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Spontaneous Cdc42 polarization independent of GDI-mediated extraction and actin-based trafficking.

Bendezú FO, Vincenzetti V, Vavylonis D, Wyss R, Vogel H, Martin SG - PLoS Biol. (2015)

Bottom Line: We show that Cdc42 is highly mobile at the membrane and accumulates at sites of activity, where it displays slower mobility.By contrast, a near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity.We propose that Cdc42 activation, enhanced by positive feedback, leads to its local accumulation by capture of fast-diffusing inactive molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland.

ABSTRACT
The small Rho-family GTPase Cdc42 is critical for cell polarization and polarizes spontaneously in absence of upstream spatial cues. Spontaneous polarization is thought to require dynamic Cdc42 recycling through Guanine nucleotide Dissociation Inhibitor (GDI)-mediated membrane extraction and vesicle trafficking. Here, we describe a functional fluorescent Cdc42 allele in fission yeast, which demonstrates Cdc42 dynamics and polarization independent of these pathways. Furthermore, an engineered Cdc42 allele targeted to the membrane independently of these recycling pathways by an amphipathic helix is viable and polarizes spontaneously to multiple sites in fission and budding yeasts. We show that Cdc42 is highly mobile at the membrane and accumulates at sites of activity, where it displays slower mobility. By contrast, a near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity. We propose that Cdc42 activation, enhanced by positive feedback, leads to its local accumulation by capture of fast-diffusing inactive molecules.

No MeSH data available.


Related in: MedlinePlus

Construction and functional testing of fluorescently tagged cdc42 sandwich alleles.(A) Structure of Cdc42 (left) and sandwich fusion (right). Note the site of green fluorescent protein (GFP) insertion at Q134 (with spacer sequences in purple) is distant from N and C termini, nucleotide binding pocket (nt) and Switch region. (B) Complementation of cdc42-1625 temperature sensitivity with pREP41-based plasmids grown on Edinburgh minimal medium (EMM). (C) Tetrad dissection of wt/cdc42-mCherrySW:kanMX diploids on YE (top) and subsequent replica plate on YE G418 (bottom). (D) 10-fold serial dilutions of indicated strains on yeast extract (YE) medium. The top three strains are prototroph, the bottom two strains are double auxotroph for leucine and uracil. (E) Growth curve of cells grown at 30°C in EMM with appropriate supplements. The top three strains are prototroph, the bottom two strains are double auxotroph for leucine and uracil. (F) Calcofluor images of cells grown to log phase at indicated temperatures. (G) Medial spinning disk confocal section of Cdc42-mCherrySW in wild type (wt; left) and following depletion of exocyst component Sec8 (right). Note localization to nuclear membrane (small arrowhead), division site (wide arrow) and enrichment at cell poles (arrows). Arrowheads (right) show sub-apical accumulation of Cdc42-mCherrySW. Bars = 5 μm.
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pbio.1002097.g001: Construction and functional testing of fluorescently tagged cdc42 sandwich alleles.(A) Structure of Cdc42 (left) and sandwich fusion (right). Note the site of green fluorescent protein (GFP) insertion at Q134 (with spacer sequences in purple) is distant from N and C termini, nucleotide binding pocket (nt) and Switch region. (B) Complementation of cdc42-1625 temperature sensitivity with pREP41-based plasmids grown on Edinburgh minimal medium (EMM). (C) Tetrad dissection of wt/cdc42-mCherrySW:kanMX diploids on YE (top) and subsequent replica plate on YE G418 (bottom). (D) 10-fold serial dilutions of indicated strains on yeast extract (YE) medium. The top three strains are prototroph, the bottom two strains are double auxotroph for leucine and uracil. (E) Growth curve of cells grown at 30°C in EMM with appropriate supplements. The top three strains are prototroph, the bottom two strains are double auxotroph for leucine and uracil. (F) Calcofluor images of cells grown to log phase at indicated temperatures. (G) Medial spinning disk confocal section of Cdc42-mCherrySW in wild type (wt; left) and following depletion of exocyst component Sec8 (right). Note localization to nuclear membrane (small arrowhead), division site (wide arrow) and enrichment at cell poles (arrows). Arrowheads (right) show sub-apical accumulation of Cdc42-mCherrySW. Bars = 5 μm.

Mentions: To better understand the mechanisms underlying Cdc42 dynamics in live cells, we set out to construct a functional fluorescent fusion protein in fission yeast. Existing fusions in various organisms fuse fluorescent proteins to Cdc42 N-terminus, as the C-terminus is subject to post-translational modification by prenylation (Fig. 1A). However, when this has been tested by gene replacement, these N-terminal fusions compromise Cdc42’s functions, yielding temperature-sensitivity and failure to associate with post-Golgi vesicles in S. cerevisiae [18,23,26] or altered cell morphology in Schizosaccharomyces pombe [27]. Indeed, the GFP-cdc42 strain displayed slow growth and aberrant morphology at all tested temperatures (see Fig. 1D–F). We chose the alternate approach of placing a fluorescent protein gene within the cdc42 reading frame, a strategy previously tried with success on other proteins [36,37].


