Goldseal

Samples are placed in optical chambers for microscopy experiments. The chamber is made from a microscope slide and a cover slip (Goldseal, Fisher Scientific) separated by a spacer consisting of melted Parafilm and sealed using ultraviolet–cured glue (Norland Optical). Glass surfaces are thoroughly cleaned with a hot 0.5% Hellmanex solution (Hellma Analytics) and coated with acrylamide brushes which prevent binding of the viruses to the glass surface61 .

Samples were stained with 1% Toluidine Blue (Fishersci.com) before being mounted on microslides (Gold‐Seal, Fishersci.com). The number of single cells and clusters of 2–4, 5–10 and >10 cells were counted from micrographs obtained using an Olympus BX43 microscope with an Olympus DP26 camera. Proportions of each cluster were determined from greater than 1,000 cells from 20 micrographs from 3 replicate experiments for each genotype and treatment.
For electron microscopy, untreated and treated samples were infiltrated with Embed‐812 epoxy resin (Electron Microscopy Sciences, Hatfield, PA) for 24 h, spun down into a pellet in a 2‐mL vial and polymerized at 70 °C overnight. Semi‐thin Toluidine Blue‐stained sections were mounted on glass slides for light microscopy (500 nm thickness), then ultrathin sections (200 nm and 100 nm) were collected on 100‐mesh formvar‐coated copper grids, stained with 4% aqueous uranyl acetate, rinsed in water, and finally stained with lead citrate and rinsed. Images were captured with a four megapixel Gatan UltraScan 1000 camera (Gatan, Pleasanton, CA) on an FEI Tecnai G2 20 Twin 200 kV LaB6 TEM (FEI, Hillsboro, OR).

Plain 75 × 25 mm glass microscope slides (VWR) and 22 × 22 mm glass coverslips (Gold Seal, ThermoFisher) were cleaned by boiling in 1% Hellmanex III (Hellma Analytics) in MilliQ water. The glass was then sonicated for 30 min at 37 °C. After washing thoroughly in MilliQ water, the slides were dried and stored at room temperature in a sealed and desiccated container.
A recording chamber was prepared by laying thin strips of Kapton Tape (DuPont) onto a pre-cleaned microscope slide, then placing and sealing the glass coverslip on top by applying pressure along the tape lines. This created several channels with a height of ~1 mm. Each channel was flushed with 5 mg mL⁻¹ Roche Blocking reagent dissolved in PBS and allowed to sit in a humidified chamber for 10–30 min at room temperature in order to block the glass surfaces and prevent bead sticking. The channel was then flushed with 10–20 chamber volumes of TBS-TB + Ca²⁺ buffer. Each chamber was used immediately after blocking and washing.
1 µL each of the coated beads containing either PCDH15 or CDH23 protein-DNA tethers was diluted 1:100 in TBS-TB + Ca²⁺ buffer and injected into the recording chamber. The chamber was then hermetically sealed with vacuum grease and locked onto the microscope stage of the instrument. For each condition, measurements were taken from experimental and sample replicates.