Snap cell tmr star

SNAPf–dynein complexes bound to IgG Sepharose 6 beads were incubated with approximately 5 μM SNAP-Cell TMR-Star or approximately 5 μM SNAP-Surface Alexa Fluor 647 (New England Biolabs) at 4°C for 40 min. Prior to TEV cleavage, excess dye was washed away with TEV buffer. Following purification of dynein complexes, labelling efficiency was determined using a Nanodrop 1000 spectrophotometer (Nanodrop Technologies) and shown to be 87–97% per dynein monomer for TMR and Alexa Fluor 647, respectively (equating to a labelling efficiency of 98.5 or 100% per dimeric dynein complex).

Samples (1 mL) were withdrawn from cultures in SMB at selected times during sporulation. Cells were collected by centrifugation (10,000 × g for 3 min), resuspended in 200 µL of phosphate saline buffer (PBS) and labeled by incubation with SNAP-cell TMR-star (New England Biolabs) for 30 min at 37 °C in the dark at a final concentration of 250 nM. This TMR-star-probed suspension was centrifuged (12,000 × g, 1 min), washed with 1 mL of PBS, suspended again in 1 mL of PBS and labeled with Mitotracker Green (MTG, Thermofischer) and/or FM4-64 Fx (Thermo Fisher Scientific) at a final concentration of 1 µg/mL or in 100 nM MV405 for simultaneous visualization of YFP protein (Supplementary Fig. S1B) for 1 min at room temperature. Cells were then washed three times in PBS and suspended in 50–200 µL PBS, depending on the concentration of sporulating cells/spores. For diffraction-limited as well as super-resolved microscopy, 3 µL of the labeled cells suspension were applied onto 1.7% agarose in PBS-coated glass slides. All experiments were done at RT. Samples were imaged with a BX-61 (Olympus), Elyra PS.1 or Elyra 7 AxioObserver (Zeiss) microscope.