Spontaneous Cdc42 polarization independent of GDI-mediated extraction and actin-based trafficking.

Bendezú FO, Vincenzetti V, Vavylonis D, Wyss R, Vogel H, Martin SG - PLoS Biol. (2015)

Construction and functional testing of fluorescently tagged cdc42 sandwich alleles.(A) Structure of Cdc42 (left) and sandwich fusion (right). Note the site of green fluorescent protein (GFP) insertion at Q134 (with spacer sequences in purple) is distant from N and C termini, nucleotide binding pocket (nt) and Switch region. (B) Complementation of cdc42-1625 temperature sensitivity with pREP41-based plasmids grown on Edinburgh minimal medium (EMM). (C) Tetrad dissection of wt/cdc42-mCherrySW:kanMX diploids on YE (top) and subsequent replica plate on YE G418 (bottom). (D) 10-fold serial dilutions of indicated strains on yeast extract (YE) medium. The top three strains are prototroph, the bottom two strains are double auxotroph for leucine and uracil. (E) Growth curve of cells grown at 30°C in EMM with appropriate supplements. The top three strains are prototroph, the bottom two strains are double auxotroph for leucine and uracil. (F) Calcofluor images of cells grown to log phase at indicated temperatures. (G) Medial spinning disk confocal section of Cdc42-mCherrySW in wild type (wt; left) and following depletion of exocyst component Sec8 (right). Note localization to nuclear membrane (small arrowhead), division site (wide arrow) and enrichment at cell poles (arrows). Arrowheads (right) show sub-apical accumulation of Cdc42-mCherrySW. Bars = 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4383620&req=5

pbio.1002097.g001: Construction and functional testing of fluorescently tagged cdc42 sandwich alleles.(A) Structure of Cdc42 (left) and sandwich fusion (right). Note the site of green fluorescent protein (GFP) insertion at Q134 (with spacer sequences in purple) is distant from N and C termini, nucleotide binding pocket (nt) and Switch region. (B) Complementation of cdc42-1625 temperature sensitivity with pREP41-based plasmids grown on Edinburgh minimal medium (EMM). (C) Tetrad dissection of wt/cdc42-mCherrySW:kanMX diploids on YE (top) and subsequent replica plate on YE G418 (bottom). (D) 10-fold serial dilutions of indicated strains on yeast extract (YE) medium. The top three strains are prototroph, the bottom two strains are double auxotroph for leucine and uracil. (E) Growth curve of cells grown at 30°C in EMM with appropriate supplements. The top three strains are prototroph, the bottom two strains are double auxotroph for leucine and uracil. (F) Calcofluor images of cells grown to log phase at indicated temperatures. (G) Medial spinning disk confocal section of Cdc42-mCherrySW in wild type (wt; left) and following depletion of exocyst component Sec8 (right). Note localization to nuclear membrane (small arrowhead), division site (wide arrow) and enrichment at cell poles (arrows). Arrowheads (right) show sub-apical accumulation of Cdc42-mCherrySW. Bars = 5 μm.
Mentions: To better understand the mechanisms underlying Cdc42 dynamics in live cells, we set out to construct a functional fluorescent fusion protein in fission yeast. Existing fusions in various organisms fuse fluorescent proteins to Cdc42 N-terminus, as the C-terminus is subject to post-translational modification by prenylation (Fig. 1A). However, when this has been tested by gene replacement, these N-terminal fusions compromise Cdc42’s functions, yielding temperature-sensitivity and failure to associate with post-Golgi vesicles in S. cerevisiae [18,23,26] or altered cell morphology in Schizosaccharomyces pombe [27]. Indeed, the GFP-cdc42 strain displayed slow growth and aberrant morphology at all tested temperatures (see Fig. 1D–F). We chose the alternate approach of placing a fluorescent protein gene within the cdc42 reading frame, a strategy previously tried with success on other proteins [36,37].

Bottom Line: We show that Cdc42 is highly mobile at the membrane and accumulates at sites of activity, where it displays slower mobility.By contrast, a near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity.We propose that Cdc42 activation, enhanced by positive feedback, leads to its local accumulation by capture of fast-diffusing inactive molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland.

ABSTRACT
The small Rho-family GTPase Cdc42 is critical for cell polarization and polarizes spontaneously in absence of upstream spatial cues. Spontaneous polarization is thought to require dynamic Cdc42 recycling through Guanine nucleotide Dissociation Inhibitor (GDI)-mediated membrane extraction and vesicle trafficking. Here, we describe a functional fluorescent Cdc42 allele in fission yeast, which demonstrates Cdc42 dynamics and polarization independent of these pathways. Furthermore, an engineered Cdc42 allele targeted to the membrane independently of these recycling pathways by an amphipathic helix is viable and polarizes spontaneously to multiple sites in fission and budding yeasts. We show that Cdc42 is highly mobile at the membrane and accumulates at sites of activity, where it displays slower mobility. By contrast, a near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity. We propose that Cdc42 activation, enhanced by positive feedback, leads to its local accumulation by capture of fast-diffusing inactive molecules.

No MeSH data available.


Related in: MedlinePlus