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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

Enrichment of GTP-Cdc42 at sites of polarity.(A) Cdc42-mCherrySW and CRIB-3GFP localization. Traces for cortical measurements are shown on the right. (B) Average profiles of fluorescence intensity along cortical traces with standard deviation (left) and after normalization to the maximum and minimum intensity values (right). (C) Representative Cdc42-mCherrySW and CRIB-3GFP images at cell poles in wt and indicated mutant cells. (D) Average profiles of cells shown in C. (E) Plot of Cdc42 versus CRIB fluorescence at the tip showing that the enrichment of total Cdc42 correlates with its activity. n ≥ 40 for each profile or data point. Bars = 2 μm.
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pbio.1002097.g002: Enrichment of GTP-Cdc42 at sites of polarity.(A) Cdc42-mCherrySW and CRIB-3GFP localization. Traces for cortical measurements are shown on the right. (B) Average profiles of fluorescence intensity along cortical traces with standard deviation (left) and after normalization to the maximum and minimum intensity values (right). (C) Representative Cdc42-mCherrySW and CRIB-3GFP images at cell poles in wt and indicated mutant cells. (D) Average profiles of cells shown in C. (E) Plot of Cdc42 versus CRIB fluorescence at the tip showing that the enrichment of total Cdc42 correlates with its activity. n ≥ 40 for each profile or data point. Bars = 2 μm.

Mentions: To quantitatively define the distribution and activity of Cdc42 at the plasma membrane, we co-imaged Cdc42-mCherrySW with CRIB-3GFP, a probe that selectively binds active Cdc42-GTP (CRIB stands for Cdc42- and Rac-interactive binding domain) [43]. The distribution of CRIB closely mirrored that of Cdc42 enrichment at cell poles (Fig. 2A). At cell poles with strong CRIB localization, Cdc42 was enriched 3-fold (± 0.7, n = 40) over its levels at cell side (Fig. 2B). At cell poles with low CRIB levels, these correlated with low Cdc42 enrichment (S2 Fig). Normalization of the Cdc42 and CRIB distribution profiles to their maximum and minimum yielded overlapping curves with identical decay rates, suggesting a very tight correlation between enrichment of Cdc42 and the active form (Fig. 2B right).


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)

Enrichment of GTP-Cdc42 at sites of polarity.(A) Cdc42-mCherrySW and CRIB-3GFP localization. Traces for cortical measurements are shown on the right. (B) Average profiles of fluorescence intensity along cortical traces with standard deviation (left) and after normalization to the maximum and minimum intensity values (right). (C) Representative Cdc42-mCherrySW and CRIB-3GFP images at cell poles in wt and indicated mutant cells. (D) Average profiles of cells shown in C. (E) Plot of Cdc42 versus CRIB fluorescence at the tip showing that the enrichment of total Cdc42 correlates with its activity. n ≥ 40 for each profile or data point. Bars = 2 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383620&req=5

pbio.1002097.g002: Enrichment of GTP-Cdc42 at sites of polarity.(A) Cdc42-mCherrySW and CRIB-3GFP localization. Traces for cortical measurements are shown on the right. (B) Average profiles of fluorescence intensity along cortical traces with standard deviation (left) and after normalization to the maximum and minimum intensity values (right). (C) Representative Cdc42-mCherrySW and CRIB-3GFP images at cell poles in wt and indicated mutant cells. (D) Average profiles of cells shown in C. (E) Plot of Cdc42 versus CRIB fluorescence at the tip showing that the enrichment of total Cdc42 correlates with its activity. n ≥ 40 for each profile or data point. Bars = 2 μm.
Mentions: To quantitatively define the distribution and activity of Cdc42 at the plasma membrane, we co-imaged Cdc42-mCherrySW with CRIB-3GFP, a probe that selectively binds active Cdc42-GTP (CRIB stands for Cdc42- and Rac-interactive binding domain) [43]. The distribution of CRIB closely mirrored that of Cdc42 enrichment at cell poles (Fig. 2A). At cell poles with strong CRIB localization, Cdc42 was enriched 3-fold (± 0.7, n = 40) over its levels at cell side (Fig. 2B). At cell poles with low CRIB levels, these correlated with low Cdc42 enrichment (S2 Fig). Normalization of the Cdc42 and CRIB distribution profiles to their maximum and minimum yielded overlapping curves with identical decay rates, suggesting a very tight correlation between enrichment of Cdc42 and the active form (Fig. 2B right).

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