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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

Cdc42 dynamics at the plasma membrane are largely independent of GDI or vesicle trafficking and strongly diffusive.(A) FRAP halftimes (t1/2) of Cdc42-mCherrySW recovery for indicated mutants and drug treatments. n ≥ 12 for wt, rdi1Δ, and rdi1Δ LatA. n ≥ 7 for all others. The asterisks indicate statistically significant differences between mutant tip or side versus wild-type tip or side, respectively, in a Student’s t test in which n.s. = p > 0.05, * is p ≤ 0.05, ** is p ≤ 0.01, *** is p ≤ 0.001, and **** is p ≤ 0.0001. (B) CRIB-3GFP in wt and rdi1Δ spores on rich YE media with either DMSO or LatA. Arrowheads indicate zones of active Cdc42. Time is shown in minutes. Scale bar = 5μm. (C) Cdc42-mCherrySW images at indicated time points relative to large cortical side bleach (dashed box). (D) Intensity profile along cell side versus time for cell in panel C. The intensity along the membrane was measured by fitting an active contour to the cell boundary and integrating the intensity within 3 pixels. Continuous lines show fit to a model of recovery with diffusion coefficient D and uniform cytoplasmic exchange with time constant τ (see Materials and Methods). (E) Same as panel D but for a smaller bleached region (0.9 μm) exhibiting faster recovery. This difference indicates that the recovery of the smaller bleached region is dominated by diffusion. (F) Normalized fluorescence correlation spectroscopy (FCS) autocorrelation curves of calibration dye Rhodamine B (green) and of Cdc42-mCherrySW at side (red) and tip locations (blue). The curve showing a slow diffusion component corresponds to the membrane-inserted prenylated species (see S5G Fig). Data were collected from a pool of eight cells and the curves were fitted with a two-component diffusion model (grey dashed lines).
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pbio.1002097.g004: Cdc42 dynamics at the plasma membrane are largely independent of GDI or vesicle trafficking and strongly diffusive.(A) FRAP halftimes (t1/2) of Cdc42-mCherrySW recovery for indicated mutants and drug treatments. n ≥ 12 for wt, rdi1Δ, and rdi1Δ LatA. n ≥ 7 for all others. The asterisks indicate statistically significant differences between mutant tip or side versus wild-type tip or side, respectively, in a Student’s t test in which n.s. = p > 0.05, * is p ≤ 0.05, ** is p ≤ 0.01, *** is p ≤ 0.001, and **** is p ≤ 0.0001. (B) CRIB-3GFP in wt and rdi1Δ spores on rich YE media with either DMSO or LatA. Arrowheads indicate zones of active Cdc42. Time is shown in minutes. Scale bar = 5μm. (C) Cdc42-mCherrySW images at indicated time points relative to large cortical side bleach (dashed box). (D) Intensity profile along cell side versus time for cell in panel C. The intensity along the membrane was measured by fitting an active contour to the cell boundary and integrating the intensity within 3 pixels. Continuous lines show fit to a model of recovery with diffusion coefficient D and uniform cytoplasmic exchange with time constant τ (see Materials and Methods). (E) Same as panel D but for a smaller bleached region (0.9 μm) exhibiting faster recovery. This difference indicates that the recovery of the smaller bleached region is dominated by diffusion. (F) Normalized fluorescence correlation spectroscopy (FCS) autocorrelation curves of calibration dye Rhodamine B (green) and of Cdc42-mCherrySW at side (red) and tip locations (blue). The curve showing a slow diffusion component corresponds to the membrane-inserted prenylated species (see S5G Fig). Data were collected from a pool of eight cells and the curves were fitted with a two-component diffusion model (grey dashed lines).

Mentions: We were surprised to discover that deletion of rdi1 did not affect Cdc42 mobility at cell tips in the experiment above (Fig. 3F, Fig. 4A). Though Rdi1 is the sole predicted GDI in S. pombe, its deletion yields only a minor morphological phenotype, with cells slightly shorter and wider than wild-type cells at division (S4A–D Fig). Disruption of actin cables in formin for3Δ mutant [51], interference with endocytosis in end4Δ mutant [52], or disruption of all actin structures by treatment with 200 μM LatA for a short time (5–10 min), also had no or minor effect on Cdc42 mobility (Fig. 4A). Remarkably, Cdc42 mobility at cell tips was even maintained in rdi1Δ mutant cells treated with LatA. Further collapse of the membrane trafficking system by treatment with Brefeldin A (BFA) also failed to slow down Cdc42 dynamics at cell tips (Fig. 4A). We note, however, that rdi1 deletion slightly slowed down Cdc42 mobility at cell sides especially in combination with actin cytoskeleton disruption, though the absence of known actin structures or trafficking pathways at cell sides suggests the effect of actin disruption may be indirect.


