, C) Cartoon of the wildtype (Cof1 wt ), floxed conditional (Cof1 fl ) and deleted (Cof1 ? ) cofilin1 alleles. CD4-cre mediated recombination will delete exon2 in thymocytes and generate the knock-out allele (Cof1 ? ); exons, loxP sites, diagnostic EcoRI sites and Southern blot probe are indicated. (D) Developmental control of CD4-cre mediated deletion in thymocyte subpopulations using a Rosa26-Stop-YFP CD4cre reporter line. The vertical line in the histogram plots separates negative cells (left) and recombined, percent of YFP-positive cells (right). CD4-cre activity is already detectable at the DN stage, recombination increases at the DP stage, in CD4SP, and CD8SP cells. (E) Southern blot of control (Cof1 wt/fl ) and Cof1 fl/fl
, Cof1 fl/fl,CD4cre thymocytes, but no recombination in tail DNA used as controls
, Equal amounts of lysates from control (ctrl.) and mutant (Cof1 fl/fl,CD4cre ) thymocytes, brain and uterus were loaded and probed with antibodies against cofilin1, cofilin2, ADF, gelsolin, CapG and GAPDH, p.4
, Figure 2: Actin filament dynamics in cofilin1 depleted thymocytes
, In cofilin1 depleted thymocytes, F-actin was significantly increased and G-actin decreased. (B) Absolute amounts of Triton insoluble F-and Triton soluble G-actin were determined by high speed sedimentation and subsequent western blot analyses of control (n=4) and Cof1 fl/fl,CD4cre (n=3) thymocytes. Cofilin1 depleted thymocytes showed increased F-actin (pellet) and decreased G-actin (spn) levels (standard deviations with *0.01<P<0.05; unpaired two-tailed Students t-Test). (C) Factin dynamics in SDF-1? stimulated total thymocytes. F-actin was monitored by phalloidin staining and FACS analysis at the indicated time points after stimulation. The control is shown as solid line, cofilin1 depleted cells as dashed line. Values were normalized to unstimulated cells. Note that the data represent biological and not technical replicas, Quantification of relative F-and G-actin levels in Cof1 fl/fl,CD4cre thymocytes with respect to control thymocytes (ctrl.) using phalloidin/DNaseI staining and FACS analysis (n=7, standard deviations are shown with **0.001<P<0.01, unpaired two-tailed Students t-Test)
, 001<P<0.01, unpaired two-tailed Students t-Test), Journal of Cell Science ? Accepted manuscript
, Single positive thymocytes were lacking in Cof1 fl/fl,CD4cre mutant mice, Figure, vol.3
, CD4cre mice (n=5). (B) FACS analysis of total control (ctrl.) and Cof1 fl/fl,CD4cre thymocytes for CD4 and CD8, showing the differentiation block from DP cells to SP cells. CD4 and CD8 single positive thymocytes were decreased in Cof1 fl/fl,CD4cre mice. Percentages of each cell population are given in the respective quadrants of the FACS plots. (C) Statistical analysis of thymocyte subpopulations. On average the number of DN and DP thymocytes were not altered in Cof1 fl/fl,CD4cre mice, while CD4SP and CD8SP cells were severely reduced when compared to control mice (n=5). (D) Expression of TCRß receptor and CD69 in total thymocytes from control (dashed line) and Cof1 fl/fl,CD4cre mice (solid lines). Histogram of FACS analysis is shown for TCRß high and CD69 + thymocytes, The total number of thymocytes was comparable in control (ctrl.) and Cof1 fl/fl
, TCRß high /CD69 + population is severely diminished in Cof1 fl/fl,CD4cre mice. The bar chart (right panel) shows statistical analysis of TCRß high CD69 + cells in the DP gated subpopulation (n=7). (E) The thymic medulla (white dotted lines) was reduced in adult
, Quantitation of the medullary area (right panel), relative to total thymus size (n=8)
, 001; unpaired two-tailed Students t-Test, Journal of Cell Science ? Accepted
, Under both conditions control thymocytes (black lines) and Cof1 fl/fl,CD4cre thymocytes (grey lines) showed comparable expansion (n=8; standard error is shown; ns p<0,5; unpaired two-tailed Students t-Test). (D) Cell in control (ctrl.) and Cof1 fl/fl,CD4cre thymocytes. (E) CD4 and CD8 surface expression on thymocytes was determined by FACS analysis before (untreated) and 14h after Pronase treatment either at 4°C or 37°C, respectively. Recovery of CD4/CD8 expression is shown in the upper right quadrant
, receptor recovery from 0 to 14h after Pronase treatment in control (ctrl., black line) and Cof1 fl/fl,CD4cre thymocytes (grey line) (n=5, diagram displays standard deviations; ns 0,05<P; unpaired two-tailed Students t-Test), Journal of Cell Science ? Accepted manuscript
, Figure 6: Adhesion and migration were impaired in Cof1 fl/fl
, Adhesion and (B) spreading of control (ctrl.) and Cof1 fl/fl, CD4cre thymocytes to ICAM
,
, Transwell migration of thymocytes from control (ctrl.) and Cof1 fl/fl,CD4cre mice in the absence (media) or presence of SDF-1? or CCL25 in the lower compartment
, Cof1 fl/fl,CD4cre thymocytes in a collagen gel either unstimulated (media) or stimulated by SDF-1? or CCL25. Migrating cells were analysed by time-lapse microscopy over 6h. (n=4). (E) Migration speed of control
, Data are presented as whisker plots. Whiskers comprise 95% of the non-parametric Mann-Whitney Rank Sum test, normality of data was tested by Shapiro-Wilk test. (F) Random migration path of SDF1? stimulated control (black) and Cof1 fl/fl,CD4cre (red) thymocytes in collagen. (G) Binning analysis of (ctrl.) and Cof1 fl/fl,CD4cre thymocytes (middle panel). Proportion of resting periods in Cof1 fl/fl, and Cof1 fl/fl,CD4cre thymocytes in collagen gel either unstimulated (media) or stimulated by SDF-1? or CCL25
, Expression of extracellular ICAM-1 receptor chains CD11a and CD18 was comparable in DP thymocytes from control (ctrl.) and Cof1 fl/fl,CD4cre mice. (B) Expression levels of chemokine receptors CD184 (CXCR4) for SDF-1a, p.199
, CD4cre mice 20,1% of DN cells were g,d positive, while in controls (ctrl.) only 6,6% g,d positive cells were found. (D) In a Rosa26-Stop-YFP CD4cre reporter mice, only 4,5% of of g,d cells were YFP positive, indicating that the CD4-cre transgene is only marginally expressed in g,d cells. Consequently the increased g,d cell number observed in (C) is due to a non-cell autonomous epiphenomenon. (E) The absolute numbers of CD19 positive B cells and CD4 positive T cells per organ were determined in lymph nodes of control (ctrl.) and Cof1 fl/fl,CD4cre mice, Cof1 fl/fl
, Supplementary information Journal of Cell Science ? Supplementary information