitored on a FACSCelesta cytometer. The basal Indo-1-AM ratio was monitored for 30 seconds, followed by stimulation with the indicated reagents. Goat anti-human F(ab′)2 fragments against human IgG or κ were obtained from Southern Biotech (Birmingham, AL, USA). NIP-16 and NIP-24-BSA were purchased from Biosearch Technologies (Petaluma, CA, USA). NIP-containing peptides were synthesized by CASLO ApS (Lyngby, Denmark) and had the sequences (Biotin)-KSK(NIP)GESKG (NIP1-peptide) and (Biotin)-KSK(NIP)GESK(NIP)G (NIP2-peptide), respectively. Anti-human CD8 (clone MEM-31) was a gift from Dr. Vaclav Horejsi (Prague, Czech Republic). Purified streptavidin was obtained from Jackson ImmunoResearch (West Grove, PA, USA). The data were analyzed via FlowJo (FlowJo LLC, Ashland, OR, USA), Microsoft Excel and GraphPad Prism.

Antigen endocytosis assay

To analyze the internalization of BCR-bound antigens, NIP-specific B cells were incubated either with biotinylated NIP peptides (10 ng/ml) or with biotinylated NIP16-BSA (500 ng/ml) in PBS for 10 min on ice to allow for antigen binding. The cells were subsequently pelleted and washed with PBS. The cell solutions were split into 6 tubes, each of which was incubated at 37 °C for the indicated period of time (unstimulated cells (time point 0) were kept on ice). The cells were subsequently stained with streptavidin-APC (BD Biosciences) for 10 min on ice, followed by another wash step with ice-cold PBS. Finally, the cells were analyzed on an LSRII flow cytometer. The MFIs of unstimulated cells were set to represent 100% bound antigen, and all other MFIs were normalized accordingly.

Biochemical assays, antibodies and reagents

Preparation of cellular lysates in 1% NP40-containing lysis buffer for affinity purification and western blot analyses was performed as previously described [9]. In brief, the cells were lysed with lysis buffer composed of 50 mM Tris-HCl (pH 7.8), 137 mM NaCl, 0.5 mM EDTA, 1 mM sodium orthovanadate, 10% (v/v) glycerol, 1% (v/v) NP40 and a protease inhibitor cocktail containing AEBSF, Aprotinin, Bestatin, E-64, Leupeptin and EDTA (Sigma Aldrich, #P2714). Lysis was performed on ice for 10 min, and the cell debris was pelleted at 20,000 × g at 4 °C for 10 min. The supernatant containing the cell lysate was mixed with reducing SDS‒PAGE sample buffer, boiled at 95 °C for 5 min and analyzed by SDS‒PAGE and immunoblotting. For affinity purification of proteins from B cells, cell lysates were prepared as described above from 3 × 107 Ramos cells and incubated with either 2 µg of monoclonal antibody 4G10 (Merck Millipore) or 2 µg of biotinylated anti-ALFA sdAb (Nanotag, Göttingen, Germany). Purification was performed via either Protein A/G PLUS agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or streptavidin-Sepharose (GE Healthcare, Chicago, IL, USA) under gentle rotation at 4 °C for 2 h. All affinity-purified samples were washed three times with lysis buffer before they were boiled in Laemmli buffer at 95 °C for 5 min and subjected to SDS‒PAGE and immunoblotting. The phospho-tyrosine antibody (clone 100) was from Cell Signaling Technology (Danvers, MA, USA), and the monoclonal anti-Syk antibody (clone 4D10) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The immunoblot images were processed via Photoshop CS4 and Corel Draw software. The band intensities were quantified with LabImage 1D software (Kapelan Bio-Imaging, Leipzig, Germany).

Generation of plasma membrane sheets

As a quality control measure, prior to each round of membrane sheet generation, the cell viability and response to antigen stimulation were assessed via Ca2+ mobilization assays, as described in the methods section. The plasma membrane sheets were prepared via a combination of methods described by Gomes de Castro et al. [27] and Erlendsson et al. [56], with a few modifications. The newly developed technique is illustrated in Supplementary Fig. S9. Briefly, ~3 × 106 cells per condition were freshly taken from the cell culture, transferred to a low-bind 1.5 ml tube (Sigma‒Aldrich, cat. no. Z666548-250EA), pelleted at 300 × g for 5 min, and washed once in PBS (Sigma, cat. no. D8537) at room temperature (RT). The cells were subsequently pelleted again and resuspended in Krebs-Ringer solution (120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 25 mM NaHCO3, and 5.5 mM D-glucose). Cell stimulation was performed at this step in Krebs-Ringer solution by adding the indicated antigens. The cells were immediately fixed either 90 s or 180 s after the addition of ligand by adding an equal volume of fixation solution (8% paraformaldehyde, 0.2% glutaraldehyde in PBS; i.e., final concentrations were 4% paraformaldehyde, 0.1% glutaraldehyde) and incubating at RT for 30 min. The fixation solution was removed after centrifugation, and the cells were washed three times with quenching buffer (0.1 M glycine in PBS). For staining, the cells were incubated with 10 nM FluoTag®-Q AbberiorStar635P anti-ALFA (NanoTag Biotechnologies, Göttingen, Germany; cat. no. N1502-Ab635P-L) for 1 h at RT, followed by three washing steps with PBS. After staining, the cells were postfixed with 4% paraformaldehyde and 0.1% glutaraldehyde in PBS for 15 min at RT, followed by three rounds of 5-minute washing steps with quenching buffer. The cells were then stained with CellBrite™ Green membrane dye (Biotium Inc., Freemont, CA, USA, cat. no. 30021) diluted 1:500 in PBS for 15 min at RT and washed three times with PBS. The cells were transferred onto poly-L-lysine (PLL)-coated coverslips (ø 18 mm) placed in 12-well plates (Thermo Scientific™, cat. no. 150200) and centrifuged at 300 × g for 5 min at RT to allow the cells to sediment onto the coverslips. A second clean coverslip was placed on top of the cells, creating a coverslip-cell-coverslip “sandwich”. A flat plunger from a 10 ml syringe was used to gently put pressure onto the coverslip while the top coverslip was rotated clockwise, thus causing cell disruption and flattening of the cell membranes on the coverslips. Finally, the coverslips were mounted in Mowiol mounting media (12 ml 0.2 M Tris buffer pH 8.5, 6 ml ddH2O, 6 g glycerol, 2.4 g Mowiol, Carl Roth, cat. no. 0713.2).

