Figure 1.
Expression of ADF and n-cofilin in myeloid cells and gene deletion in macrophages.
(A) Western blot analysis of different myeloid cell types. In the middle panel, the filter was probed with n-cofilin and ADF specific antibodies to allow a comparison of relative amounts. Comparable levels of n-cofilin were found in total bone marrow (bm), bone marrow derived macrophages (BMM), peritoneal exudate cells (PEC), dendritic cells (DC) and granulocytes. ADF is down-regulated in BMM and PEC. Actin is shown as loading control; (B) Distribution of ADF and n-cofilin in wildtype BMM. ADF and n-cofilin (green, antibodies) co-localize with F-actin rich domains (red, phalloidin for ADF staining, and anti-actin antibody for n-cofilin staining, see arrows); (C) Time course of n-cofilin deletion in bone marrow derived macrophages after poly(I-C) treatment. Deletion on day 0, 3 and 6 of culture. Upper panel: Southern blot analysis for n-cofilin deletion as indicated by the loss of the flx-allele. Wild type (wt), conditional (flox-cof) and the deleted alleles (Δcof) are indicated. Lower panels: Western blot analysis of the respective cell lysates after n-cofilin deletion. The amount of n-cofilin strongly decreases on day 3 and slightly increases again at day 6, due to cells, which escaped the deletion. Actin and tubulin are shown as loading controls; (D) Upregulation of the macrophage marker CapG during BMM differentiation, ADF and gelsolin expression is not altered upon deletion of n-cofilin; (E, F) Cell shape of n-cofnull and ADF−/− macrophages. (E) N-cofnull BMM can be identified in n-cofflx/Δ,Mx1-cre cultures, using an anti-n-cofilin antibody (green). N-cofnull BMM had a distinct round shape and showed a high actin signal (red, anti-actin antibody). N-cofilin expressing escaper cells have a regular shape and much lower F-actin content. (F) ADF−/− macrophages (ADF antibody staining, green) have regular morphology, cell polarity and F-actin content (phalloidin, red).
Figure 2.
Characterization of n-cofilin null macrophages.
(A) Confocal sections through an adherent n-cofnull macrophage show the unusual cytoskeletal structure and the impaired leading edge formation. F-actin (in red) is shown by phalloidin staining. Note the short extensions emerging from the cell body and the lack of a lamellipodium; (B) Scanning EM of saponin extracted macrophages illustrate a loose network of short filaments in the lamellipodium of control cells, while n-cofnull BMM show densly packed cortical F-actin bundles; (C) Biochemical fractionation showed increased Triton X-100 stable F-actin levels in n-cofflx/Δ,Mx1-cre macrophage cultures (nculture = 6, p<0.05). ADF−/− macrophages showed normal F-actin levels; (D) FACS overlay of phalloidin stained macrophages from a day 6 control (grey histogram) and n-cofflx/Δ,Mx1-cre (black line histogram) culture. About 10-fold increase in phalloidin signal intensity was observed in n-cofnull cells, while the escaper cells are represented in the low intensity F-actin peak, similar to the control cells. (E) Cell adhesion on laminin is not altered in n-cofilin mutant macrophage cultures (n-cofflx/Δ,Mx1-cre) at 4 to 24 hours after plating. The average of three independent experiments performed in quadruplicates is shown; (F) Cell spreading was significantly impaired in n-cofilin mutant macrophages (n-cofnull). The spreading area was compared in control (n-cofwt/wt,Mx1-cre), ADF-/-, ADF+/− and n-cofilin mutant BMM (ncells>100, student's test, n-cofnull:control p<0,0001; ADF−/−:ADF+/− p = 0,15); (G) N-cofnull BMM fail to polarize. The shape of more than 90% of n-cofnull BMM was apolar, while 75% of control and ADF+/− cells were either bipolar or multipolar (ncells>130). ADF−/− BMM showed a noticeable tendency to increase cell polarization.
Figure 3.
Cell proliferation and cytokinesis is impaired in n-cofilin null macrophages.
