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Rac1 dynamics in the human opportunistic fungal pathogen Candida albicans.

Vauchelles R, Stalder D, Botton T, Arkowitz RA, Bassilana M - PLoS ONE (2010)

Bottom Line: Furthermore, we show that C. albicans Rac1 dynamics, both at the plasma membrane and in the nucleus, are dependent on its activation state and in particular that the inactive form accumulates faster in the nucleus.Heterologous expression of human Rac1 in C. albicans also results in nuclear accumulation, yet accumulation is more rapid than that of C. albicans Rac1.Taken together our results indicate that Rac1 nuclear accumulation is an inherent property of this G-protein and suggest that the requirements for its nucleo-cytoplasmic shuttling are conserved from fungi to humans.

View Article: PubMed Central - PubMed

Affiliation: Institute of Developmental Biology and Cancer, Centre National de la Recherche Scientifique UMR 6543, Université de Nice, Faculté des Sciences-Parc Valrose, Nice, France.

ABSTRACT
The small Rho G-protein Rac1 is highly conserved from fungi to humans, with approximately 65% overall sequence identity in Candida albicans. As observed with human Rac1, we show that C. albicans Rac1 can accumulate in the nucleus, and fluorescence recovery after photobleaching (FRAP) together with fluorescence loss in photobleaching (FLIP) studies indicate that this Rho G-protein undergoes nucleo-cytoplasmic shuttling. Analyses of different chimeras revealed that nuclear accumulation of C. albicans Rac1 requires the NLS-motifs at its carboxyl-terminus, which are blocked by prenylation of the adjacent cysteine residue. Furthermore, we show that C. albicans Rac1 dynamics, both at the plasma membrane and in the nucleus, are dependent on its activation state and in particular that the inactive form accumulates faster in the nucleus. Heterologous expression of human Rac1 in C. albicans also results in nuclear accumulation, yet accumulation is more rapid than that of C. albicans Rac1. Taken together our results indicate that Rac1 nuclear accumulation is an inherent property of this G-protein and suggest that the requirements for its nucleo-cytoplasmic shuttling are conserved from fungi to humans.

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Rac1 dynamics at the plasma membrane depend on its activation state.(A) Localization of Rac1, activated (Rac1[G12V]) or inactivated (Rac[T17N]) forms. Confocal microscopy images of budding rac1Δ/rac1Δ PADH1GFPRAC1 (PY205), rac1Δ/rac1Δ PADH1GFPrac1[G12V] (PY209), and rac1Δ/rac1Δ PADH1GFPrac1[T17N] (PY212) cells were taken; both the maximal projection and central section are shown. (B) Inactivated and activated forms of Rac1 have different plasma membrane distributions. Graph of signal intensity along plasma membrane perimeter of the indicated cells, as shown in the bottom panel of Figure 6A. (C) FRAP analysis of cells expressing Rac1 (n = 17), its activated (Rac1[G12V]) (n = 20) or inactivated (Rac[T17N]) (n = 17) forms. FRAP t½ values (means ± standard deviation) are determined from single-phase exponential curve fit of fluorescence recovery after photobleaching. The two-tailed P-values of the indicated mean FRAP t½'s (*) are less than 0.006 compared with the FRAP t½ of GFP-Rac1.
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pone-0015400-g006: Rac1 dynamics at the plasma membrane depend on its activation state.(A) Localization of Rac1, activated (Rac1[G12V]) or inactivated (Rac[T17N]) forms. Confocal microscopy images of budding rac1Δ/rac1Δ PADH1GFPRAC1 (PY205), rac1Δ/rac1Δ PADH1GFPrac1[G12V] (PY209), and rac1Δ/rac1Δ PADH1GFPrac1[T17N] (PY212) cells were taken; both the maximal projection and central section are shown. (B) Inactivated and activated forms of Rac1 have different plasma membrane distributions. Graph of signal intensity along plasma membrane perimeter of the indicated cells, as shown in the bottom panel of Figure 6A. (C) FRAP analysis of cells expressing Rac1 (n = 17), its activated (Rac1[G12V]) (n = 20) or inactivated (Rac[T17N]) (n = 17) forms. FRAP t½ values (means ± standard deviation) are determined from single-phase exponential curve fit of fluorescence recovery after photobleaching. The two-tailed P-values of the indicated mean FRAP t½'s (*) are less than 0.006 compared with the FRAP t½ of GFP-Rac1.

