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Systematic definition of protein constituents along the major polarization axis reveals an adaptive reuse of the polarization machinery in pheromone-treated budding yeast.

Narayanaswamy R, Moradi EK, Niu W, Hart GT, Davis M, McGary KL, Ellington AD, Marcotte EM - J. Proteome Res. (2009)

Bottom Line: We further trained a machine learning algorithm to refine the cell imaging screen, identifying additional shmoo-localized proteins.In all, we identified 74 proteins that specifically localize to the mating projection, including previously uncharacterized proteins (Ycr043c, Ydr348c, Yer071c, Ymr295c, and Yor304c-a) and known polarization complexes such as the exocyst.Functional analysis of these proteins, coupled with quantitative analysis of individual organelle movements during shmoo formation, suggests a model in which the basic machinery for cell polarization is generally conserved between processes forming the bud and the shmoo, with a distinct subset of proteins used only for shmoo formation.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems and Synthetic Biology, Departments of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712.

ABSTRACT
Polarizing cells extensively restructure cellular components in a spatially and temporally coupled manner along the major axis of cellular extension. Budding yeast are a useful model of polarized growth, helping to define many molecular components of this conserved process. Besides budding, yeast cells also differentiate upon treatment with pheromone from the opposite mating type, forming a mating projection (the 'shmoo') by directional restructuring of the cytoskeleton, localized vesicular transport and overall reorganization of the cytosol. To characterize the proteomic localization changes accompanying polarized growth, we developed and implemented a novel cell microarray-based imaging assay for measuring the spatial redistribution of a large fraction of the yeast proteome, and applied this assay to identify proteins localized along the mating projection following pheromone treatment. We further trained a machine learning algorithm to refine the cell imaging screen, identifying additional shmoo-localized proteins. In all, we identified 74 proteins that specifically localize to the mating projection, including previously uncharacterized proteins (Ycr043c, Ydr348c, Yer071c, Ymr295c, and Yor304c-a) and known polarization complexes such as the exocyst. Functional analysis of these proteins, coupled with quantitative analysis of individual organelle movements during shmoo formation, suggests a model in which the basic machinery for cell polarization is generally conserved between processes forming the bud and the shmoo, with a distinct subset of proteins used only for shmoo formation. The net effect is a defined ordering of major organelles along the polarization axis, with specific proteins implicated at the proximal growth tip.

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Protocol for quantifying organellar localization following pheromone treatment. Synthetic alpha factor was added to vegetatively growing cells containing GFP tagged Fus1 and one of nine different RFP tagged organellar marker proteins. Computational image analysis was used to measure the subcellular distribution of the tagged organellar marker as illustrated. (A) Image of shmoo tip marker Fus1 labeled with GFP, clathrin marker Chc1 labeled with RFP, cell nuclei stained with DAPI. (B) Binary thresholded image. (C) Binary image of nuclei used to seed watershed segmentation. (D) Result of watershed segmentation. (E) Shmooing cells were identified and cell-by-cell thresholding on the Fus1-GFP, Chc1-RFP, and nuclear (DAPI) images was overlaid on the DIC image. (F) Result of rotating and overlaying thresholded fluorescent intensity distributions for all shmooing cells (n = 324) from multiple still images, measuring the distribution of the RFP-tagged organellar protein (here, Chc1) throughout the observed cells and plotted relative to the shmoo tip and nuclear markers. In this case, clathrin (as localized by Chc1) shows a definite bias toward the shmoo tip and lies predominantly between the shmoo tip and nucleus.
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fig1: Protocol for quantifying organellar localization following pheromone treatment. Synthetic alpha factor was added to vegetatively growing cells containing GFP tagged Fus1 and one of nine different RFP tagged organellar marker proteins. Computational image analysis was used to measure the subcellular distribution of the tagged organellar marker as illustrated. (A) Image of shmoo tip marker Fus1 labeled with GFP, clathrin marker Chc1 labeled with RFP, cell nuclei stained with DAPI. (B) Binary thresholded image. (C) Binary image of nuclei used to seed watershed segmentation. (D) Result of watershed segmentation. (E) Shmooing cells were identified and cell-by-cell thresholding on the Fus1-GFP, Chc1-RFP, and nuclear (DAPI) images was overlaid on the DIC image. (F) Result of rotating and overlaying thresholded fluorescent intensity distributions for all shmooing cells (n = 324) from multiple still images, measuring the distribution of the RFP-tagged organellar protein (here, Chc1) throughout the observed cells and plotted relative to the shmoo tip and nuclear markers. In this case, clathrin (as localized by Chc1) shows a definite bias toward the shmoo tip and lies predominantly between the shmoo tip and nucleus.

Mentions: It has been previously observed that organelles move during the pheromone-induced, polarized growth of yeast.(18) To establish conditions for screening for genes involved in polarized growth, we used fluorescence microscopy to identify reproducible trends in organellar movements. We systematically measured the subcellular localization of 9 major organelles via organelle-specific fluorescent protein fusion markers. For each organelle, we constructed a yeast strain expressing both a red fluorescent protein (RFP)-tagged organelle marker, and an orientation marker, a green fluorescent protein (GFP)-tagged shmoo tip marker, Fus1. The positions of the two fluorescent protein markers and the nucleus (detected by staining with DAPI dye) were determined before and after treatment with the mating pheromone alpha factor. Image analysis was used to quantify each organelle’s spatial distribution across >100 individual cells in an image (Figure 1). Organellar positions were calculated after reorienting and scaling each individual cell in the image to a common reference frame (the axis from the nucleus to the shmoo tip).


