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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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C. albicans Rac1 accumulates in the nucleus.(A) Intracellular accumulation of GFP-Rac1 in the absence of cell agitation. DIC and fluorescence images of rac1Δ/rac1Δ PADH1GFPRAC1 (PY205) cells from shaking cultures, after 1 h without agitation, followed by 1 h of shaking. Bar, 5 µm. (B) Rac1 accumulates in the nucleus in the absence of cells agitation. Intracellular GFP-Rac1 co-localizes with 4′,6-diamidino-2-phenylindole (DAPI). (C) Time-course of Rac1 nuclear accumulation. Cells with nuclear fluorescent signal were counted after the indicated times in the absence of agitation. The average of four experiments is shown with approximately 50 cells counted at each time point in each experiment (bars indicate standard deviation). (D) Rac1 accumulates in the nucleus of serum-induced hyphal cells. Hyphal growth in PY205 cells was induced in rich (YEPD) liquid media containing 50% FCS, for the indicated times, in the presence or the absence of agitation at 37°C.
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pone-0015400-g001: C. albicans Rac1 accumulates in the nucleus.(A) Intracellular accumulation of GFP-Rac1 in the absence of cell agitation. DIC and fluorescence images of rac1Δ/rac1Δ PADH1GFPRAC1 (PY205) cells from shaking cultures, after 1 h without agitation, followed by 1 h of shaking. Bar, 5 µm. (B) Rac1 accumulates in the nucleus in the absence of cells agitation. Intracellular GFP-Rac1 co-localizes with 4′,6-diamidino-2-phenylindole (DAPI). (C) Time-course of Rac1 nuclear accumulation. Cells with nuclear fluorescent signal were counted after the indicated times in the absence of agitation. The average of four experiments is shown with approximately 50 cells counted at each time point in each experiment (bars indicate standard deviation). (D) Rac1 accumulates in the nucleus of serum-induced hyphal cells. Hyphal growth in PY205 cells was induced in rich (YEPD) liquid media containing 50% FCS, for the indicated times, in the presence or the absence of agitation at 37°C.

Mentions: In mammalian cells, Rac1 accumulates in the nucleus [12], [13] where it is proposed to have different functions, such as activating proteins of the STAT family [11] or promoting cell division [10]. Intriguingly, in C. albicans we observed that when cells sediment, Rac1 also accumulates in an organelle, which is likely to be the nucleus. Specifically, we observed that while GFP-Rac1 localized to the plasma membrane in exponentially growing cells under agitation (Figure 1A, left panel), this fusion protein accumulated in an intracellular compartment in the absence of agitation (Figure 1A, middle panel). If these cells were then subsequently agitated, accumulation in this intracellular compartment was no longer observed (Figure 1A, right panel). To confirm that Rac1 is indeed in the nucleus, we examined GFP fluorescence in cells grown in low levels of 4′,6-diamidino-2-phenylindole (DAPI), which labels DNA. The GFP and DAPI signals co-localized as illustrated in Figure 1B. When the nuclear accumulation of Rac1 was followed over time, we observed ∼100% cells with nuclear fluorescence after 80–90 min in the absence of agitation (Figure 1C). Rac1 nuclear accumulation was independent of the cell cycle stage and of the culture media conditions, including the presence of serum, which induces hyphal growth (Figure 1D).


Rac1 dynamics in the human opportunistic fungal pathogen Candida albicans.

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

C. albicans Rac1 accumulates in the nucleus.(A) Intracellular accumulation of GFP-Rac1 in the absence of cell agitation. DIC and fluorescence images of rac1Δ/rac1Δ PADH1GFPRAC1 (PY205) cells from shaking cultures, after 1 h without agitation, followed by 1 h of shaking. Bar, 5 µm. (B) Rac1 accumulates in the nucleus in the absence of cells agitation. Intracellular GFP-Rac1 co-localizes with 4′,6-diamidino-2-phenylindole (DAPI). (C) Time-course of Rac1 nuclear accumulation. Cells with nuclear fluorescent signal were counted after the indicated times in the absence of agitation. The average of four experiments is shown with approximately 50 cells counted at each time point in each experiment (bars indicate standard deviation). (D) Rac1 accumulates in the nucleus of serum-induced hyphal cells. Hyphal growth in PY205 cells was induced in rich (YEPD) liquid media containing 50% FCS, for the indicated times, in the presence or the absence of agitation at 37°C.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2965673&req=5

pone-0015400-g001: C. albicans Rac1 accumulates in the nucleus.(A) Intracellular accumulation of GFP-Rac1 in the absence of cell agitation. DIC and fluorescence images of rac1Δ/rac1Δ PADH1GFPRAC1 (PY205) cells from shaking cultures, after 1 h without agitation, followed by 1 h of shaking. Bar, 5 µm. (B) Rac1 accumulates in the nucleus in the absence of cells agitation. Intracellular GFP-Rac1 co-localizes with 4′,6-diamidino-2-phenylindole (DAPI). (C) Time-course of Rac1 nuclear accumulation. Cells with nuclear fluorescent signal were counted after the indicated times in the absence of agitation. The average of four experiments is shown with approximately 50 cells counted at each time point in each experiment (bars indicate standard deviation). (D) Rac1 accumulates in the nucleus of serum-induced hyphal cells. Hyphal growth in PY205 cells was induced in rich (YEPD) liquid media containing 50% FCS, for the indicated times, in the presence or the absence of agitation at 37°C.
Mentions: In mammalian cells, Rac1 accumulates in the nucleus [12], [13] where it is proposed to have different functions, such as activating proteins of the STAT family [11] or promoting cell division [10]. Intriguingly, in C. albicans we observed that when cells sediment, Rac1 also accumulates in an organelle, which is likely to be the nucleus. Specifically, we observed that while GFP-Rac1 localized to the plasma membrane in exponentially growing cells under agitation (Figure 1A, left panel), this fusion protein accumulated in an intracellular compartment in the absence of agitation (Figure 1A, middle panel). If these cells were then subsequently agitated, accumulation in this intracellular compartment was no longer observed (Figure 1A, right panel). To confirm that Rac1 is indeed in the nucleus, we examined GFP fluorescence in cells grown in low levels of 4′,6-diamidino-2-phenylindole (DAPI), which labels DNA. The GFP and DAPI signals co-localized as illustrated in Figure 1B. When the nuclear accumulation of Rac1 was followed over time, we observed ∼100% cells with nuclear fluorescence after 80–90 min in the absence of agitation (Figure 1C). Rac1 nuclear accumulation was independent of the cell cycle stage and of the culture media conditions, including the presence of serum, which induces hyphal growth (Figure 1D).

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