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)

Cdc42 dynamics at the plasma membrane are largely independent of GDI or vesicle trafficking and strongly diffusive.(A) FRAP halftimes (t1/2) of Cdc42-mCherrySW recovery for indicated mutants and drug treatments. n ≥ 12 for wt, rdi1Δ, and rdi1Δ LatA. n ≥ 7 for all others. The asterisks indicate statistically significant differences between mutant tip or side versus wild-type tip or side, respectively, in a Student’s t test in which n.s. = p > 0.05, * is p ≤ 0.05, ** is p ≤ 0.01, *** is p ≤ 0.001, and **** is p ≤ 0.0001. (B) CRIB-3GFP in wt and rdi1Δ spores on rich YE media with either DMSO or LatA. Arrowheads indicate zones of active Cdc42. Time is shown in minutes. Scale bar = 5μm. (C) Cdc42-mCherrySW images at indicated time points relative to large cortical side bleach (dashed box). (D) Intensity profile along cell side versus time for cell in panel C. The intensity along the membrane was measured by fitting an active contour to the cell boundary and integrating the intensity within 3 pixels. Continuous lines show fit to a model of recovery with diffusion coefficient D and uniform cytoplasmic exchange with time constant τ (see Materials and Methods). (E) Same as panel D but for a smaller bleached region (0.9 μm) exhibiting faster recovery. This difference indicates that the recovery of the smaller bleached region is dominated by diffusion. (F) Normalized fluorescence correlation spectroscopy (FCS) autocorrelation curves of calibration dye Rhodamine B (green) and of Cdc42-mCherrySW at side (red) and tip locations (blue). The curve showing a slow diffusion component corresponds to the membrane-inserted prenylated species (see S5G Fig). Data were collected from a pool of eight cells and the curves were fitted with a two-component diffusion model (grey dashed lines).
© Copyright Policy
Related In: Results  -  Collection

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

pbio.1002097.g004: Cdc42 dynamics at the plasma membrane are largely independent of GDI or vesicle trafficking and strongly diffusive.(A) FRAP halftimes (t1/2) of Cdc42-mCherrySW recovery for indicated mutants and drug treatments. n ≥ 12 for wt, rdi1Δ, and rdi1Δ LatA. n ≥ 7 for all others. The asterisks indicate statistically significant differences between mutant tip or side versus wild-type tip or side, respectively, in a Student’s t test in which n.s. = p > 0.05, * is p ≤ 0.05, ** is p ≤ 0.01, *** is p ≤ 0.001, and **** is p ≤ 0.0001. (B) CRIB-3GFP in wt and rdi1Δ spores on rich YE media with either DMSO or LatA. Arrowheads indicate zones of active Cdc42. Time is shown in minutes. Scale bar = 5μm. (C) Cdc42-mCherrySW images at indicated time points relative to large cortical side bleach (dashed box). (D) Intensity profile along cell side versus time for cell in panel C. The intensity along the membrane was measured by fitting an active contour to the cell boundary and integrating the intensity within 3 pixels. Continuous lines show fit to a model of recovery with diffusion coefficient D and uniform cytoplasmic exchange with time constant τ (see Materials and Methods). (E) Same as panel D but for a smaller bleached region (0.9 μm) exhibiting faster recovery. This difference indicates that the recovery of the smaller bleached region is dominated by diffusion. (F) Normalized fluorescence correlation spectroscopy (FCS) autocorrelation curves of calibration dye Rhodamine B (green) and of Cdc42-mCherrySW at side (red) and tip locations (blue). The curve showing a slow diffusion component corresponds to the membrane-inserted prenylated species (see S5G Fig). Data were collected from a pool of eight cells and the curves were fitted with a two-component diffusion model (grey dashed lines).
Mentions: We were surprised to discover that deletion of rdi1 did not affect Cdc42 mobility at cell tips in the experiment above (Fig. 3F, Fig. 4A). Though Rdi1 is the sole predicted GDI in S. pombe, its deletion yields only a minor morphological phenotype, with cells slightly shorter and wider than wild-type cells at division (S4A–D Fig). Disruption of actin cables in formin for3Δ mutant [51], interference with endocytosis in end4Δ mutant [52], or disruption of all actin structures by treatment with 200 μM LatA for a short time (5–10 min), also had no or minor effect on Cdc42 mobility (Fig. 4A). Remarkably, Cdc42 mobility at cell tips was even maintained in rdi1Δ mutant cells treated with LatA. Further collapse of the membrane trafficking system by treatment with Brefeldin A (BFA) also failed to slow down Cdc42 dynamics at cell tips (Fig. 4A). We note, however, that rdi1 deletion slightly slowed down Cdc42 mobility at cell sides especially in combination with actin cytoskeleton disruption, though the absence of known actin structures or trafficking pathways at cell sides suggests the effect of actin disruption may be indirect.

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