Mentions: To address whether Rac1 dynamics depend on its activation state, we compared the localization and FRAP dynamics of Rac1 and its derivatives, which mimic either the activated form (Rac1[G12V]) or the inactivated form (Rac1[T17N]). Figure 6A illustrates the localization of GFP-Rac1, GFP-Rac1[G12V] or GFP-Rac1[T17N] in exponentially growing cells, showing both the maximal projections and the central optical sections. We observed that Rac1 and Rac1[G12V] were distributed similarly at the plasma membrane. In contrast, Rac1[T17N] was localized to clusters at the plasma membrane (Figures 6A and 6B). The FRAP t½ of Rac1[G12V] was slightly slower compared to Rac1 (1.41±0.99 sec and 1.15±0.23 sec, respectively), whereas that of Rac1[T17N] was substantially slower (13.00±3.38 sec) (Figure 6C). During the time course of the FRAP experiments (∼2 min) we did not observe movement of the Rac1[T17N] clusters at the plasma membrane. These results indicate that Rac1[T17N] is substantially less mobile at the plasma membrane than Rac1 or Rac1[G12V], and suggest that the rapid dynamics of this Rho G-protein at the plasma membrane requires GTPase cycling. Similarly, we previously showed that the fluorescence recovery of GFP-Rac1 was slower in cells lacking the Rac1 specific activator, Dck1, or the scaffold protein Lmo1 [39]. Furthermore, we examined whether these different mutant forms of Rac1 could accumulate in the nucleus. Figure 7A shows that both Rac1[G12V] and Rac1[T17N] can accumulate in the nucleus, indicating that GTPase cycling is not required for this nuclear import. Compared to the FRAP measurements of nuclear Rac1 (t½ = 34.21±12.02 sec), Rac1[G12V] had a significantly slower recovery of nuclear fluorescence (t½ = 57.82±24.86 sec) and Rac1[T17N] a significantly faster recovery of nuclear fluorescence (t½ = 23±6.8 sec) (Figure 7B). Furthermore, although there was no significant difference between t½ recovery of nuclear fluorescence of Rac1 in wild-type cells (t½ = 35.54±11.70 sec, n = 18) and rac1Δ/rac1Δ cells (t½ = 34.21±12.02 sec, n = 25), the t½ recovery of nuclear fluorescence of Rac1 in dck1Δ/dck1Δ cells was significantly faster (t½ = 23.88±7.73 sec, n = 30; Figure 8B), consistent with the results from the rac1[T17N] strain. These data suggest that while active and inactive forms of Rac1 can accumulate in the nucleus, the latter is imported into the nucleus roughly 2.5 times faster than the former. Together these results show that Rac1 dynamics both at the plasma membrane as well as shuttling in and out of the nucleus depend on the activation state of this G-protein.


Rac1 dynamics in the human opportunistic fungal pathogen Candida albicans.

Vauchelles R, Stalder D, Botton T, Arkowitz RA, Bassilana M - PLoS ONE (2010)

Rac1 dynamics at the plasma membrane depend on its activation state.(A) Localization of Rac1, activated (Rac1[G12V]) or inactivated (Rac[T17N]) forms. Confocal microscopy images of budding rac1Δ/rac1Δ PADH1GFPRAC1 (PY205), rac1Δ/rac1Δ PADH1GFPrac1[G12V] (PY209), and rac1Δ/rac1Δ PADH1GFPrac1[T17N] (PY212) cells were taken; both the maximal projection and central section are shown. (B) Inactivated and activated forms of Rac1 have different plasma membrane distributions. Graph of signal intensity along plasma membrane perimeter of the indicated cells, as shown in the bottom panel of Figure 6A. (C) FRAP analysis of cells expressing Rac1 (n = 17), its activated (Rac1[G12V]) (n = 20) or inactivated (Rac[T17N]) (n = 17) forms. FRAP t½ values (means ± standard deviation) are determined from single-phase exponential curve fit of fluorescence recovery after photobleaching. The two-tailed P-values of the indicated mean FRAP t½'s (*) are less than 0.006 compared with the FRAP t½ of GFP-Rac1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965673&req=5

