Subcellular localization of GFP-PfSec22p. GFP-expressing parasites were fixed and stained with anti-PfERD2 or anti-PfBip antibodies. Blue arrowheads indicate co-localization sites between the fusion protein GFP-PfSec22p (green) and the cis-Golgi marker PfERD2 (red, panel a) or the ER marker PfBip (red, panel b). The inserted red arrows show GFP fluorescence in locations different from cis-Golgi or ER compartments.Ayong L, Pagnotti G, Tobon AB, Chakrabarti D. Identification of Plasmodium falciparum family of SNAREs. Mol Biochem Parasitol. 2007 152:113-22. Copyright Elsevier 2009.

Immunoelectron micrographs depicting the association of GFP-PfSec22 with transition vesicles and tubovesicular elements. Ultrathin cryosections of the GFP-PfSec22 expressing cells were probed with monoclonal anti-GFP antibodies followed by immunogold detection using gold (12nm)-labeled anti-mouse secondary antibodies. (A) Association of GFP-PfSec22 with ER-derived transition vesicles (black arrowhead). (B) Association of GFP-PfSec22 with membrane-limited vesicles (white arrowheads) in the host cell compartment. (C) Association of GFP-PfSec22 with the TVN-like extension (dotted arrow) and membrane-bound vesicles (white arrowhead) in the infected 7 erythrocyte cytosol. N: nucleus, MC: Maurer’s cleft, RBC: red blood cell cytosol. Scale bars are 8 500 nm in (A), and 100 nm in (B) and (C).Ayong L, Raghavan A, Schneider TG, Taraschi TF, Fidock DA, Chakrabarti D. The Longin domain regulates the steady-state dynamics of Sec22 in P. falciparum. Eukaryot Cell. 2009 8(9):1330-40

Co-localization of GFP-PfSec22 with ER and cis-Golgi markers. Immunoflorescence micrographs indicating co-localization of the GFP-PfSec22 and anti-PfSec22 signals in transgenic parasites (top row), and co-localization of GFP-PfSec22 with the ER marker PfBip PFI0875w (middle row), and Golgi marker PfErd2 PF13_0280 (bottom row). Scale bars, 2μm.Ayong L, Raghavan A, Schneider TG, Taraschi TF, Fidock DA, Chakrabarti D. The Longin domain regulates the steady-state dynamics of Sec22 in P. falciparum. Eukaryot Cell. 2009 8(9):1330-40.

Expression analyses of PfSec22 Antibodies were generated against a peptide sequence in the non-conserved region of PfSec22 and affinity purified as described in the ‘Materials and Methods’ section. (A) Immunoblot analysis of parasite extracts showing expression of endogenous PfSec22 in ring (R), trophozoite (T), and schizont (S) stage parasites. Absence of antibody reaction with the uninfected erythrocyte lysate (UE) indicates high specificity of the antibodies to the parasite protein. (B) Immunofluorescence microscopy of fixed cells using anti-PfSec22 antibodies and goat anti16 rabbit Alexa Fluor-555 secondary antibodies. An intense ring of PfSec22 fluorescence is visible in ring and trophozoite infected cells. Isolated foci of PfSec22 fluorescence (black arrow) are also detected in the host cell compartment in trophozoite-infected cells suggesting export of PfSec22 into the erythrocyte cytosol. Scale bar, 2μm Ayong L, Raghavan A, Schneider TG, Taraschi TF, Fidock DA, Chakrabarti D. The Longin domain regulates the steady-state dynamics of Sec22 in P. falciparum. Eukaryot Cell. 2009 8(9):1330-40

Live-cell imaging of GFP-tagged PfSec22 chimeras. (B) Live-cell imaging of parasites expressing the C-terminal GFP-tagged PfSec22 (PfSec22-GFP) showing diffuse localization of the protein throughout the parasite cytoplasm and occasionally in the host cell compartment. (C) Confocal micrographs showing localization of the N-terminal GFP-tagged protein (GFP-PfSec22) in earlytrophozoite (top row) and mid-trophozoite (bottom row) stage parasites. In addition to the ER-like profiles, GFP-PfSec22 associates with tubovesicular elements in the infected host cell. The micrographs (left to right) represent differential interference contrast, GFP fluorescence, and a merge of the two. Scale bars, 2 mm. Ayong L, Raghavan A, Schneider TG, Taraschi TF, Fidock DA, Chakrabarti D. The Longin domain regulates the steady-state dynamics of Sec22 in P. falciparum. Eukaryot Cell. 2009 8(9):1330-40.

Immunofluorescence microscopy of fixed cells using anti-PfSec22 antibodies and goat anti-rabbit Alexa Fluor 555 secondary antibodies. An intense ring of PfSec22 fluorescence is visible in ring- and trophozoite-infected cells. Isolated foci of PfSec22 fluorescence (arrow) are also detected in the host cell compartment in trophozoite-infected cells, suggesting export of PfSec22 into the erythrocyte cytosol. Scale bars, 2 mm.Bottom row: Coimmunolocalization and membrane association of endogenous PfSec22. The association of PfSec22 with the parasite ER was investigated by coimmuno-fluorescence assays using rabbit anti-PfSec22 and rat anti-PfBip antibodies, as indicated. Both proteins colocalized significantly within the parasite. Scale bars, 2 mm.Ayong L, Raghavan A, Schneider TG, Taraschi TF, Fidock DA, Chakrabarti D. The Longin domain regulates the steady-state dynamics of Sec22 in P. falciparum. Eukaryot Cell. 2009 8(9):1330-40.