Figure 2. Pom1 clusters are stable structures that can be isolated in vitro.

(A) TIRF microscopy image of Pom1-mNG clusters in the extruded cytoplasm (white dotted line) of a lysed cell (yellow dotted line). Scale bar 5 µm. (B) TIRF microscopy images of cell extracts prepared from wild-type (no tag) or Pom1-mNG cells. Images are 50 frame sum projections of continuous 200 ms time-lapse exposures. The two images were contrasted equally. Scale bars 1 µm. (C) Cytoplasmic extracts of pom1-3HA cells were subjected to velocity sucrose gradient sedimentation, and fractions were probed against the HA tag (upper blot). Fraction one corresponds to the top of the gradient and contains smaller structures; fraction nine corresponds to bottom of the gradient and contains larger structures. Fractions 6–8 were pooled, sucrose was removed by dialysis, and then the sample was subjected to a second identical round of sucrose gradient sedimentation and western blotting of the resulting fractions (lower blot). (D) TIRF microscopy of Pom1-mNG clusters from cytoplasmic extracts on supported lipid bilayers. Scale bar 1 µm. Left panel is single time point image. Right panel is kymograph taken from a line scan of time-lapse TIRF experiment. (E) Quantification of binding duration of Pom1-mNG clusters on supported lipid bilayers imaged by TIRF microscopy as in panel (D). Values are compared to cellular measurements of Pom1 clusters on cell sides (n.s., p=0.05954, n = 713 in vivo, 421 in vitro). (F) Quantification of life-time displacement of Pom1-mNG clusters diffusing on supported lipid bilayers imaged by TIRF microscopy as in panel (D). Values are compared to cellular measurements of Pom1 clusters on cell sides (****p<0.0001, n = 713 in vivo, 421 in vitro). For (E) and (F), statistical significance was tested using a Student’s T-test. (G) Purified GST-Pom1 was subjected to sucrose gradient sedimentation and the fractions were probed against the GST tag (upper blot). Purified GST-Pom1 was also added to wild-type or pom1∆ cell extracts and incubated for 1 hr at 4 ˚C in the presence of ATP before velocity sucrose sedimentation and western blotting (bottom blots).

Figure 2—figure supplement 1. Controls and supporting in vitro analysis of Pom1 clusters.

(A) Fluorescence intensity trace of a single Pom1-mNG cluster binding to the cell cortex, measured by high speed TIRF microscopy (~50 frames per second with continuous acquisition). Rapid intensity changes indicating binding and unbinding are marked by green upward and magenta downward pointing arrows respectively. (B) FRAP analysis of supported lipid bilayers. Upper panel: A circular ROI (teal dashed line) was used to bleach fluorescent PE in the bilayers. Lower panel: Kymograph at white dashed line in upper panel of fluorescence recovery as monitored by TIRF microscopy, indicating fluidity. (C) Quantification and comparison of binding duration of either wild-type or kinase-dead Pom1-mNG clusters to supported lipid bilayers in TIRF microscopy movies (***p=<0.0001, n = 1526 wild-type, 1993 kinase-dead). Statistical significance was tested with a Student’s T-test. (D) TIRF microscopy images of cell extracts prepared from cells co-expressing Pom1-mNG and Cdr2-tagRFP-t. Pom1 clusters are shown in green and Cdr2 nodes are shown in magenta. Images are three frame sum projections of continuous 200 ms time-lapse exposures. Scale bar 1 µm.

Figure 2—figure supplement 2. Quantification of Pom1 sedimentation in velocity sucrose gradients and size standards.

(A) Quantification of Pom1-3HA band intensities from western blot of yeast extracts in main (Figure 2C). (B) Size standards for velocity sucrose gradients, as in main (Figure 2C,G). Data are quantified from single Coomassie-stained gels or using spectrophotometry data. (C) Plot of S-value versus sedimentation peak. Line is the linear regression of the three size standards, with the S-value for Pom1 peaks interpolated.

Figure 2—figure supplement 3. Analysis of Pom1 cluster diffusion on supported lipid bilayers.

(A) Pom1-mNG clusters diffusing on supported lipid bilayers imaged by high-speed (100 ms acquisition) TIRF microscopy. (B) Single-particle diffusion traces of Pom1-mNG clusters on supported lipid bilayers. (C) Mean squared displacement (MSD) curves calculated from traces of individual Pom1-mNG cluster diffusion as in panel (B). (D) Mean MSD curves generated from averaging of 421 single-particle traces. Weighted linear regression of the curve (red line) provides the diffusion coefficient D = 0.168 ± 0.018 µm²/s. Note that the weighted regression best fits the initial region of the curve because few clusters bind longer than 10 s. Black line represents weighted mean of all traces, gray lines indicate standard deviation, and error bars represent weighted standard deviation.