Halo-Cnp1 Strain Construction and Fluorescence Labeling

The construction of the N-terminal Halo-cnp1 strain is described in Vojnovic (2016) . An overnight Halo-cnp1 YES culture was harvested by centrifugation, resuspended in fresh YES, and fixed with 3.7% PFA at RT for 10 min. Afterwards, the sample was washed 3× and residual PFA was quenched using 1× PEM (100 mM Pipes [#P1851-100G; Sigma-Aldrich], pH 6.9, 1 mM EGTA [#EDS-100G; Sigma-Aldrich], and 1 mM MgSO₄ [#M2643-500G; Sigma-Aldrich]) containing 50 mg/ml NH₄Cl (#P726.1; Carl Roth). Next, the sample was permeabilized using 1.25 mg/ml Zymolyase (#320921; MP Biomedicals) in 1× PEM at 37°C for 10 min and subsequently washed 3× with 1× PEM for 10 min. The sample was incubated using a few drops of Image-iT FX signal enhancer (#I36933; Invitrogen) at RT for 1 h to prevent non-specific staining and afterwards stained with 50 nM Halo-CF647 in PEMBAL buffer (1× PEM containing 3% BSA [#A8549-10MG; Sigma-Aldrich], 0.1% NaN₃ [#4221.1; Carl Roth], and 100 mM lysine hydrochloride [#L5626; Sigma-Aldrich]) at RT for 3 h. The stained cells were then washed four times for 10 min with alternating 1× PEM and 1× PBS and incubated at RT for 15 min on a previously washed and with poly-L-lysine–coated Ibidi 8-well glass bottom slide. Finally, the attached cells were washed twice with 1× PEM for 10 min.