Replication Data for: Dynamic actin-mediated nano-scale clustering of CD44 regulates its meso-scale organization at the plasma membrane

<b>Plasmids, cell lines, and antibodies</b><br> CD44-GFP, CD44ECDTm-GFP, and CD44TmICD-GFP cloned in p-EGFP N1 vector were gifts from Rob Parton at the University of Queensland, Australia. CD44ΔERM-GFP, CD44ΔAnk-GFP, CD44ΔEA-GFP, and CD44Δ15-GFP constructs were generated by site-directed mutagenesis using CD44-GFP as the template in the same backbone. SNAP- and FR-tagged CD44 constructs were designed and cloned into a lentiviral pHR transfer backbone and cloned between MluI and BamHI/NotI sites using the Gibson Assembly method. All constructs were sequenced and verified using appropriate primers (Supplemental Table S1). SNAP CD59 GPI was obtained from Addgene (Addgene #50374). Sequences and primer sequences will be made available on request. Cell line-expressing FR-CD44TmICD and FR-CD44Tm were generated by transfecting and selecting transfected cells by staining for FR-expressing cells with anti-FR MOV19 antibody using fluorescence-assisted cell sorting. CHO cells were cultured in Ham’s F12 media (HiMedia, Mumbai, India); MCF-7, COS-7 (African green monkey kidney cells), and MEFs were cultured in DMEM high glucose (Gibco, 21720-024). The media was supplemented with fetal bovine serum (FBS) (Gibco, 16000044) and a cocktail of penicillin, streptomycin, and l-glutamine (Sigma; G1146-100 ml). MEFS, MCF-7, COS-7, or CHO cells were seeded sparsely and grown for 2 d on 35-mm cell culture dishes fitted with a glass bottom coverslip for imaging. Cells were transfected with the different CD44 plasmids, 12–16 h before imaging, using FuGENE 6 Transfection Reagent (E2692; Promega). <br><br>

<b>Antibody labeling and expression level estimation</b><br>
Endogenous and overexpressed CD44 on the cell surface in the different cell lines, plated on cover slip bottom 35-mm dishes, after 2 d of plating, were labeled using IM7 antibody (14-0441-82; eBioscience) on ice for 1 h followed by incubation with anti-Rat secondary antibody tagged to Alexa 633 (A21094; Life Technologies) on ice for 1 h. The antibodies were diluted in 10% FBS containing culture media (DMEM). The cells were washed and imaged in HEPES buffer and imaged using a 20× objective on a spinning-disk microscope. Mean intensity from ROIs drawn around cells was quantified using ImageJ. <br><br>
<b>Actomyosin perturbation</b><br>
Blebs were generated using 14 µM Jas (Thermo Fisher, Invitrogen; Cat. No. J7473) for 15 min. Formin perturbation was carried out using 10–25 µM SMI-FH2 (Calbiochem; Cat. No. S4826-5MG) for 15 min–1 h based on experimental requirement. Arp2/3 inhibition was carried out using 200 µM CK-666 (Sigma-Aldrich; Cat. No. SML0006 5MG) treatment for 3 h. Ezrin perturbation was carried out using the inhibitor NSC668394 purchased from EMD Millipore (Cat. No. 341216-10MG). Cells were treated with 25 µM of the drug for 1 h. Myosin II perturbation was carried out using a cocktail of ML-7 (Sigma-Aldrich; Cat. No. I2764) and Y27632 (Sigma-Aldrich; Cat. No. Y0503-1MG) or H1152 purchased from Tocris (Cat. No. 2414). Cells were treated with a cocktail of the ML-7 and Y27632/ H1152 at a final concentration of 20 µM of each for 1 h. Owing to the reversible nature of the drugs acting on the target, imaging was carried out in the presence of the drug except in the case of Jas treatment. All drug treatments were carried out in HEPES buffer saline containing 2 mg/ml glucose at 37°C for the indicated time periods. <br><br><b>
STORM sample preparation and imaging</b><br>
CHO cells were plated on an eight-well Lab-Tek #1 chamber slide system (Nunc) at a density of 30,000 cells/well. Cells were incubated at 37°C for 24 h. After incubation, the samples were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 20 min. After fixation, blocking solution (3% wt/vol bovine serum albumin in PBS) was applied for 30 min. Cells were labeled with rat-anti-mouse–anti-CD44 primary antibody (Clone KM114; BD Pharmingen #558739) at a concentration of 5 µg/ml for 1 h at room temperature. The corresponding secondary antibody (anti-rat) was tagged with Alexa Fluor 647 (Invitrogen) as a reporter and with Alexa Fluor 405 as an activator. The secondary antibody was incubated for 1 h at room temperature. Cells were stored in 1% PFA in PBS. The STORM buffer used was the same as that of Gómez-García et al. (2018): Glox solution (40 mg/ml Catalase [Sigma Aldrich], 0.5 mg/ml glucose oxidase, 10% glucose in PBS) and MEA 10 mM (Cysteamine MEA [Sigma-Aldrich; #30070-50G] in 360 mM Tris-HCl). The imaging for STORM on endogenous CD44 from top surface in CHO cells is from one experiment. <br>To study the nearest-neighbor distribution of clusters, we identif ied the clusters of localizations based on intensity (i.e., high density of localizations) and determined the position of the center of mass. With this information, we calculate the NND for the experimental set. For the simulations, we take the same identified clusters (keeping their size) and reshuffle them in space. We repeat this process many times (100×) to get more robust information on the simulated NND. <br><br><b>

