Num1 interacts with PIK patches and is involved in PI(4)P homeostasis. (A) Scs2 is a novel component of MECA. A version of the MECA cartoon shown in Fig. 1 A depicting Scs2 as the molecular link between Num1 and the ER. (B) IP-MS pulldown analysis of Num1-GFP. Each dot represents one identified protein and proteins of interest are highlighted by various colors. The vertical dashed line is centered at 0 to enable easy visual comparison of enriched hits while the horizontal dashed line represents a significance threshold of P = 0.05. (C) A cartoon representation of phosphoinositide kinase (PIK) patches. PIK patches consist of Efr3, Ypp1, and Stt4 (the PI4Kinase), and catalyze the formation of PI(4)P (depicted as green lipids) from precursor PI (depicted as magenta lipids) on the PM. Efr3 interacts with Scs2 via a FFAT motif which helps control the localization of PIK patches to ER–PM contact sites. PI(4)P generated at the PM is presented to Sac1, the ER-localized PI(4)P phosphatase, via members of the Osh/ORP protein family and is converted back to PI. This cartoon was based on models and work presented in Omnus et al. (2020). (D) Num1 localizes adjacent to the PIK patch component Ypp1. Super resolution fluorescence micrographs of cells expressing Ypp1-GFP and either Num1-Halo or Num1∆FFAT-Halo. Individual channels are shown in grayscale. Images are single slices from the center of a cell. Yellow arrows point to Num1 foci that are adjacent to Ypp1 foci. Blue arrows point to Num1∆FFAT foci that are not adjacent to a Ypp1 focus. Scale bar, 2 µm. (E) Quantification of the percentage of Num1 or Num1∆FFAT foci that contain overlapping Ypp1 signal. The Ypp1 signal was thresholded so that only punctate fluorescence was visible and Num1 foci were manually scored for the presence or absence of Ypp1 signal. Each dot represents the percentage of Num1 or Num1∆FFAT foci containing Ypp1 signal per imaging replicate with each replicate containing at least 50 foci. The bars represent the mean and the error bars represent the SEM of the three replicates. To determine statistical significance, an unpaired t test was used (*** = P < 0.001). (F) Coimmunoprecipitation of Num1 with Efr3. Cells expressing genomically tagged Efr3-GFP with or without Num1-HA were lysed and subjected to affinity purification with an α-GFP antibody followed by SDS-PAGE and Western blot analysis. A representative blot of three independent experiments is shown. (G) Array of representative fluorescence micrographs showing the observed localization patterns for the PI(4)P biosensor GFP-PHOsh2. The Bud enriched + puncta image is from a wild type strain, the PM + puncta and Puncta images are from a num1∆ strain, and the PM image is from a sac1∆ strain. Images are single slices from the center of the cell. Dashed yellow lines indicate cell outlines. Scale bar, 2 µm. (H) Quantification of the percentage of cells in the indicated genetic backgrounds showing the GFP-PHOsh2 localization patterns depicted in G. Each portion of the stacked bar graph represents the average percentage of cells displaying the indicated GFP-PHOsh2 localization pattern of three biological replicates. Error bars represent SEM. Imaging replicates consisted of at least 38 cells for a total of at least 130 cells per condition. See Materials and methods for a complete description of the quantification methodology. To determine statistical significance, an ordinary one-way ANOVA with multiple comparisons was used (*** = P < 0.001, **** = P < 0.0001). All statistical analyses are comparing the Bud enriched + puncta category to the WT condition. (I) Quantification strategy to measure the fold enrichment of PI(4)P on the daughter cell PM compared to the mother cell PM. The image is a single slice from the center of a wild type cell expressing GFP-PHOsh2. The fluorescence channel is merged with a bright field image to show the cell boundaries. “Fd” and “Fm” refer to the measured GFP-PHOsh2 PM signal intensity in the daughter and mother cell, respectively. See Materials and methods for a complete description of the quantification methodology. Scale bar, 2 µm. (J) Quantification of the ratio of GFP-PHOsh2 enrichment in daughter cells compared to mother cells using the strategy depicted in I. Each dot represents the PM GFP-PHOsh2 ratio measured from a single cell. Imaging replicates are depicted as different colors and the average of each replicate is shown as a circle of the appropriate color with a black outline. Each replicate contains 20 cells for a total of 60 cells measured per condition. The horizontal line indicates the mean of the three imaging replicates with error bars representing SEM. To determine statistical significance, an ordinary one-way ANOVA with multiple comparisons was used (**** = P < 0.0001). All statistical analyses are in comparison to the WT condition. The dashed black line depicts the average GFP-PHOsh2 enrichment in WT cells. Source data are available for this figure: SourceData F5.
Figure 5.

