Polybead

For all observations, animals were mounted on 2% agarose dry pads with 4% polystyrene beads (Polybeads, Polysciences) in M9 buffer. For images corresponding to Figures 1a, 2 and 5, and Figure S1, worms were observed using an Andor spinning disk system (Oxford Instruments) installed on a Nikon‐IX86 microscope (Olympus) equipped with a 40x/NA 1.3 and a 60×/NA 1.42 oil‐immersion objectives and an Evolve electron‐multiplying charge‐coupled device camera. Each animal was imaged with IQ software (APIS Informationstechnologien) as a stack of optical sections (0.3 μm apart) across the whole thickness of the worm (Figure 1a and Figure S1a) or across a specific tissue (Figures 2 and 4). All images were processed using the Fiji (ImageJ) software and correspond to the sums of the same number of slices (for each strain with or without auxin) except for images of the intestine (Figure 2g and Figure S1h) which correspond to z projection of maximum intensity. For fluorescence quantification, images covering the whole head including the most posterior pharyngeal bulb were summed (85 slices) and a ROI (180 × 230 pixels) was positioned relative to the middle of the most posterior part of the pharynx.

First 1-μm polystyrene beads containing a reactive amine blue dye were purchased from a commercial source (polybeads; Polysciences, Warrington, PA). In pilot experiments, it was discovered that simple adsorption provided comparable adhesion compared to attempted carboxylate/phosphate coupling using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. For the reactions shown in Fig. 5, microspheres were diluted to 1/10th their stock concentration in CA buffer, and 200 μl were mixed with 1 ml of CAP dialysates, incubated overnight at room temperature, and then vortexed with an additional 1 ml of CA buffer prior to centrifugation. Subsequent dilutions in CA buffer were used to assess any influence of sample viscosity.

One-day-old adult hermaphrodites were mounted for imaging by placing a single worm on an agar pad (10% agarose dissolved in M9 buffer) on a microscope slide with 1.6 µL of a solution containing equal measures of polystyrene beads (Polybead, Cat# 00876-15, Polysciences Inc) and 30 mM muscimol (Cat# 195336, MP Biomedicals). Once the muscimol slowed worm movement (~5 min), a coverslip was dropped onto the agar pad, physically restraining the worm. The worm’s orientation was manually adjusted by sliding the coverslip to reorient the positioning of the AVA interneurons for imaging (Doser et al., 2023 (link)).