Live cell imaging for fluorescence emission anisotropy and cartography experiments</b><br>
All live imaging were interchangeably carried out, based on requirement, in one of the following setups: 1) confocal spinning disk microscope (for imaging blebs in 3D) equipped with a Yokogawa CSU22 unit and 100×, 1.4NA Nikon oil objective; Andor technologies laser combiner emitting 488 and 561 nm wavelengths, amongst others; and Andor ixon+897 EMCCD cameras. Images were acquired using Andor iQ2 software. 2) Total internal reflection fluorescence (TIRF) microscope setup was equipped with Nikon Eclipse Ti body; a 100×, 1.45NA Nikon oil objective; photometrics Evolve EMCCD cameras; an Agilent laser combiner MCL400 (Agilent Technologies) whose 488, 561, and 640 nm excitation wavelengths were used as necessary; and µManager for image acquisition. 3) TIRF microscope setup was equipped with Nikon TE2000 body; a 100×, 1.49NA Nikon oil objective; EMCCD Cascade 512 cameras (Photometrics, Tuscon, AZ); a home-built laser combiner equipped with 488 and 561 nm lasers; and Metamorph/µManager for image acquisition. Wherever necessary, live imaging was performed in a temperature-controlled stage-top incubator chamber with immersion thermostat, ECO Silver, from Lauda Brinkmann.<br><br>

<b>Fluorescence emission anisotropy measurements</b><br>

We measure emission anisotropy of our protein of interest by labeling them with GFP or PLB, both of which are suitable for fluorescence anisotropy measurement to report on Homo-FRET (Sinnecker et al., 2005; Ghosh et al., 2012). Cells were treated with 50–100 µg/l cycloheximide in complete media for 2.5–3 h prior to imaging for anisotropy measurement of GFP-based constructs, in order to prevent signal from GFP from the ER/Golgi-based internal pool contaminating the fluorescence signal from the plasma membrane pool. This is in accordance with anisotropy measurements of GFPtagged membrane proteins conducted in the lab in the past (Sharma et al., 2004). Cells were imaged in HEPES buffer containing 2 mg/ml glucose on an inverted TIRF microscope using a polarized excitation light source. Emission was split into orthogonal polarization components using a polarization beam splitter and collected simultaneously by two EM CCD cameras to detect polarization of emitted f luorescence. Fluorescence emission anisotropy measurements were interchangeably carried out, based on requirements, in one of the dual camera-equipped imaging systems described before. Steady-state fluorescence emission anisotropy was calculated as elaborated in Ghosh et al. (2012).The absolute value of anisotropy is a function of the effective numerical aperture of the imaging system (Ghosh et al., 2012). Since the effective numerical aperture is determined by the combinatorial effect of individual lenses in the light path of the microscope system, the absolute anisotropy value of the same protein varied from one system to another. Also, since the different experiments reported here have been conducted over several years, absolute values of anisotropy for the same constructs would have varied based on the status of the optics in a given microscope system. Hence, the measurements typically contained an internal control for sensitivity of anisotropy change, which was generally a measurement of the extent of aniso tropy change between the wild-type CD44-GFP and CD44ECDTm-GFP (or CD44-TmICDGFP and CD44-Tm-GFP). <br><br><b>Fluorescence anisotropy image analysis </b><br>
Image analysis was carried out using using imaging software: ImageJ or Metamorph. Fluorescence emission anisotropy of GFP- and PLB-tagged proteins was calculated using images from the two cameras, which were individually background corrected, and the perpendicular image was additionally G-Factor corrected (Ghosh et al., 2012) to rectify effects of inherent polarization bias of the imaging system. Regions of interest (ROIs) of size 20 × 20 or 30 × 30 pixels were drawn to sample the cell membrane, and anisotropy values from these regions were obtained. Anisotropy maps were generated after aligning the images from the two cameras and calculating pixelwise anisotropy value as described in Ghosh et al. (2012) using a custom code written in MATLAB (MathWorks, Natick, MA). Code will be available on request. For data plotting, intensity was binned for appropriate intensity range, and each data point represents mean, and an error bar represents SD of anisotropy corresponding to the intensity bin. We ensured that data comparisons were done between conditions across similar intensity ranges. Intensity range chosen was decided based on different microscope properties, especially the bit depth and noise levels of the cameras. For representation calculated anisotropy values from the intensity images of the parallel and perpendicular cameras have been plotted on the y-axis as a function of the expression level, which is described as “Total intensity in arbitrary units” on the x-axis. Here, the total intensity is computed as a summation of the intensity recorded in the parallel image and two times the intensity recoded in the perpendicular image as described in Ghosh et al. (2012).