Live cell microscopy

For TIRF imaging, we used an Eclipse Ti-U microscope (Nikon) equipped with 60× NA1.49 TIRF objective and through-the-objective TIRF illumination provided by a 40 mW 488 nm and a 75 mW 561 nm diode laser (Spectraphysics) as previously described (Lechtreck, 2013; Lechtreck, 2016). The excitation lasers were cleaned up with a Nikon GFP/mCherry TIRF filter cube and the emission was separated using an Image Splitting Device (Photometrics DualView2 with filter cube 11-EM) supplemented with an et595/33m filter to reduced chlorophyll autofluorescence and a meniscus lens to adjust for focus. Images were recorded at 10 fps using an iXON3 (Andor) and the NIS-Elements Advanced Research software (Nikon). The optical set-up for the focused laser beam used for FRAP analysis was previously described by Wingfield et al., 2017. FIJI (National Institutes of Health) was used to generate kymograms using either the build-in Multi Kymogram tool or the KymoResliceWide plugin (https://imagej.net/KymoResliceWide). The Plot Profile tool was used to analyze signal intensity and Microsoft Excel was used for statistical analysis. Adobe Photoshop was used to adjust image contrast and brightness, and figures were prepared in Adobe Illustrator.

Observation chambers for Chlamydomonas reinhardtii were constructed by applying a ring of vacuum grease or petroleum jelly to a 24 × 60 mm² No. 1.5 coverslip; 10 μl of cell suspension were applied and allowed to settle for ~1 min. Then, the chamber was closed by inverting a 22 × 22 mm² No. 1.5 cover glass with ~5–10 μl of 5 mM HEPES, pH 7.3 supplemented with 3–5 mM EGTA onto the larger cover glass. Cells were imaged through the large cover glass at room temperature. Regenerating cells were obtained as follows: Cells in fresh M medium were deflagellated by a pH shock, sedimented, resuspended in a small volume of M medium, and stored on ice for 15 min or until needed. Then, cells were diluted with M medium and agitated in bright light at room temperature. Aliquots were analyzed by TIRF microscopy at various time points. To generate long-short cells, cells in M medium were chilled on ice for ~3 min and then passed repeatedly (5–8×) through a 26 G½ needle using a 1 ml syringe (Ludington et al., 2012). The presence of spinning cells, indicative or the loss of just one flagellum, was verified by microscopy and the cells we allowed to regenerate for 5–10 min prior to mounting for TIRF microscopy.

To determine the swimming velocity, cells in fresh M medium were placed in a chambered plastic slide (Fisherbrand, 14-377-259) and observed using an inverted cell culture microscope (Nikon,TMS). Using a constant exposure time of 1 s, images were taken using a MU500 camera (Amscope) and Topview software. The length of the trajectories resulting from the cells’ movements was analyzed using ImageJ and converted into μm/s.