PAE (A) or NIH 3T3 (B) cells stably expressing GFP-Rac1 at levels below endogenous were examined by time-lapse confocal microscopy over one division cycle

Arrowheads indicate representative parent and daughter cells. Note that nuclear Rac1 is high immediately preceding mitosis and that GFP-Rac1 is excluded from the nuclei of the daughter cells immediately after cell division. See Videos 1 and 2 (available at ). Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division"</p><p></p><p>The Journal of Cell Biology 2008;181(3):485-496.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364699.</p><p></p

COS-1 cells were separated into nuclear and nonnuclear fractions as described in Materials and methods

Equal cell equivalents of each fraction were analyzed by SDS-PAGE and immunoblots (inset) for the indicated proteins. Immunoprecipitated proteins were detected and quantified with [I]protein A and phosphorimaging, and the percentage of total protein in the nuclear fraction was calculated (mean ± SEM; = 3).<p><b>Copyright information:</b></p><p>Taken from "Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division"</p><p></p><p>The Journal of Cell Biology 2008;181(3):485-496.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364699.</p><p></p

(A) PAE cells stably expressing GFP-Rac1 at levels below endogenous Rac1 (Fig

S1, available at ) were scored for the percentage of cells showing strong nuclear fluorescence before and 16 h after the addition of increasing amounts of the indicated compounds. Representative cells at the indicated dose are shown with the percentage of cells showing each phenotype indicated (left), and cumulative dose-response data are shown on the right (mean ± SEM; = 3). Bars, 10 μm. (B) Endogenous Rac1, Ras, and RCC1 were measured in the nuclear fractions as described in before and after the addition of 50 μM apigenin for 24 h (mean ± SEM; = 4).<p><b>Copyright information:</b></p><p>Taken from "Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division"</p><p></p><p>The Journal of Cell Biology 2008;181(3):485-496.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364699.</p><p></p

(A) PAE cells stably expressing GFP-Rac1 at levels below endogenous were synchronized in G1/S by serum starvation followed by hydroxyurea and then released

The percentage of cells with nuclear Rac1 was determined hourly and plotted as mean ± SEM ( = 4). (B) Aliquots of the cells analyzed in A were scraped from plates at the indicated times and analyzed for stage of the cell cycle by propidium iodide and cytofluorimetry. (C) Unsynchronized COS-1 cells were transfected with GFP-Rac1 and, after 16 h, fixed and stained for cyclin A. Although the cell expressing GFP-Rac1 in the nucleus (arrows) stained for cyclin A, a marker of G2/M, those excluding the protein from the nucleus (arrowheads) did not. This correlation held for 93% of transfected cells examined (>100). Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division"</p><p></p><p>The Journal of Cell Biology 2008;181(3):485-496.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364699.</p><p></p

(A) Nuclear and nonnuclear fractions were prepared as described in Materials and methods

After separation, each fraction was brought to 1% Triton X-114 and phase separation was initiated by heating the samples to 37°C. The aqueous and detergent phases of each fraction were analyzed for Rac1, RhoGDI, and lamin B by immunoblotting. Immunoblots were quantified with [I]protein A and phosphorimaging, and the percentage of total protein in each fraction in the detergent phase was calculated (mean ± SEM; = 3). (B) Triton X-114 partition as shown in A was performed on the nuclear fractions of COS-1 cells treated overnight with or without 10 μM simvistatin. Aq, aqueous; Det, detergent phases. (C) Selected images of GFP-Rac1 in COS-1 and ECV cells showing prominent decoration of the nuclear envelope (arrowhead). Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division"</p><p></p><p>The Journal of Cell Biology 2008;181(3):485-496.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364699.</p><p></p

(A) Confocal images of asynchronous T98G cells expressing GFP-Rac1 with the percent of the transfected population represented by each pattern indicated

T98G (B) or IMR-90 (C) cells were synchronized by serum deprivation for 72 h and then induced to cycle by refeeding with 10% FBS. Aliquots of cells were harvested at the times indicated and assayed for Rac1 and the indicated control proteins by immunoblotting. Bar, 5 μm.<p><b>Copyright information:</b></p><p>Taken from "Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division"</p><p></p><p>The Journal of Cell Biology 2008;181(3):485-496.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364699.</p><p></p

Localized diacylglycerol-dependent stimulation of Ras and Rap1 during phagocytosis

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcgamma receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcgamma receptor-mediated phagocytosis to enhance the innate immune response.  </p

Single-Walled Carbon Nanotubes Deliver Peptide Antigen into Dendritic Cells and Enhance IgG Responses to Tumor-Associated Antigens

We studied the feasibility of using single-wall carbon nanotubes (SWNTs) as antigen carriers to improve immune responses to peptides that are weak immunogens, a characteristic typical of human tumor antigens. Binding and presentation of peptide antigens by the MHC molecules of antigen presenting cells (APCs) is essential to mounting an effective immune response. The Wilm’s tumor protein (WT1) is upregulated in many human leukemias and cancers and several vaccines directed at this protein are in human clinical trials. WT1 peptide 427 induces human CD4 T cell responses in the context of multiple human HLA-DR.B1 molecules, but the peptide has a poor binding affinity to BALB/c mouse MHC class II molecules. We used novel, spectrally quantifiable chemical approaches to covalently append large numbers of peptide ligands (0.4 mmol/g) onto solubilized SWNT scaffolds. Peptide-SWNT constructs were rapidly internalized into professional APCs (dendritic cells and macrophages) within minutes <i>in vitro</i>, in a dose dependent manner. Immunization of BALB/c mice with the SWNT–peptide constructs mixed with immunological adjuvant induced specific IgG responses against the peptide, while the peptide alone or peptide mixed with the adjuvant did not induce such a response. The conjugation of the peptide to SWNT did not enhance the peptide-specific CD4 T cell response in human and mouse cells, <i>in vitro</i>. The solubilized SWNTs alone were nontoxic <i>in vitro</i>, and we did not detect antibody responses to SWNT <i>in vivo</i>. These results demonstrated that SWNTs are able to serve as antigen carriers for delivery into APCs to induce humoral immune responses against weak tumor antigens