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Figure 8. Quantification of spontaneous synaptic efficacy fluctuations due to the fast exchange of receptors between synapses and the receptor pool. ; (A) Example simulation of a piece of dendrite with 7 synapses during 10 min of simulated time. The number of receptor slots in each synapse is given in the legend. The relative pool size was set to ϕ=2.67 and the filling fraction was set to F=0.5 by choosing the binding rate to receptor slots α via Equation 13. (B) Same as A. but for a higher filling fraction of F=0.9. (C) Size of synaptic efficacy fluctuations as measured by the coefficient of variation (CV) as a function of the steady state number of receptors in each synapse, which is given by the product of the filling fraction F and the number of slots si in synapse i. The relative pool size was set to ϕ=2.67. Data points represent averages over 10 simulations of 30 min simulated time each. Lines represent linear fits through the data points in double log space. (D) CV as a function of steady state number of receptors for different relative pool sizes ϕ achieved by holding the binding rate to receptor slots α fixed and varying the externalization rate γ.

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Figure 4. Exposure of PS is dependent on CD8+ T cells and FasL. ; (A) Experimental protocol for the evaluation of CD8+-T-cell-dependent PS externalization in parasitized cells in vitro. Splenic TER119+ cells containing RBC, pRBC, erythroblasts (EB) and pEB (3 × 105) isolated from gld mice 17 days after infection with PyNL–GFP were cultured for 4 hr with CD8+ T cells from WT or gld mice 17 days after PyNL infection, at the indicated ratios. (B) Cultured TER119+ cells with CD8+ T cells from the indicated mice were stained with annexin V, and GFP+ cells were analyzed for PS expression. Numbers in histograms indicate percentages of annexin V+ cells in the gated cells. (C) Values are means ± SD from triplicate cultures in one experiment, representative of the four performed. **p < 0.01, Mann–Whitney U-test. (D) Experimental protocol for the evaluation of FasL-dependent PS externalization in parasitized cells in vitro. TER119+ cells isolated from spleens and peripheral blood of gld mice 7 days after infection with PyNL–GFP were cultured for 4 hr with the indicated amounts of FasL–Strep or bovine serum albumin (negative control). (E) Cultured cells were collected and stained with annexin V, and annexin V+ cells among the GFP+ parasitized cells were quantified. Values are means ± SD of triplicate cultures in one experiment, representative of the four performed. *p < 0.05 and **p < 0.01, Mann–Whitney U-test. (F) Annexin V-positive or -negative GFP+ parasitized cells were analyzed for the expression of MHC class I antigens, as in Figure 3C.

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Figure 3. Infection with PyNL induces externalization of phosphatidylserine (PS) on parasitized cells. ; (A) Spleen cells and peripheral blood obtained from the indicated mice 8 days after infection with PyNL–GFP were stained with anti-TER119 antibody and annexin V. Infected GFP+ or uninfected GFP− TER119+ cells were analyzed for the expression of PS. (B) Percentages of TER119+ GFP+ PS+ cells in the TER119+GFP+ cells in the control (open symbols) and CD8+-depleted mice (closed symbols) are shown as means ± SD from one experiment (N = 4), representative of the three performed. (C) Those in the gld mice were also analyzed. **p < 0.01, Mann–Whitney U-test. (D) Splenic TER119+ cells infected (right panel) or uninfected (left panel) obtained from mice 8 days after infection with PyNL–GFP were analyzed for the expression of PS and Fas. Numbers indicate the percentages of the gated cells in each quadrant.

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Figure 6. Externalization of PS in parasitized cells requires contact with CD8+ T cells. ; (A) Protocol of the contact dependence assay using Transwell cultures. Splenic TER119+ cells from gld mice infected with PyNL–GFP and CD8+ T cells from WT mice infected with PyNL were placed into the upper and/or lower wells and cultured for 6 hr. GFP+ parasitized cells were analyzed for PS expression, as in Figure 4B. The ratio of the percentages of PS+ cells in the GFP+ cells in the upper (B) and lower wells (C) was calculated as (% PS+ GFP+ of GFP+ cells in each test)/(% PS+GFP+ in GFP+ cells in the absence of cell components in the lower well) in (B), and as (% PS+ GFP+ in GFP+ cells in the presence of CD8+ T cells)/(% PS+ GFP+ in GFP+ cells in the absence of CD8+ T cells in the lower well) in (C). Values shown are the means ± SD of triplicate cultures in one experiment, representative of the three performed. **p < 0.01, Mann–Whitney U-test.

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Figure 5. Externalization of PS in pRBCs was not induced in vitro. ; Peripheral blood cells obtained from gld mice infected with PyNL–GFP were cultured with CD8+ T cells (A) or FasL–Strep (B) and analyzed as in Figure 4.

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• Authors : Ko, Kwang Woo; Devault, Laura; Sasaki, Yo ...

• Source: eLife ; volume 10 ; ISSN 2050-084X

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• Source: eLife ; volume 4 ; ISSN 2050-084X

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