All micrographs in the main text were acquired on a CSU-X spinning-disk confocal microscopy system (Yokogawa, Intelligent Imaging Innovations) with a DMI6000B microscope (Leica), 100 × 1.45 NA objective (Leica), and an Evolve 512Delta EMCCD (Photometrics) with a 2× magnifying lens (Yokogawa) for a final resolution of 0.084 µm/pixel. All images and videos shown are transverse, single-plane cross-sections, unless stated otherwise. For live-cell imaging of yeast, cells in mid–log phase were adhered to a glass-bottomed dish (CellVis) coated with concanavalin A (EY laboratories) and washed with the respective cell medium. Molecule counting for Rgd3 was performed by comparison to Cse4 puncta intensity at anaphase, as described previously (Donovan and Bretscher, 2012 (link)). Imaging at elevated temperatures was performed in an environmental chamber (Okolab) following 1-h incubations in a 37°C water bath, except for the tpm1-2 tpm2∆ experiment, which was performed in a CherryTemp chamber (Cherry Biotech) for rapid temperature shift. Images were analyzed and processed with Slidebook 6.0 software (Intelligent Imaging Innovations) or FIJI. Images and figures were assembled in Illustrator (Adobe).