Rac1/WAVE2 and Cdc42/N-WASP Participation in Actin-Dependent Host Cell Invasion by Extracellular Amastigotes of Trypanosoma cruzi

Figures

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  Figure 1. Rac1, Cdc42 and RhoA are recruited to actin-rich cup-like structures induced by EAs. HeLa cells were transfected with Cdc42, Rac1 (GFP tagged) or RhoA (c-myc tagged) and interactions with EAs were evaluated by live confocal microscopy. (A) Frames from time-lapse movies showing the moments of initial contact, beginning of actin recruitment and EA (arrowheads) internalization with recruitment and colocalization of Rac1-GFP (green) and LifeAct-RFP (red). (B) Frames from time-lapse movies showing Cdc42-GFP (green) recruited along with LifeAct-RFP (red) to EA (arrowheads) invasion sites during internalization. (C) Recruitment and colocalization of RhoA-c-myc (green) with actin (red) at EA invasion sites (arrowheads) was observed in fixed cells stained with phalloidin-TRITC. Nuclei and kinetoplasts were stained with Hoechst (A,B) or DAPI (C). Bar = 20 μm.

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  Figure 2. Depletion of Rac1, but not RhoA, reduces EA internalization by host cells. HeLa cells depleted of Rho-family GTPases were incubated with EA for 2 h, fixed with Bouin, and stained with Giemsa, and internalized parasites were quantified by optical microscopy. (A) Lysates of HeLa cells transduced by lentivirus containing shRNAi sequences and treated with puromycin for 2 weeks were evaluated for Rho GTPases expression by western blotting using actin or GAPDH as loading controls (detailed in Materials and Methods). (B) EA internalization was reduced approximately 35 and 76% in Rac1 depleted cell lineages (R30 and R75, respectively) when compared to the non-transduced group (-). (C) Cells depleted for RhoA had similar EA internalization when compared to the non-transduced groups (-). (D) EA invasion in cells transduced with scramble RNA sequences displayed no difference when compared to a non-transduced group (-). Results represent mean of at least three independent experiments in triplicate, and each dot represents one replicate (One-Way ANOVA, ***P < 0.001, **P < 0.01, n ≥ 9).

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  Figure 3. Depletion of Rac1 delays EA internalization. (A) Depleted HeLa lineages incubated with EA for the indicated times points were fixed and stained for filamentous actin, and the proportion of EA-induced actin recruitment was quantified by epifluorescence microscopy. Approximately 80% of interacting EAs induced actin recruitment in cells depleted of Cdc42, Rac1 or RhoA (C31, R75 or R11, respectively) and in the control [scramble (Scr) and non-transduced (-)] cell lineages at 30 or 120 min of interaction. Results represent mean of two independent experiments in triplicate, and each dot represents a replicate (One-Way ANOVA, no statistical differences, n ≥ 6). (B) EA-induced actin recruitment and internalization were assessed in the indicated depleted lineages by live time-lapse confocal microscopy. In Rac1 depleted cells (left panel), EA (white arrowheads) attaches and induces actin polymerization but fails to invade even after 30 min of initial contact. In contrast, in the non-transduced control group (right panel) and in the Cdc42 or RhoA depleted HeLa cells (central panels), EAs (white arrowheads) attach, induce actin (red) polymerization and successfully invade (blue arrowhead)…

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  Figure 4. Expression of activation constructs of Rac1 and RhoA altered the morphology of EA-induced actin recruitment. HeLa cells were transfected with WT, DN or CA mutated GTPases (green) and incubated with EAs for 1 h, and their recruitment (arrowheads) along with actin (red) was evaluated by confocal microscopy. (A) Recruitment and colocalization with actin to EA invasion sites of the indicated Rac1 constructs with morphological alterations only in the CA-Rac1 transfected group (single asterisk) compared to the DN and WT control groups. (B) Recruitment and colocalization with actin of the Cdc42 constructs (GFP tagged; green) without drastic morphology alterations when comparing the DN and CA groups to the WT control group. (C) Recruitment and colocalization with actin of WT and DN RhoA constructs (His-tagged, Alexa Flour 488 secondary; green). CA-RhoA expression induced discrete actin recruitment, leading to its partial colocalization with actin (double asterisk). (D) No alteration in EA-induced actin-rich cup-like structures in cells transfected with cytosolic GFP alone (GFP) or in non-transfected cells (−). Single focal plane images with phalloidin-TRITC (red) staining filamentous…

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  Figure 5. EA internalization is altered by the expression of activation mutated Rho-family GTPases. HeLa cells transfected with WT, DN or CA mutated GTPases were incubated with EAs (MOI 10) for 2 h, and intracellular parasites were quantified under epifluorescence microscope in transfected cells. Cells transfected with cytosolic GFP (GFP) were used as a control group and displayed similar EA internalization as non-transfected cells (-). (A) Cells expressing CA-Rac1 displayed increased EA internalization, and cells expressing DN-Rac1 displayed reduced EA internalization compared to the GFP control group. (B) For Cdc42, cells expressing WT construct had increased EA internalization and cells expressing CA construct had reduced EA internalization compared to the GFP control group. (C) Cells transfected with WT-RhoA or CA displayed reduced EA internalization compared to the GFP control group. Results represent mean of two independent experiments in triplicate or duplicate, and each dot represents one replicate (One-Way ANOVA, ***P < 0.001, **P < 0.01, n ≥ 4).

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  Figure 6. Rho-family GTPase effector proteins, N-WASP and WAVE2, participate in EA invasion of HeLa cells. (A) HeLa cells were transfected with N-WASP-GFP or WAVE2-YFP and incubated with EAs for 1 h and interactions were evaluated by confocal microscopy. Both N-WASP (green) and WAVE2 (green) are recruited and colocalize with actin (red) at EA invasion sites (arrowheads). (B) Lysates of HeLa cells transduced by lentivirus containing shRNAi sequences and treated with puromycin for 2 weeks were evaluated for N-WASP and WAVE2 expression by western blotting using GAPDH as loading controls (detailed in Materials and Methods). (C) EA internalization was reduced in cells transduced with the N-WASP shRNAi N61 sequence (with confirmed N-WASP depletion, B) but not with the N62 sequence (no N-WASP depletion, B). EA invasion was also reduced in cells depleted of WAVE2 (depletion in both transduced sequences, W27 and W85, C) when compared to the non-transduced control group. Results represent mean of at least three independent experiments in triplicate, and each dot represents one replicate (One-Way ANOVA, ***P < 0.001, **P < 0.01, n ≥ 9). (D) Depleted HeLa lineages incubated with EA for the indicated…

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  Figure 7. Involvement of Rho-family GTPases and their effector proteins in actin mediated EA host cell invasion. Our work showed the recruitment of Rho-family GTPases, N-WASP and WAVE2 after EA contact with the host cell. None of these proteins alone is required for F-actin polymerization, but Cdc42 cycling between active/inactive states and Rac1 activation are important for EA internalization. RhoA promotes actin polymerization in stress fibers but inhibits this process at EA invasion sites, which abolishes parasite entry and reveals the negative role of RhoA in this process. Dashed lines represent the possible joint participation of Rac1/WAVE2 and Cdc42/N-WASP and the possible negative regulation mechanisms of Cdc42 and RhoA over Rac1 activity.