Trans Europ Express VT 11.5: The Ultimate Guide to Download and Play
We were surprised to find that a major proportion of Zera fusions were still retained in the ER (Figure 2A, lane 3), suggesting that human cells could contain a large population of ER-derived organelles resembling natural PBs. This result was unexpected and suggests that ER-derived organelles are not restricted to higher metazoa and may be shared by lower eukaryotes. In order to investigate the biology of these unexpected ER-derived organelles, we investigated whether such PB-like organelles were induced by other recombinant proteins expressed in the ER in mammalian cells. Zera-hGH which is present in both natural and induced PBs (Figure 2A, lane 5) were both secreted and was retained in ER-derived organelles (Figure 2A, lane 3) while Zera-EGF, which is mainly in natural PBs (Figure 2A, lane 6) was distributed between the ER and the secretory pathway. In addition, we investigated whether the C-terminal KDEL C-terminal ER retention signal was essential for formation of PB-like organelles (Figure 2A, lane 7) since previous studies have suggested that an ER-derived lipid rich environment is required to form PB-like organelles [ 29 ]. Zera-Ct (without KDEL), which is mostly retained in the ER (Figure 2A, lane 3), was secreted. We also investigated whether ER-derived organelles induced by Zera fusion proteins could be efficiently targeted to the plasma membrane and characterized their size. HEK293T and Cos1 cells were transfected with Zera-Ct or Zera-hGH, fixed and labeled with anti-Ct antibodies to reveal natural PBs (green) and the ER (red) by immunocytochemistry (Figure 2E, 2F ). Contrary to the ER-derived organelles (Figure 2A, lane 3), natural PBs were successfully secreted in both HEK293T and Cos1 cells (Figure 2E, 2F ). Zera fusions containing the KDEL C-terminal ER retention sequence were present in the ER (Figure 2A, lane 7), but neither secreted nor targeted to the plasma membrane (Figure 2E, 2F ). Therefore, we concluded that PBs and PB-like organelles may be biochemically different and may have distinct biogenesis pathways. We next expressed a Zera-EGF chimera containing the HA epitope tag in the cytosol of mammalian cells to determine whether the ER-derived organelles were induced by Zera fusions (Figure 2A, lane 8). Zera-EGF was mainly secreted (Figure 2A, lane 2) and in the cytosol (Figure 2A, lane 4) or was retained in the ER (Figure 2A, lane 3). Interestingly, the HA epitope was also detected within the PB-like organelles (Figure 2A, lane 5). To test whether human PB-like organelles contained recombinant protein, Zera-Ct fused to a fluorophore (mCherry) was expressed in Cos1 cells and then fixed and immunostained with anti-Ct antibodies (Figure 2G ). PB-like organelles were positive for mCherry (green) (Figure 2G ). These data suggest that PB-like organelles could contain proteins other than Zera fusion proteins.
Trans Europ Express VT 11.5 Torrent Download [full Version]