Limits...
Synthetic morphology using alternative inputs.

Tanaka H, Yi TM - PLoS ONE (2009)

Bottom Line: Following up this lead, we demonstrated that AI-Ste4p and AI-Ste5p were sufficient to produce multiple projections when combined.Further, we showed that overexpression of a membrane-targeted form of Ste5p alone could also induce multiple projections.Thus, we successfully re-engineered the multiple projections mating morphology using alternative inputs without alpha-factor.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Cell Biology, University of California Irvine, Irvine, California, USA.

ABSTRACT
Designing the shape and size of a cell is an interesting challenge for synthetic biology. Prolonged exposure to the mating pheromone alpha-factor induces an unusual morphology in yeast cells: multiple mating projections. The goal of this work was to reproduce the multiple projections phenotype in the absence of alpha-factor using a gain-of-function approach termed "Alternative Inputs (AIs)". An alternative input is defined as any genetic manipulation that can activate the signaling pathway instead of the natural input. Interestingly, none of the alternative inputs were sufficient to produce multiple projections although some produced a single projection. Then, we extended our search by creating all combinations of alternative inputs and deletions that were summarized in an AIs-Deletions matrix. We found a genetic manipulation (AI-Ste5p ste2Delta) that enhanced the formation of multiple projections. Following up this lead, we demonstrated that AI-Ste4p and AI-Ste5p were sufficient to produce multiple projections when combined. Further, we showed that overexpression of a membrane-targeted form of Ste5p alone could also induce multiple projections. Thus, we successfully re-engineered the multiple projections mating morphology using alternative inputs without alpha-factor.

Show MeSH

Related in: MedlinePlus

Two different outputs produced by alternative inputs to α-factor.(A) Transcriptional activation induced by alternative inputs. Either α-factor (1 µM) or the alternative inputs were added and transcriptional activation was measured at an early time point (t = 4 hours, white bars) and a late time point (t = 24 hours, black bars) using the PFUS1-GFP reporter. GFP fluorescence (arbitrary units) was normalized by dividing by the cell density (OD600 units). The control was cells unstimulated by α-factor or an alternative input. PFUS1-GFP/OD600 values were averaged from at least three measurements, and bar graphs show mean±SEM. (B) Morphologies induced by α-factor (1 µM) or alternative inputs. Bright field images taken at t = 24 h of a typical set of cells for each AI. The morphologies of AI-Ste2p (PGAL1-STE2P258L S259L) and AI-Fus3p (PGAL1-FUS3I161L) are not shown; they resembled the control cells. The scale bar represents 10 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2735001&req=5

pone-0006946-g002: Two different outputs produced by alternative inputs to α-factor.(A) Transcriptional activation induced by alternative inputs. Either α-factor (1 µM) or the alternative inputs were added and transcriptional activation was measured at an early time point (t = 4 hours, white bars) and a late time point (t = 24 hours, black bars) using the PFUS1-GFP reporter. GFP fluorescence (arbitrary units) was normalized by dividing by the cell density (OD600 units). The control was cells unstimulated by α-factor or an alternative input. PFUS1-GFP/OD600 values were averaged from at least three measurements, and bar graphs show mean±SEM. (B) Morphologies induced by α-factor (1 µM) or alternative inputs. Bright field images taken at t = 24 h of a typical set of cells for each AI. The morphologies of AI-Ste2p (PGAL1-STE2P258L S259L) and AI-Fus3p (PGAL1-FUS3I161L) are not shown; they resembled the control cells. The scale bar represents 10 µm.

Mentions: A natural stimulus activates signaling molecules in a pathway resulting in an output response. We define any genetic manipulation (i.e. overexpressing wild-type or constituitively active forms) that can activate the signaling pathway in lieu of the natural input as “Alternative Inputs or (AIs)”. Here, we set the goal to induce the natural output using alternative inputs. In this study, we constructed alternative inputs to the yeast mating pheromone α-factor in the pathway leading from α-factor to the transcription of pheromone-inducible genes (Figure 1A). The signaling proteins were overexpressed from the PGAL1 promoter on a multi-copy 2μ plasmid. After inducing expression of the alternative input with galactose, we monitored two different outputs, transcriptional activation of the reporter PFUS1-GFP and cell morphology, at an early (4 hours) and a late time point (24 hours) (Figure 2). We quantified transcription in terms of GFP fluorescence per unit of cell density (PFUS1-GFP/OD600).