Num1 interacts with PIK patches and is involved in PI(4)P homeostasis. (A) Scs2 is a novel component of MECA. A version of the MECA cartoon shown in Fig. 1 A depicting Scs2 as the molecular link between Num1 and the ER. (B) IP-MS pulldown analysis of Num1-GFP. Each dot represents one identified protein and proteins of interest are highlighted by various colors. The vertical dashed line is centered at 0 to enable easy visual comparison of enriched hits while the horizontal dashed line represents a significance threshold of P = 0.05. (C) A cartoon representation of phosphoinositide kinase (PIK) patches. PIK patches consist of Efr3, Ypp1, and Stt4 (the PI4Kinase), and catalyze the formation of PI(4)P (depicted as green lipids) from precursor PI (depicted as magenta lipids) on the PM. Efr3 interacts with Scs2 via a FFAT motif which helps control the localization of PIK patches to ER–PM contact sites. PI(4)P generated at the PM is presented to Sac1, the ER-localized PI(4)P phosphatase, via members of the Osh/ORP protein family and is converted back to PI. This cartoon was based on models and work presented in Omnus et al. (2020). (D) Num1 localizes adjacent to the PIK patch component Ypp1. Super resolution fluorescence micrographs of cells expressing Ypp1-GFP and either Num1-Halo or Num1∆FFAT-Halo. Individual channels are shown in grayscale. Images are single slices from the center of a cell. Yellow arrows point to Num1 foci that are adjacent to Ypp1 foci. Blue arrows point to Num1∆FFAT foci that are not adjacent to a Ypp1 focus. Scale bar, 2 µm. (E) Quantification of the percentage of Num1 or Num1∆FFAT foci that contain overlapping Ypp1 signal. The Ypp1 signal was thresholded so that only punctate fluorescence was visible and Num1 foci were manually scored for the presence or absence of Ypp1 signal. Each dot represents the percentage of Num1 or Num1∆FFAT foci containing Ypp1 signal per imaging replicate with each replicate containing at least 50 foci. The bars represent the mean and the error bars represent the SEM of the three replicates. To determine statistical significance, an unpaired t test was used (*** = P < 0.001). (F) Coimmunoprecipitation of Num1 with Efr3. Cells expressing genomically tagged Efr3-GFP with or without Num1-HA were lysed and subjected to affinity purification with an α-GFP antibody followed by SDS-PAGE and Western blot analysis. A representative blot of three independent experiments is shown. (G) Array of representative fluorescence micrographs showing the observed localization patterns for the PI(4)P biosensor GFP-PHOsh2. The Bud enriched + puncta image is from a wild type strain, the PM + puncta and Puncta images are from a num1∆ strain, and the PM image is from a sac1∆ strain. Images are single slices from the center of the cell. Dashed yellow lines indicate cell outlines. Scale bar, 2 µm. (H) Quantification of the percentage of cells in the indicated genetic backgrounds showing the GFP-PHOsh2 localization patterns depicted in G. Each portion of the stacked bar graph represents the average percentage of cells displaying the indicated GFP-PHOsh2 localization pattern of three biological replicates. Error bars represent SEM. Imaging replicates consisted of at least 38 cells for a total of at least 130 cells per condition. See Materials and methods for a complete description of the quantification methodology. To determine statistical significance, an ordinary one-way ANOVA with multiple comparisons was used (*** = P < 0.001, **** = P < 0.0001). All statistical analyses are comparing the Bud enriched + puncta category to the WT condition. (I) Quantification strategy to measure the fold enrichment of PI(4)P on the daughter cell PM compared to the mother cell PM. The image is a single slice from the center of a wild type cell expressing GFP-PHOsh2. The fluorescence channel is merged with a bright field image to show the cell boundaries. “Fd” and “Fm” refer to the measured GFP-PHOsh2 PM signal intensity in the daughter and mother cell, respectively. See Materials and methods for a complete description of the quantification methodology. Scale bar, 2 µm. (J) Quantification of the ratio of GFP-PHOsh2 enrichment in daughter cells compared to mother cells using the strategy depicted in I. Each dot represents the PM GFP-PHOsh2 ratio measured from a single cell. Imaging replicates are depicted as different colors and the average of each replicate is shown as a circle of the appropriate color with a black outline. Each replicate contains 20 cells for a total of 60 cells measured per condition. The horizontal line indicates the mean of the three imaging replicates with error bars representing SEM. To determine statistical significance, an ordinary one-way ANOVA with multiple comparisons was used (**** = P < 0.0001). All statistical analyses are in comparison to the WT condition. The dashed black line depicts the average GFP-PHOsh2 enrichment in WT cells. Source data are available for this figure: SourceData F5.

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