Figure 1.
Annotated proteins in the contractile vacuole proteome belong to a variety of metabolic groups.
Table 1.
Proteins identified as potentially present in the contractile vacuole complex, showing localizations confirmed in this study or in other organisms.
Figure 2.
Fluorescence microscopy and western blot analysis of V-H+-ATPase subunit B-, AP180-, and VAMP1-GFP fusion proteins in live T. cruzi epimastigotes.
V-H+-ATPase subunit B (A), AP180 (B), and VAMP1 (C) localize to the bladder under hyposmotic conditions. Brightness and contrast of panels was adjusted, and fluorescence images in C were deconvolved. Scale bars: 10 µm. Confirmation of tagging by western blot analyses with polyclonal anti-GFP (dilution 1∶5,000-1∶10,000, Invitrogen) in epimastigotes. HRP-conjugated goat anti-rabbit was used as a secondary antibody. Magic Mark XP (Invitrogen) was used as a molecular weight marker. Arrows indicate bands of interest. A, V-H+-ATPase subunit B, expected size of fusion protein = 82 kDa. B, AP-180, expected size of fusion protein = 81 kDa. A 100 kDa cross-reacting band is only detected in the supernatant. C, VAMP1 expected size = 52 kDa. P, membrane pellet, S, soluble fraction, H, homogenate of whole parasites, WT, wild-type epimastigotes (negative control).
Figure 3.
AP180 and SNARE 2.1 immuno-electron microscopy.
GFP fusion proteins were detected in epimastigotes with anti-GFP polyclonal antibodies and gold-conjugated anti-rabbit secondary antibody. AP180 localizes mainly in the bladder of the CV (A and B) while SNARE 2.1 clearly localizes in the vesicular structures of the spongiome (C and D) although, some labeling can be observed is Golgi-like structures (arrow in D). CV: contractile vacuole bladder; S: spongiome; K: kinetoplast; N: nucleus. Bars: 0.5 µm.
Figure 4.
Rab-GFP fusion proteins localize in T. cruzi contractile vacuole.
Rab11 (green) (A) land Rab32 (green) (B) localize to the bladder under hyposmotic conditions. Rab11 (C) and Rab32 (D) partially co-localize with BODIPY-ceramide (red). DNA is stained with DAPI (blue). Brightness and contrast of panels was adjusted, and fluorescence images in C-D were deconvolved. Inset in C shows one cell (dotted rectangle) at higher magnification. Scale bars: A, B and C = 10 µm; D = 5 µm. E, confirmation of tagging by western blot analyses with anti GFP shows the expected size for both fusion proteins (50 kDa). Wild-type epimastigotes were used as negative control (WT).
Figure 5.
SNARE-GFP fusion proteins localize in the contractile vacuole spongiome.
SNARE2.1-GFP co-localizes with calmodulin (CaM) (A) and BODIPY-ceramide (B). SNARE2.2-GFP co-localizes with CaM (C) but localizes to a compartment that does not stain with BODIPY-ceramide (D). DNA is stained with DAPI (blue). Brightness and contrast of panels was adjusted, and fluorescence images in B and D were deconvolved. Scale bars = 10 µm. E, F, western blot analyses reveal the expected size for GFP tagged SNARE proteins (50 kDa). P, membrane pellet; S, soluble fraction, H, homogenate of whole parasites; WT, wild-type epimastigotes (negative control); GFP, epimastigotes overexpressing GFP (positive control).
Figure 6.
CaM- and TcPho1-GFP fusion proteins localization.
CaM-GFP overexpressing parasites (A) showed a localized signal to the contractile vacuole (green) but also cytosolic distribution. This localization was confirmed with anti-GFP (A, in red). A monoclonal antibody against human CaM was used to perform IFA (B). Green corresponds to the CaM-GFP overexpressed protein and in red the specific localization for the anti-human CaM can be observed in the spongiome of the CV. TcPho1 is localized at the bladder of the CV (C) both by direct GFP signal (green) and labeled by anti-GFP (red). Under hyposmotic conditions (D, hypo), TcPho1 bladder localization becomes more evident. The expected molecular weight for fusion proteins is 44 kDa for CaM (E) and 107 kDa for TcPho1 (F). P, membrane pellet; S, soluble fraction; H, homogenate whole parasites; WT, wild-type epimastigotes (negative control); GFP, epimastigotes overexpressing GFP (positive control).