(A) Cell proliferation was impaired in n-cofflx/Δ,Mx1-cre cultures. Within 120 hours after plating, n-cofflx/Δ,Mx1-cre bm cells did not expand (average of three independent experiments is shown); (B) Cytokinesis defect in n-cofnull cells and accumulation of multinucleated cells (ncells>150, 2-way ANOVA analysis: Mx1-cre:n-cofnull p<0.0001). ADF is not required for cytokinesis; (C) In n-cofflx/Δ,Mx1-cre cultures DNA synthesis decreased during the first days of bone marrow culture (ncells>200); (D) TUNEL staining showed slightly increased apoptosis in n-cofflx/Δ,Mx1-cre cultures (ncells>100).
Figure 4.
In vitro and in vivo migration of macrophages lacking n-cofilin.
(A) Random motility of n-cofnull BMM is impaired, ADF−/− macrophages behave normally. Velocity of center movement over a period of 20 min is shown (ncells>60, student's test n-cofnull:control p<0,0001); (B) Over a period of 8 hours n-cofnull cells show no significant net translocation, cell protrusions collapse back onto the cell body (see arrows). Migratory tracks are shown by white lines and dots, for two cells each (see also videos S1, S2, S3); (C) In vivo recruitment of n-cofnull cells to sites of inflammation is impaired. Thioglycollate injection (TG) into n-cofflx/Δ,Mx1-cre mice does not lead to recruitment of mutant macrophages (Δcof) to the peritoneum (PEC). N-cofilin depleted cells remain in the bone marrow (bm). Southern blot of the respective mutant cell fractions (n-cofflx/Δ,Mx1-cre) is shown in the left panel, and the corresponding western blot in the right panel. PEC mainly consists of escaper cells as indicated by the equal Δcof/flox-cof signals in Southern blot and the presence of n-cofilin protein. Western blot analysis of control mice (n-cofwt/wt,Mx1-cre), is shown on the right panel to illustrate the normal expression levels of n-cofilin in bm and PEC under these conditions; (D) FACS analysis of bone marrow (bm) and PEC from the experiment in (C). Bone marrow composition after TG injection (TG) and n-cofilin deletion (poly(I−C)+TG) was comparable (left panels). The main portion of bm cells were Mac1/Gr1 positive, few B cells (B220) and T cells (CD3) were detected. By FACS the Mac1 and Gr1 positive cells in bm appear as one population. 3 days after TG treatment, the majority of PECs in controls are positive for the macrophage marker Mac1 (upper right panel). Upon deletion of n-cofilin (poly(I−C)+TG), the PEC population increases in Gr1 (granulocytes) and decreases in Mac1 (macrophages).
Figure 5.
Immunological responses of n-cofilin mutant dendritic cells.
(A) Phagocytosis of FITC-labelled E. coli in n-cofflx/Δ,Mx1-cre, ADF-/-, ADF+/-, and control macrophage cultures (n-cofwt/wt,Mx1-cre); (B) Phagocytosis of FITC-zymosan by individual macrophages was scored (ncells>100). Phagocytosis in ADF−/− cells was not changed; (C) TNF-α production induced by Zymosan phagocytosis. Macrophages were allowed to ingest zymosan particles and after 16 hours, TNF-α levels were determined in the cell supernatant; (D-E) Impaired T-cell stimulation by n-cofilin mutant dendritic cells. Serial dilutions of OVA323-339 peptide were presented by n-coffwt/wt,Mx1-cre or n-cofflx/Δ,Mx1-cre dendritic cells to OT-II T-cells. T cell stimulation was measured by production of IL-2 (D) and IFN-γ (E) after 24 hours; (F-G) T-cell stimulation of ADF−/− dendritic cells was similar to ADF+/− controls; (H) MHC class II-induced contact formation is impaired in n-cofnull macrophages. A mix of control macrophages (escaper cells) and n-cofnull macrophages, preincubated with anti-MHC class II antibody were allowed to form contacts with the beads. Control cells form F-actin free contact zones (arrows) with anti-mouse-IgG-coated beads (FITC labeled, green), while n-cofnull macrophages showed a F-actin rich domain (phalloidin, red) at the interface with the bead (arrows).