Bar: 10 μm. The PVM-localized Rho and Rac GTPases do not respond to epithelial growth factor (EGF) activation Rho GTPases control cell motility by regulating the reorganization of the cytoskeleton in response to EGF . Rho and Rac GTPases translocated from the cytosol to the cell membrane upon EGF activation . To study whether the Rho and Rac GTPases accumulated on the PVM would translocate following EGF activation, MAPK inhibitor the COS-7 cells overexpressing CFP-tagged Rho and Rac1 were starved overnight, infected with T. gondii RH tachyzoites and then activated with
EGF. The result showed that the recruited Rho and Rac GTPases on the PVM did not change in fluorescence brightness, unlike the fluorescence brightness in the cytosol that became faint because of the translocation of RhoA and Rac1 from the cytosol to the cell membrane towards the EGF activation spot (Figure 6). More photographs showing the RhoA and Rac1 sequestered on the PVM regardless the activation of EGF are provided in Additional file 4: Data S4. Figure 6 The CFP-tagged Rho and Rac1 GTPases AC220 accumulated on the parasitophorous vacuole membrane (PVM) do not translocate toward epithelial growth factor (EGF) activation. Two paralleled groups of COS-7 cells were grown on coverslips and transfected with BIX 1294 mw pECFP-RhoA and pECFP-Rac1 respectively.
Resveratrol Forty-eight hr post-transfection, cells were starved overnight in serum-free DMEM. One group of cells was infected with T. gondii tachyzoites and the other group was kept uninfected. One hr post-infection, the infected cells were washed 3× with PBS to remove the unrecruited tachyzoites. Cells were site-activated with EGF for 5 min. (A) In uninfected cells, the CFP-tagged RhoA and Rac1 GTPases in the
cytosol translocated to the host cell membrane (white arrowhead) in response to EGF activation. (B) In infected cells, the CFP-tagged RhoA and Rac1 were sequestered on the PVM without translocation toward the EGF, while the unassociated RhoA and Rac1 in the cytosol still translocated toward the EGF as in uninfected cells. More photographs provided in Additional file 4: Data S4 showing the RhoA and Rac1 sequestered on the PVM regardless the activation of EGF. Bar: 10 μm. Interference with RhoA and Rac1 endogenous activity affects tachyzoite infection To study the role of host cell RhoA and Rac1 GTPases during the tachyzoites invasion, COS-7 cells were over-expressed with RhoA-WT, RhoA-N19, Rac1-WT, and Rac1-N17. The endogenous expression of RhoA or Rac1 was inhibited by siRNA targeted towards either RhoA or Rac1 separately or towards both in human 16-HBE cells and then infected with RH tachyzoites. The infection rate was determined for each group.