pone-0015400-g006: Rac1 dynamics at the plasma membrane depend on its activation state.(A) Localization of Rac1, activated (Rac1[G12V]) or inactivated (Rac[T17N]) forms. Confocal microscopy images of budding rac1Δ/rac1Δ PADH1GFPRAC1 (PY205), rac1Δ/rac1Δ PADH1GFPrac1[G12V] (PY209), and rac1Δ/rac1Δ PADH1GFPrac1[T17N] (PY212) cells were taken; both the maximal projection and central section are shown. (B) Inactivated and activated forms of Rac1 have different plasma membrane distributions. Graph of signal intensity along plasma membrane perimeter of the indicated cells, as shown in the bottom panel of Figure 6A. (C) FRAP analysis of cells expressing Rac1 (n = 17), its activated (Rac1[G12V]) (n = 20) or inactivated (Rac[T17N]) (n = 17) forms. FRAP t½ values (means ± standard deviation) are determined from single-phase exponential curve fit of fluorescence recovery after photobleaching. The two-tailed P-values of the indicated mean FRAP t½'s (*) are less than 0.006 compared with the FRAP t½ of GFP-Rac1.
Mentions: To address whether Rac1 dynamics depend on its activation state, we compared the localization and FRAP dynamics of Rac1 and its derivatives, which mimic either the activated form (Rac1[G12V]) or the inactivated form (Rac1[T17N]). Figure 6A illustrates the localization of GFP-Rac1, GFP-Rac1[G12V] or GFP-Rac1[T17N] in exponentially growing cells, showing both the maximal projections and the central optical sections. We observed that Rac1 and Rac1[G12V] were distributed similarly at the plasma membrane. In contrast, Rac1[T17N] was localized to clusters at the plasma membrane (Figures 6A and 6B). The FRAP t½ of Rac1[G12V] was slightly slower compared to Rac1 (1.41±0.99 sec and 1.15±0.23 sec, respectively), whereas that of Rac1[T17N] was substantially slower (13.00±3.38 sec) (Figure 6C). During the time course of the FRAP experiments (∼2 min) we did not observe movement of the Rac1[T17N] clusters at the plasma membrane. These results indicate that Rac1[T17N] is substantially less mobile at the plasma membrane than Rac1 or Rac1[G12V], and suggest that the rapid dynamics of this Rho G-protein at the plasma membrane requires GTPase cycling. Similarly, we previously showed that the fluorescence recovery of GFP-Rac1 was slower in cells lacking the Rac1 specific activator, Dck1, or the scaffold protein Lmo1 [39]. Furthermore, we examined whether these different mutant forms of Rac1 could accumulate in the nucleus. Figure 7A shows that both Rac1[G12V] and Rac1[T17N] can accumulate in the nucleus, indicating that GTPase cycling is not required for this nuclear import. Compared to the FRAP measurements of nuclear Rac1 (t½ = 34.21±12.02 sec), Rac1[G12V] had a significantly slower recovery of nuclear fluorescence (t½ = 57.82±24.86 sec) and Rac1[T17N] a significantly faster recovery of nuclear fluorescence (t½ = 23±6.8 sec) (Figure 7B). Furthermore, although there was no significant difference between t½ recovery of nuclear fluorescence of Rac1 in wild-type cells (t½ = 35.54±11.70 sec, n = 18) and rac1Δ/rac1Δ cells (t½ = 34.21±12.02 sec, n = 25), the t½ recovery of nuclear fluorescence of Rac1 in dck1Δ/dck1Δ cells was significantly faster (t½ = 23.88±7.73 sec, n = 30; Figure 8B), consistent with the results from the rac1[T17N] strain. These data suggest that while active and inactive forms of Rac1 can accumulate in the nucleus, the latter is imported into the nucleus roughly 2.5 times faster than the former. Together these results show that Rac1 dynamics both at the plasma membrane as well as shuttling in and out of the nucleus depend on the activation state of this G-protein.

Bottom Line: Furthermore, we show that C. albicans Rac1 dynamics, both at the plasma membrane and in the nucleus, are dependent on its activation state and in particular that the inactive form accumulates faster in the nucleus.Heterologous expression of human Rac1 in C. albicans also results in nuclear accumulation, yet accumulation is more rapid than that of C. albicans Rac1.Taken together our results indicate that Rac1 nuclear accumulation is an inherent property of this G-protein and suggest that the requirements for its nucleo-cytoplasmic shuttling are conserved from fungi to humans.

View Article: PubMed Central - PubMed

Affiliation: Institute of Developmental Biology and Cancer, Centre National de la Recherche Scientifique UMR 6543, Université de Nice, Faculté des Sciences-Parc Valrose, Nice, France.

ABSTRACT
The small Rho G-protein Rac1 is highly conserved from fungi to humans, with approximately 65% overall sequence identity in Candida albicans. As observed with human Rac1, we show that C. albicans Rac1 can accumulate in the nucleus, and fluorescence recovery after photobleaching (FRAP) together with fluorescence loss in photobleaching (FLIP) studies indicate that this Rho G-protein undergoes nucleo-cytoplasmic shuttling. Analyses of different chimeras revealed that nuclear accumulation of C. albicans Rac1 requires the NLS-motifs at its carboxyl-terminus, which are blocked by prenylation of the adjacent cysteine residue. Furthermore, we show that C. albicans Rac1 dynamics, both at the plasma membrane and in the nucleus, are dependent on its activation state and in particular that the inactive form accumulates faster in the nucleus. Heterologous expression of human Rac1 in C. albicans also results in nuclear accumulation, yet accumulation is more rapid than that of C. albicans Rac1. Taken together our results indicate that Rac1 nuclear accumulation is an inherent property of this G-protein and suggest that the requirements for its nucleo-cytoplasmic shuttling are conserved from fungi to humans.

Show MeSH
Related in: MedlinePlus