Synthetic morphology using alternative inputs.

Tanaka H, Yi TM - PLoS ONE (2009)

Two different outputs produced by alternative inputs to α-factor.(A) Transcriptional activation induced by alternative inputs. Either α-factor (1 µM) or the alternative inputs were added and transcriptional activation was measured at an early time point (t = 4 hours, white bars) and a late time point (t = 24 hours, black bars) using the PFUS1-GFP reporter. GFP fluorescence (arbitrary units) was normalized by dividing by the cell density (OD600 units). The control was cells unstimulated by α-factor or an alternative input. PFUS1-GFP/OD600 values were averaged from at least three measurements, and bar graphs show mean±SEM. (B) Morphologies induced by α-factor (1 µM) or alternative inputs. Bright field images taken at t = 24 h of a typical set of cells for each AI. The morphologies of AI-Ste2p (PGAL1-STE2P258L S259L) and AI-Fus3p (PGAL1-FUS3I161L) are not shown; they resembled the control cells. The scale bar represents 10 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2735001&req=5

pone-0006946-g002: Two different outputs produced by alternative inputs to α-factor.(A) Transcriptional activation induced by alternative inputs. Either α-factor (1 µM) or the alternative inputs were added and transcriptional activation was measured at an early time point (t = 4 hours, white bars) and a late time point (t = 24 hours, black bars) using the PFUS1-GFP reporter. GFP fluorescence (arbitrary units) was normalized by dividing by the cell density (OD600 units). The control was cells unstimulated by α-factor or an alternative input. PFUS1-GFP/OD600 values were averaged from at least three measurements, and bar graphs show mean±SEM. (B) Morphologies induced by α-factor (1 µM) or alternative inputs. Bright field images taken at t = 24 h of a typical set of cells for each AI. The morphologies of AI-Ste2p (PGAL1-STE2P258L S259L) and AI-Fus3p (PGAL1-FUS3I161L) are not shown; they resembled the control cells. The scale bar represents 10 µm.
Mentions: A natural stimulus activates signaling molecules in a pathway resulting in an output response. We define any genetic manipulation (i.e. overexpressing wild-type or constituitively active forms) that can activate the signaling pathway in lieu of the natural input as “Alternative Inputs or (AIs)”. Here, we set the goal to induce the natural output using alternative inputs. In this study, we constructed alternative inputs to the yeast mating pheromone α-factor in the pathway leading from α-factor to the transcription of pheromone-inducible genes (Figure 1A). The signaling proteins were overexpressed from the PGAL1 promoter on a multi-copy 2μ plasmid. After inducing expression of the alternative input with galactose, we monitored two different outputs, transcriptional activation of the reporter PFUS1-GFP and cell morphology, at an early (4 hours) and a late time point (24 hours) (Figure 2). We quantified transcription in terms of GFP fluorescence per unit of cell density (PFUS1-GFP/OD600).

Bottom Line: Following up this lead, we demonstrated that AI-Ste4p and AI-Ste5p were sufficient to produce multiple projections when combined.Further, we showed that overexpression of a membrane-targeted form of Ste5p alone could also induce multiple projections.Thus, we successfully re-engineered the multiple projections mating morphology using alternative inputs without alpha-factor.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Cell Biology, University of California Irvine, Irvine, California, USA.

ABSTRACT
Designing the shape and size of a cell is an interesting challenge for synthetic biology. Prolonged exposure to the mating pheromone alpha-factor induces an unusual morphology in yeast cells: multiple mating projections. The goal of this work was to reproduce the multiple projections phenotype in the absence of alpha-factor using a gain-of-function approach termed "Alternative Inputs (AIs)". An alternative input is defined as any genetic manipulation that can activate the signaling pathway instead of the natural input. Interestingly, none of the alternative inputs were sufficient to produce multiple projections although some produced a single projection. Then, we extended our search by creating all combinations of alternative inputs and deletions that were summarized in an AIs-Deletions matrix. We found a genetic manipulation (AI-Ste5p ste2Delta) that enhanced the formation of multiple projections. Following up this lead, we demonstrated that AI-Ste4p and AI-Ste5p were sufficient to produce multiple projections when combined. Further, we showed that overexpression of a membrane-targeted form of Ste5p alone could also induce multiple projections. Thus, we successfully re-engineered the multiple projections mating morphology using alternative inputs without alpha-factor.

Show MeSH
Related in: MedlinePlus