Csu10

Confocal DIC and fluorescent images were acquired on an AxioImager A1 microscope (Carl Zeiss) equipped with an EMCCD camera (Hamamatsu Photonics), a 100x or 40x Plan-Apochromat (1.4 NA) objective, and a spinning disc confocal scan head (CSU-10; Yokogawa Electric Corporation) driven by μManager software (Edelstein et al., 2010 (link)) at 20°C, except Figure 7D and E, which were acquired on a Leica DMI8 with an xLIGHT V3 confocal spinning disk head (89 North) with a 63x Plan-Apochromat (1.4 NA) objective and an ORCA‐Fusion Gen‐III sCMOS camera (Hamamatsu Photonics). Worms were mounted on 4% noble agar pads containing 0.01 M sodium azide for imaging during endpoint experiments. Images were processed with FIJI 2.0 and Photoshop CC (Adobe Systems Inc). Images are displayed as single confocal z-slices or maximum intensity projections generated in FIJI, as noted. Supplemental videos were generated with FIJI. Graphs generated by R and MS Excel were refined using Illustrator CC (Adobe Systems Inc).

Budding yeast was grown in standard media and then manipulated and transformed by standard methods [40 ]. GFP-Tub1 fusions were integrated into the genome and expressed ectopically, in addition to the native $\alpha$ -tubulin genes TUB1 and TUB3 [41 (link)]. We estimate that GFP-Tub1 comprises approximately $25\%$ of the total $\alpha$ -tubulin expressed in these cells [42 (link)]. Cells were grown asynchronously to the early log phase in a nonfluorescent medium and adhered to slide chambers coated with concanavalin A [43 ]. Images were collected on a Nikon Ti-E microscope equipped with a 1.45 NA 100× CFI Plan Apo objective, piezoelectric stage (Physik Instrumente; Auburn, MA, USA), spinning disk confocal scanner unit (CSU10; Yokogawa, Musashino, Tokyo), 488 nm laser (Agilent Technologies; Santa Clara, CA, USA), and an EMCCD camera (iXon Ultra 897; Andor Technology; Belfast, UK) using NIS Elements software (Nikon, Minato City, Tokyo). During imaging, sample temperature was maintained at $37{}^{°}$ C as indicated using the CherryTemp system (CherryBiotech; Rennes, France). Z-stacks consisting of 12 images separated by 0.45 µm were collected at 5 second intervals for 10 minutes. All analyses were conducted in pre-anaphase cells, which typically exhibit one or two individual astral microtubules extending from each SPB [44 (link)].

Intact isolated islets were incubated with Fura-8 AM (Invitrogen, UK) and incubated in KHB containing glucose (3–17 mmol/l) or KCl (30 mmol/l). Ca²⁺-dependent fluorescence was imaged using a Nipkow spinning disk head (Yokogawa CSU-10; Runcorn, UK).

Ca²⁺ imaging of whole islets was performed after loading with R-GECO (48 h post-isolation) in Krebs-Ringer bicarbonate buffer or cytosolic Cal-520 acetoxymethyl (AM; 2 μmol/l; 24 h post-isolation; Stratech, Cambridge, UK) in modified Krebs-Ringer bicarbonate buffer (140 mmol/l NaCl, 3.6 mmol/l KCl, 0.5 mmol/l NaH₂PO₄, 2 mmol/l NaHCO₃ [saturated with CO₂], 1.5 mmol/l CaCl₂, 0.5 mmol/l MgSO₄, 10 mmol/l HEPES; pH 7.4) containing 3 mmol/l or 17 mmol/l glucose, 17 mmol/l glucose with 0.1 mmol/l diazoxide (Sigma-Aldrich, Dorset, UK), or 20 mmol/l KCl. Images were captured at 0.5 Hz on a Zeiss Axiovert microscope equipped with a ×10 0.3–0.5 NA objective, a Hamamatsu image-EM camera coupled to a Nipkow spinning-disk head (Yokogawa CSU-10; Runcorn, UK) and illuminated at 490 nm or 530 nm. Data were analysed using ImageJ (https://imagej.nih.gov/ij/download.html, accessed 15 February 2020) with a purpose-designed macro (available upon request).