STED imaging

Images were acquired via an inverted Abberior STED Expert line microscope (Abberior Instruments, Göttingen, Germany) equipped with a UPLSAPO 100×1.4 NA oil immersion objective (Olympus). To ensure comparability of the data for quantitative analyses, the microscope settings were kept constant throughout all the experiments. The excitation lasers at 488 nm and 640 nm were set at a power of 1 μW, and the 775 nm depletion laser was set at 5 mW, which was measured at the sample. The laser power was measured via a microscope slide power sensor with a power and energy meter interface (S170C, Thorlabs; PM100USB, Thorlabs). The imaging settings were as follows: the pixel size was set to 20 nm with a dwell time of 10 μs and signal accumulation over 2 lines. Both confocal and STED images were collected from the 640 nm channel, whereas only confocal images were acquired from the 488 nm channel.

Membrane sheet image analyses

All data analyses were performed via a custom-written GUI and scripts in MATLAB 2017a. Peak detection: For each acquired STED image \(a\), regions of interest (ROIs) were manually selected to contain intact membrane patches and exclude high-intensity contamination (giving a binary mask \(m\)). To remove-of-focus signals and membrane autofluorescence, the images were high-pass filtered by subtracting a Gaussian-filtered version of the image excluding regions outside the selected ROIs:

$$_}}=a\cdot m-\frac_\left(a\cdot m\right)}_\left(m\right)},$$

where \(_\) is a 2D Gaussian filter with standard deviation σ = 8 pixels. This method constrains the filter inside the masked area and prevents high-intensity signals outside of the mask from influencing the subtracted background. In the resulting high-pass images \(_}}\), local intensity maxima above a noise threshold were detected with subpixel accuracy, generating an initial list of \(N\) peak center coordinates \(\left(_,\,_\right)\).

Peak amplitude estimation by two-pass sparse deconvolution: Due to possible overlaps between nearby peaks, each pixel intensity inside the mask was modeled as a sum of intensity contributions from individual peak Gaussian point spread functions (PSFs):

$$_}}(x,y) = _^\frac_\left(}}}\left(\frac_-x+1/2}\right)-}}}\left(\frac_-x-1/2}\right)\right)\\ \cdot \left(}}}\left(\frac_-y+1/2}\right)-}}}\left(\frac_-y-1/2}\right)\right),$$

where \(_\) represents the amplitude of an individual peak, \(s\) represents the Gaussian PSF standard deviation, which is linearly related to the PSF’s full width at half maximum (FWHM), and erf() represents the error function used to calculate the pixel intensity integral. All valid pixel positions \((x,y)\) thus form an overdetermined system of linear equations with \(N\) unknown amplitudes, solved by least-squares fit. Note that the PSF is assumed to be constant across all images.

As the overlap of multiple close-proximity peaks could make some of them nondetectable by the initial local maximum algorithm, a second pass of the above method was applied by first subtracting the modeled image \(_}}\) from \(_}}\) (revealing unaccounted interpeak intensity), performing local maximum detection again on the difference image and fitting the amplitudes a second time using the original \(_}}\) but with the combined set of peak candidates from the first and second passes.

Estimation of the monomer amplitude: Assuming a linear relationship between the total peak amplitude \(A\) and the number of underlying labeled receptors r, \(A=_\), the monomer amplitude \(_\) was fit for the peak amplitudes \(_\) by minimizing the cost function

$$}}\left(_\right)=_^_\left(\left|_-r\cdot _\right|\right)$$

represents the sum of the distances of all measured amplitudes to their respective nearest multiple monomer amplitudes (choosing from a total of \(\) possible receptor numbers).

Estimation of receptor number distribution: The number of receptors associated with each peak was estimated probabilistically. With the fitted monomer amplitude \(_\), the peak amplitude \(_\) was attributed to all receptor numbers \(r=1\) to \(R\) according to a Gaussian density \(_\) centered at the expected value \(r\cdot _\) and a standard deviation equal to half of the monomer intensity quanta \(_\):

$$_=\exp \left(-_-r\cdot _\right|}_/2}\right)}^\right).$$

The overall receptor number distribution \(_\) is then formed by the sum of these weights, normalized per peak:

$$_=\frac_^\frac_}_^_}.$$

To display the relative contribution of individual receptor numbers to the total fluorescence amplitude, the receptor number distribution was then simply multiplied by its respective amplitude:

$$_^}}=_\cdot r\cdot _.$$