Journal: Journal of Virology
Article Title: Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells
Figure Lengend Snippet: Effect of siRNA-mediated depletion of Rac1 on intracellular PI(4,5)P 2 levels in Jurkat T cells. (A) Rac1-dependent cell signaling pathways having an effect on actin filament dynamics. In T cells, Rac1 GTPase plays an important role in the regulation of actin cytoskeleton rearrangement. Indeed, on one hand, in response to extracellular signals, Rac1 is activated and could stimulate actin filament turnover by the intermediate of the Pak1 (or Pak2)-LIMK-cofilin pathway. On the other hand, activated Rac1 could also stimulate actin filament branching in lamellipodia. In the latter case, IRSp53 is recruited by Rac1 and binds the proline-rich region of Wave2. As a result, Wave2 is activated and induces actin branching via Arp2/3 complex recruitment. In a third case, Rac1-GTP could also activate the phosphatidylinositol-4-phosphate 5-kinase (PIP5K), which catalyzes the synthesis of PI(4,5)P 2 at the cell plasma membrane. (B to D) Effect of siRNA-mediated depletion of Rac1 on intracellular PI(4,5)P 2 levels. (B) Cells were transfected with pCMV-LacZ, p8.2 (Gag, Gag-Pol, and viral accessory proteins), or PH-phospholipase C delta (PLCδ)-GFP, together with the siRNA control or siRNA against Rac1, as indicated. At 48 h posttransfection, cells were fixed, permeabilized, and stained with anti-MAp17 and anti-PI(4,5)P 2 antibodies. The colocalization of the GFP-tagged PH domain of phospholipase C delta and the PI(4,5)P2 antibody is indicated as a control for PI(4,5)P2-enriched membrane domains (white arrows). (C) The ratio of the PI(4,5)P 2 signal intensity to the cell area was measured by image analysis (ImageJ) ( n = 20 cells). (D) Immunoblot analysis of Rac1 GTPase and HIV-1 Gag in cell lysates and/or in virus particles. Tubulin was used as a loading control.
Article Snippet: Plasmids pCMV-LacZ and pCMV-GFP were used as control plasmids and were derived from a pCMV tag plasmid (Stratagene, Agilent Technologies, Madrid, Spain) that expresses green fluorescent protein (GFP) or LacZ under the control of the cytomegalovirus (CMV) promoter.
Techniques: Transfection, Staining