Systematic definition of protein constituents along the major polarization axis reveals an adaptive reuse of the polarization machinery in pheromone-treated budding yeast.

Narayanaswamy R, Moradi EK, Niu W, Hart GT, Davis M, McGary KL, Ellington AD, Marcotte EM - J. Proteome Res. (2009)

Protocol for quantifying organellar localization following pheromone treatment. Synthetic alpha factor was added to vegetatively growing cells containing GFP tagged Fus1 and one of nine different RFP tagged organellar marker proteins. Computational image analysis was used to measure the subcellular distribution of the tagged organellar marker as illustrated. (A) Image of shmoo tip marker Fus1 labeled with GFP, clathrin marker Chc1 labeled with RFP, cell nuclei stained with DAPI. (B) Binary thresholded image. (C) Binary image of nuclei used to seed watershed segmentation. (D) Result of watershed segmentation. (E) Shmooing cells were identified and cell-by-cell thresholding on the Fus1-GFP, Chc1-RFP, and nuclear (DAPI) images was overlaid on the DIC image. (F) Result of rotating and overlaying thresholded fluorescent intensity distributions for all shmooing cells (n = 324) from multiple still images, measuring the distribution of the RFP-tagged organellar protein (here, Chc1) throughout the observed cells and plotted relative to the shmoo tip and nuclear markers. In this case, clathrin (as localized by Chc1) shows a definite bias toward the shmoo tip and lies predominantly between the shmoo tip and nucleus.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651748&req=5

fig1: Protocol for quantifying organellar localization following pheromone treatment. Synthetic alpha factor was added to vegetatively growing cells containing GFP tagged Fus1 and one of nine different RFP tagged organellar marker proteins. Computational image analysis was used to measure the subcellular distribution of the tagged organellar marker as illustrated. (A) Image of shmoo tip marker Fus1 labeled with GFP, clathrin marker Chc1 labeled with RFP, cell nuclei stained with DAPI. (B) Binary thresholded image. (C) Binary image of nuclei used to seed watershed segmentation. (D) Result of watershed segmentation. (E) Shmooing cells were identified and cell-by-cell thresholding on the Fus1-GFP, Chc1-RFP, and nuclear (DAPI) images was overlaid on the DIC image. (F) Result of rotating and overlaying thresholded fluorescent intensity distributions for all shmooing cells (n = 324) from multiple still images, measuring the distribution of the RFP-tagged organellar protein (here, Chc1) throughout the observed cells and plotted relative to the shmoo tip and nuclear markers. In this case, clathrin (as localized by Chc1) shows a definite bias toward the shmoo tip and lies predominantly between the shmoo tip and nucleus.
Mentions: It has been previously observed that organelles move during the pheromone-induced, polarized growth of yeast.(18) To establish conditions for screening for genes involved in polarized growth, we used fluorescence microscopy to identify reproducible trends in organellar movements. We systematically measured the subcellular localization of 9 major organelles via organelle-specific fluorescent protein fusion markers. For each organelle, we constructed a yeast strain expressing both a red fluorescent protein (RFP)-tagged organelle marker, and an orientation marker, a green fluorescent protein (GFP)-tagged shmoo tip marker, Fus1. The positions of the two fluorescent protein markers and the nucleus (detected by staining with DAPI dye) were determined before and after treatment with the mating pheromone alpha factor. Image analysis was used to quantify each organelle’s spatial distribution across >100 individual cells in an image (Figure 1). Organellar positions were calculated after reorienting and scaling each individual cell in the image to a common reference frame (the axis from the nucleus to the shmoo tip).

Bottom Line: We further trained a machine learning algorithm to refine the cell imaging screen, identifying additional shmoo-localized proteins.In all, we identified 74 proteins that specifically localize to the mating projection, including previously uncharacterized proteins (Ycr043c, Ydr348c, Yer071c, Ymr295c, and Yor304c-a) and known polarization complexes such as the exocyst.Functional analysis of these proteins, coupled with quantitative analysis of individual organelle movements during shmoo formation, suggests a model in which the basic machinery for cell polarization is generally conserved between processes forming the bud and the shmoo, with a distinct subset of proteins used only for shmoo formation.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems and Synthetic Biology, Departments of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712.

ABSTRACT
Polarizing cells extensively restructure cellular components in a spatially and temporally coupled manner along the major axis of cellular extension. Budding yeast are a useful model of polarized growth, helping to define many molecular components of this conserved process. Besides budding, yeast cells also differentiate upon treatment with pheromone from the opposite mating type, forming a mating projection (the 'shmoo') by directional restructuring of the cytoskeleton, localized vesicular transport and overall reorganization of the cytosol. To characterize the proteomic localization changes accompanying polarized growth, we developed and implemented a novel cell microarray-based imaging assay for measuring the spatial redistribution of a large fraction of the yeast proteome, and applied this assay to identify proteins localized along the mating projection following pheromone treatment. We further trained a machine learning algorithm to refine the cell imaging screen, identifying additional shmoo-localized proteins. In all, we identified 74 proteins that specifically localize to the mating projection, including previously uncharacterized proteins (Ycr043c, Ydr348c, Yer071c, Ymr295c, and Yor304c-a) and known polarization complexes such as the exocyst. Functional analysis of these proteins, coupled with quantitative analysis of individual organelle movements during shmoo formation, suggests a model in which the basic machinery for cell polarization is generally conserved between processes forming the bud and the shmoo, with a distinct subset of proteins used only for shmoo formation. The net effect is a defined ordering of major organelles along the polarization axis, with specific proteins implicated at the proximal growth tip.

Show MeSH
Related in: MedlinePlus