Subcellular localization of three ESV candidates: Gl14458HA, Gl32419HA, and Gl25205HA.

<p>Representative localization of C-terminally HA-tagged variants after inducible (Gl14458, Gl32419) or constitutive expression (Gl25205) by confocal microscopy at 13h p.i. Anti-CWP1 was used to detect ESV organelles. <b>A)</b> Wild type (WB) encysting trophozoite at 13h p.i.: CWP1 localized to “doughnut-shaped” ESVs which is typical for this time point <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094089#pone.0094089-Konrad1" target="_blank">[28]</a>. No CWP1-signal in the perinuclear ER was visible. <b>B</b>) Gl14458HA at 13h p.i.: Gl14458HA was detected primarily in the perinuclear ER and overlaps with CWP1 in ESVs. Note the retention of CWP1 in the perinuclear ER and reduction of ESV numbers. In fact, the majority of induced cells in the population did not produce ESVs at all. <b>C, D, E</b>) Representative subcellular localizations of Gl32419-HA at 13h p.i. <b>C)</b> Partial signal overlap of Gl32419HA (green) with CWP1 (red) in knob-like structures, reminiscent of ESV neogenesis at ER exit sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094089#pone.0094089-Faso1" target="_blank">[27]</a>. Alternatively, Gl32419HA localized to the ER <b>D</b>) co-localizing with CWP1 primarily in the perinuclear ER and in ESVs suggesting delayed export of CWM to ESVs. <b>E</b>) In cells with canonical mature ESVs no signal overlap of Gl32419HA and CWP1 was observed. <b>F</b>) Gl25205HA at 13h p.i. was detected in the ER and in ESVs with occasional signal overlap with CWP1. <i>Antibodies:</i> anti-HA high affinity from rat, Alexa488-conjugated goat anti-rat (green), and Texas red-conjugated anti-CWP1 (red). Nuclear DNA was labeled with DAPI. <i>pCWP1:</i> inducible CWP1 promoter; <i>pendo:</i> endogenous promoter; <i>int:</i> stable integration into the genome; <i>epi:</i> episomal maintenance of the plasmid, <i>WB:</i> wildtype. Scale bar: 1.5 µm.