Figure S3.
SAS4 knockout, sas4Δ growth, and BB loss during G1 arrest at elevated temperature, and Mob1 localization. (A) Schematic of SAS4 knockout construct. The schematic shows that the cassette replaces the entire SAS4 locus with a neomycin resistance cassette. Bottom left panel, PCR of genomic DNA showing integration of the sas4Δ cassette at the SAS4 locus. Bottom right panel, RT-PCR from WT and sas4Δ cells showing loss of SAS4 transcript. ACT1 (actin) is a control for RNA quality. Numbering on molecular weight ladders is in base pairs. (B) Timeline shows mating timeline to induce SAS4 knockout. “0 h” is +17 h from time of mating initiation and is the time of drug and media addition in most experiments. “−9.5 h” is +7.5 h from mating initiation and is the time when most cells enter the second phase of macronuclear development, leading to the destruction of the parental macronucleus driving parental expression of SAS4 (Martindale et al., 1982). (C) Measurement of total cell Sas4 protein loss after SAS4 knockout. Protein levels were measured from at least six cells per time point. Error bars denote SEM. Representative images of two BBs at −17 h and two BBs at 0 h are shown at right. Enhanced scaling/contrast to allow for optimal visualization (left panels) shows that BBs can be identified at the cell cortex, while equal scaling between images (right panels) shows that the BBs at 0 h have drastically reduced Sas4 protein levels. (D) Representative cell growth curve of WT versus sas4Δ cells. Experiment was performed in triplicate. (E) WT and sas4Δ cells (BB, centrin, grayscale) in G1 arrest at 38°C for 24 h and 48 h after SAS4 knockout. Scale bar, 5 µm. (F) Chart showing the fate of 34 single hammerhead cells isolated at 24 h after SAS4 knockout and followed for an additional 24 h. (G) Image averaging of BBs from WT cells expressing Poc1:mCherry and GFP:Mob1. Each BB was centered on the centroid of the Poc1:mCherry signal and oriented relative to the BB just anterior in the ciliary row. Only BBs in the posterior half of the cell were used for analysis. Image is an average of 43 BBs. Note that Mob1 localizes strongly to BB-proximal regions of the striated fiber and distal regions of the transverse MTs, as well as to the BB itself. Scale bar, 1 µm. (H) Comparison of Sas4 and Mob1 average localization by overlay of averaged images from G and Fig. 1 C. BB alignment was determined using the centroid of either Sas6A or Poc1 signal. Merged image of Sas4 and Mob1 (magenta and green, respectively) shows that Mob1 localizes to BBs and striated fiber regions proximal to the BB, similar to where Sas4 localizes, and to distal regions of the transverse MTs. Sas4 also localizes to transverse MTs, but at a BB proximal location. (I) Images of GFP:Mob1 localization in nondividing WT, bld10Δ, and sas4Δ cells at 24 h post-knockout. Scale bar, 5 µm. Enlarged images show Mob1 localizes to the cell cortex in WT and bld10Δ cells but not in sas4Δ. Images are from the same experiments described in Fig. 4 F. Scale bar, 1 µm. (J) Left panels, DIC images of dividing WT and sas4Δ cells. Right panels, Mob1 localizes as a gradient to the posterior ends of both daughter cells in WT, but not in sas4Δ cells. Images are from the same experiments described in Fig. 4 F. Scale bar, 5 µm. (K) Cells at 6 h after BLD10 knockout at 30°C (left) or 37°C (right), stained for BBs (centrin, grayscale). Cells shifted to 37°C display greater BB loss. Scale bar, 5 µm.

SAS4 knockout, sas4Δ growth, and BB loss during G1 arrest at elevated temperature, and Mob1 localization. (A) Schematic of SAS4 knockout construct. The schematic shows that the cassette replaces the entire SAS4 locus with a neomycin resistance cassette. Bottom left panel, PCR of genomic DNA showing integration of the sas4Δ cassette at the SAS4 locus. Bottom right panel, RT-PCR from WT and sas4Δ cells showing loss of SAS4 transcript. ACT1 (actin) is a control for RNA quality. Numbering on molecular weight ladders is in base pairs. (B) Timeline shows mating timeline to induce SAS4 knockout. “0 h” is +17 h from time of mating initiation and is the time of drug and media addition in most experiments. “−9.5 h” is +7.5 h from mating initiation and is the time when most cells enter the second phase of macronuclear development, leading to the destruction of the parental macronucleus driving parental expression of SAS4 (Martindale et al., 1982). (C) Measurement of total cell Sas4 protein loss after SAS4 knockout. Protein levels were measured from at least six cells per time point. Error bars denote SEM. Representative images of two BBs at −17 h and two BBs at 0 h are shown at right. Enhanced scaling/contrast to allow for optimal visualization (left panels) shows that BBs can be identified at the cell cortex, while equal scaling between images (right panels) shows that the BBs at 0 h have drastically reduced Sas4 protein levels. (D) Representative cell growth curve of WT versus sas4Δ cells. Experiment was performed in triplicate. (E) WT and sas4Δ cells (BB, centrin, grayscale) in G1 arrest at 38°C for 24 h and 48 h after SAS4 knockout. Scale bar, 5 µm. (F) Chart showing the fate of 34 single hammerhead cells isolated at 24 h after SAS4 knockout and followed for an additional 24 h. (G) Image averaging of BBs from WT cells expressing Poc1:mCherry and GFP:Mob1. Each BB was centered on the centroid of the Poc1:mCherry signal and oriented relative to the BB just anterior in the ciliary row. Only BBs in the posterior half of the cell were used for analysis. Image is an average of 43 BBs. Note that Mob1 localizes strongly to BB-proximal regions of the striated fiber and distal regions of the transverse MTs, as well as to the BB itself. Scale bar, 1 µm. (H) Comparison of Sas4 and Mob1 average localization by overlay of averaged images from G and Fig. 1 C. BB alignment was determined using the centroid of either Sas6A or Poc1 signal. Merged image of Sas4 and Mob1 (magenta and green, respectively) shows that Mob1 localizes to BBs and striated fiber regions proximal to the BB, similar to where Sas4 localizes, and to distal regions of the transverse MTs. Sas4 also localizes to transverse MTs, but at a BB proximal location. (I) Images of GFP:Mob1 localization in nondividing WT, bld10Δ, and sas4Δ cells at 24 h post-knockout. Scale bar, 5 µm. Enlarged images show Mob1 localizes to the cell cortex in WT and bld10Δ cells but not in sas4Δ. Images are from the same experiments described in Fig. 4 F. Scale bar, 1 µm. (J) Left panels, DIC images of dividing WT and sas4Δ cells. Right panels, Mob1 localizes as a gradient to the posterior ends of both daughter cells in WT, but not in sas4Δ cells. Images are from the same experiments described in Fig. 4 F. Scale bar, 5 µm. (K) Cells at 6 h after BLD10 knockout at 30°C (left) or 37°C (right), stained for BBs (centrin, grayscale). Cells shifted to 37°C display greater BB loss. Scale bar, 5 µm.

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