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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.

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Multiple projections induced by membrane targeting of Ste5p.(A) Transcriptional activation induced by Ste5p-CTM. Ste5p (AI-Ste5p, black) and Ste5p-CTM (white) were overexpressed in a wild-type strain and the seven deletion strains of the mating pathway. Transcriptional activation was measured at t = 24 h. PFUS1-GFP/OD600 values were averaged from at least three measurements, and bar graphs show mean±SEM. (B) Numbers of projections produced by AI-Ste4p+AI-Ste5p and Ste5p-CTM cells in a wild-type background and Ste5p-CTM cells in ste2Δ, ste4Δ, and ste5Δ strains. For each of the inputs and strain backgrounds, we determined the percent of cells with 0 or 1, 2, 3, and 4 or greater projections (t = 24 h, at least 100 responding cells). (C) Correlation between transcriptional activation and numbers of projections in Ste5p strains. Transcriptional activation (PFUS1-GFP/OD600 values) and numbers of multiple projections (“+++” indicates WT levels of projections, “++” indicates more projections than AI-Ste5p (indicated as “+”) but fewer projections than WT) were summarized for each genetic manipulation with Ste5p that produced multiple projections. (D) Time-course of number of projections produced by Ste5p-CTM. For each time point, we determined the percent of cells with 0 or 1, 2, 3, and 4 or greater projections (t = 8, 16, 24 h, at least 100 responding cells). At t = 0 h, we observed more than 400 cells, and responding cells were less than 1%.
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pone-0006946-g007: Multiple projections induced by membrane targeting of Ste5p.(A) Transcriptional activation induced by Ste5p-CTM. Ste5p (AI-Ste5p, black) and Ste5p-CTM (white) were overexpressed in a wild-type strain and the seven deletion strains of the mating pathway. Transcriptional activation was measured at t = 24 h. PFUS1-GFP/OD600 values were averaged from at least three measurements, and bar graphs show mean±SEM. (B) Numbers of projections produced by AI-Ste4p+AI-Ste5p and Ste5p-CTM cells in a wild-type background and Ste5p-CTM cells in ste2Δ, ste4Δ, and ste5Δ strains. For each of the inputs and strain backgrounds, we determined the percent of cells with 0 or 1, 2, 3, and 4 or greater projections (t = 24 h, at least 100 responding cells). (C) Correlation between transcriptional activation and numbers of projections in Ste5p strains. Transcriptional activation (PFUS1-GFP/OD600 values) and numbers of multiple projections (“+++” indicates WT levels of projections, “++” indicates more projections than AI-Ste5p (indicated as “+”) but fewer projections than WT) were summarized for each genetic manipulation with Ste5p that produced multiple projections. (D) Time-course of number of projections produced by Ste5p-CTM. For each time point, we determined the percent of cells with 0 or 1, 2, 3, and 4 or greater projections (t = 8, 16, 24 h, at least 100 responding cells). At t = 0 h, we observed more than 400 cells, and responding cells were less than 1%.

Mentions: Ste4p recruits Ste5p to the plasma membrane in response to α-factor, and forced membrane targeting of Ste5p using a C-terminal membrane tag (Ste5p-CTM) activates the MAPK cascade without Ste4p [3]. We hypothesized that in the (AI-Ste4p+AI-Ste5p) cells Ste4p performed the role of recruiting Ste5p to the plasma membrane. To test this hypothesis, we overexpressed Ste5p-CTM instead of Ste5p. Indeed, Ste5p-CTM enhanced the transcriptional response and produced more second projections even in the complete absence of Ste4p (Figures 7A and 7B).


Synthetic morphology using alternative inputs.

Tanaka H, Yi TM - PLoS ONE (2009)

Multiple projections induced by membrane targeting of Ste5p.(A) Transcriptional activation induced by Ste5p-CTM. Ste5p (AI-Ste5p, black) and Ste5p-CTM (white) were overexpressed in a wild-type strain and the seven deletion strains of the mating pathway. Transcriptional activation was measured at t = 24 h. PFUS1-GFP/OD600 values were averaged from at least three measurements, and bar graphs show mean±SEM. (B) Numbers of projections produced by AI-Ste4p+AI-Ste5p and Ste5p-CTM cells in a wild-type background and Ste5p-CTM cells in ste2Δ, ste4Δ, and ste5Δ strains. For each of the inputs and strain backgrounds, we determined the percent of cells with 0 or 1, 2, 3, and 4 or greater projections (t = 24 h, at least 100 responding cells). (C) Correlation between transcriptional activation and numbers of projections in Ste5p strains. Transcriptional activation (PFUS1-GFP/OD600 values) and numbers of multiple projections (“+++” indicates WT levels of projections, “++” indicates more projections than AI-Ste5p (indicated as “+”) but fewer projections than WT) were summarized for each genetic manipulation with Ste5p that produced multiple projections. (D) Time-course of number of projections produced by Ste5p-CTM. For each time point, we determined the percent of cells with 0 or 1, 2, 3, and 4 or greater projections (t = 8, 16, 24 h, at least 100 responding cells). At t = 0 h, we observed more than 400 cells, and responding cells were less than 1%.
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Related In: Results  -  Collection

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pone-0006946-g007: Multiple projections induced by membrane targeting of Ste5p.(A) Transcriptional activation induced by Ste5p-CTM. Ste5p (AI-Ste5p, black) and Ste5p-CTM (white) were overexpressed in a wild-type strain and the seven deletion strains of the mating pathway. Transcriptional activation was measured at t = 24 h. PFUS1-GFP/OD600 values were averaged from at least three measurements, and bar graphs show mean±SEM. (B) Numbers of projections produced by AI-Ste4p+AI-Ste5p and Ste5p-CTM cells in a wild-type background and Ste5p-CTM cells in ste2Δ, ste4Δ, and ste5Δ strains. For each of the inputs and strain backgrounds, we determined the percent of cells with 0 or 1, 2, 3, and 4 or greater projections (t = 24 h, at least 100 responding cells). (C) Correlation between transcriptional activation and numbers of projections in Ste5p strains. Transcriptional activation (PFUS1-GFP/OD600 values) and numbers of multiple projections (“+++” indicates WT levels of projections, “++” indicates more projections than AI-Ste5p (indicated as “+”) but fewer projections than WT) were summarized for each genetic manipulation with Ste5p that produced multiple projections. (D) Time-course of number of projections produced by Ste5p-CTM. For each time point, we determined the percent of cells with 0 or 1, 2, 3, and 4 or greater projections (t = 8, 16, 24 h, at least 100 responding cells). At t = 0 h, we observed more than 400 cells, and responding cells were less than 1%.
Mentions: Ste4p recruits Ste5p to the plasma membrane in response to α-factor, and forced membrane targeting of Ste5p using a C-terminal membrane tag (Ste5p-CTM) activates the MAPK cascade without Ste4p [3]. We hypothesized that in the (AI-Ste4p+AI-Ste5p) cells Ste4p performed the role of recruiting Ste5p to the plasma membrane. To test this hypothesis, we overexpressed Ste5p-CTM instead of Ste5p. Indeed, Ste5p-CTM enhanced the transcriptional response and produced more second projections even in the complete absence of Ste4p (Figures 7A and 7B).

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