Analysis of cortactin- and HS1/cortactin-deficient primary

In several cell types like osteoclasts, smooth muscle cells and macrophages, a special actin-rich structure is assembled. When cells are plated on rigid surfaces like glass coverslips, so-called podosomes are formed on the ventral side of the cell that undergo rapid turnover. These structures support the adherence of cells on a substratum, but at the same time they allow fast migration, as the lifetime of podosomes is very short compared e.g. to focal contacts (see 1.1.5). The core of podosomes is built by a dense network of actin filaments, which is surrounded by a characteristic ring of proteins that are also part of the protein composition of focal contacts, such as vinculin, talin and zyxin. At variance to focal contacts, the Arp2/3 complex localizes to the actin rich core of podosomes. For this reason it is assumed that the Arp2/3 complex polymerizes the F-actin core. Support for this theory is given by the fact that also Arp2/3 activators like the class I NPF WASP and the class II NPF cortactin are present at the core of podosomes. Studies with WASP-deficient primary macrophages from transgenic mice revealed an essential role for this NPF in the formation of podosomes (Linder et al., 1999). Although cells were still able to adhere, no podosomes could be formed in the absence of WASP. In addition, RNAi studies indicated that cortactin is a major player in podosome formation, at least in osteoclasts and smooth muscle cells (Tehrani et al., 2006a; Webb, Eves, and Mak, 2006). Therefore, it was intriguing to ask, whether macrophages from cortactin-deficient mice or from mice lacking both cortactin and HS1, the hematopoietic homolog of cortactin, still displayed podosomes and if so, whether they were normal regarding their shape, protein composition and frequency of occurrence.

For these experiments, primary macrophages from control and cortactin KO mouse littermates from heterozygous crossings of mice carrying the deleted cortactin allele were isolated from the peritoneum, seeded on coverslips coated with fibronectin and stained for F-actin, vinculin and cortactin. In control cells, podosomes were visible as actin-rich dots surrounded by a ring composed of vinculin, and the actin cores co-localized with cortactin (Figure 3-13), as expected. Podosomes could also be seen in macrophages from cortactin KO mice, in spite of the complete loss of cortactin staining (Figure 3-13). When comparing the podosomes of macrophages with and without cortactin, no apparent differences were discernable regarding size and morphology in the conditions used here.

Figure 3-13: Organization of the actin cytoskeleton, and localization of vinculin and cortactin in control and cortactin KO macrophages.

Immunofluorescence stainings of peritoneal macrophages harvested from WT or cortactin KO littermates derived from heterozygous crossings of mice with the deleted cortactin allele. Both macrophage populations displayed podosomes, F-actin-rich dot-like structures (red in merge) characteristically surrounded by a ring of focal adhesion proteins like vinculin (green in merge).

In WT macrophages cortactin (green in merge) co-localized with the F-actin-rich core of podosomes, whereas no cortactin staining is visible in cortactin KO macrophages. Boxed regions in merges are displayed as insets in higher magnification. Bar, 10 µm.

However, it was not clear whether the number of cells displaying podosomes was reduced in cortactin KO macrophages. To test this, macrophages from WT and KO mice were co-stained with phalloidin and vinculin and the number of cells displaying podosomes determined. Again, no significant difference in the number of cells with podosomes was discernible between cells with and without cortactin (Figure 3-14). In conclusion, cortactin is dispensable for podosome formation in peritoneal macrophages. Nevertheless, preliminary results from a collaboration with Christiane Wiesner and Stefan Linder (UKE Hamburg) revealed that although the podosome formation upon cortactin depletion was normal, gelatin degradation was drastically reduced in macrophages from cortactin KO mice (unpublished data). Matrix degradation assays have to be repeated to confirm this phenotype.

Figure 3-14: Quantification of frequency of WT and cortactin KO macrophages displaying podosomes.

Peritoneal macrophages from WT and cortactin KO mice were stained for F-actin with fluorescently-labeled phalloidin and vinculin. Cells were categorized into cells with podosomes (“Podosomes”), without podosomes (“no Podosomes”) or with an ambiguous morphology (“amb.”). At least 100 cells were counted for each condition and the experiment was performed in triplicate. Error bars correspond to standard errors of means.

To check the expression levels of HS1 in macrophages, western blot analysis with extracts from control and cortactin KO macrophages was performed (Figure 3-15). An extract of B16-F1 cells transiently expressing HS1-EGFP served as control that the antibody recognizes HS1. In the extract from fibroblast cells no HS1 band was detectable, because HS1 is only expressed in cells of the hematopoietic lineage.

Figure 3-15: HS1 is expressed in cortactin WT and KO macrophages.

Western blot analysis of extracts from B16-F1 cells transfected with HS1-EGFP, macrophages from cortactin control (WT) and cortactin-depleted (KO) mice, Raw cells and fibroblasts with an anti-HS1 antibody (upper panel). The same blot was reprobed with an anti-tubulin antibody as loading control (lower panel). Numbers correspond to protein size in kDa.

In cortactin WT and KO macrophages as well as in the macrophage cell line Raw the antibody recognized one or two HS1 bands demonstrating that these cells express HS1. In analogy to cortactin (Huang et al., 1997), the two bands might correspond to different phosphorylation states of HS1. The HS1 band in cortactin KO macrophages is stronger compared to cortactin WT macrophages, but the protein concentration of the samples was not measured due to the low number of isolated cells, and the anti-α-tubulin blot indicates that more extract from KO macrophages was loaded (Figure 3-15). Thus, HS1 does not seem to be upregulated in cortactin-deficient macrophages in order to compensate the loss of cortactin.

In analogy to the experiments with cortactin-deficient macrophages, cells isolated from mice lacking both cortactin and its hematopoietic form, HS1, were examined in fluorescence stainings regarding the existence of podosomes. As shown in Figure 3-16, cells lacking both cortactin and HS1 were also able to form podosomes with an actin rich core and a surrounding vinculin ring.

Figure 3-16: HS1/Cttn double KO macrophages are able to form podosomes on fibronectin.

Epifluorescence images of peritoneal macrophages from HS1^del/delCortactin^WT/del and HS1^del/delCortactin^del/del mice stained for F-actin with phalloidin and with an anti-vinculin antibody.

The macrophages can form podosomes consisting of an F-actin core (red in merged insets) and a ring structure rich in vinculin (green in merged insets), as expected. Boxed regions are shown as merged inset in the right images. Bar, 10 µm.

The quantification of double KO macrophages displaying podosomes (Figure 3-17) revealed that about 85% of cells formed podosomes, irrespective of their genotype. So again no decrease in podosome formation frequency could be observed in double KO macrophages demonstrating that also the combined loss of cortactin and HS1 does not hinder cells to form podosomes.

Figure 3-17: Depletion of both HS1 and cortactin does not alter the frequency of macrophages forming podosomes.

Macrophages from HS1^del/delCttn^WT/del and HS1^del/delCttn^del/del mice were stained for F-actin. Cells were classified as with podosomes (“Podosomes”), without podosomes (“no Podosomes”) or with ambiguous phenotype (amb.). The data summarizes one experiment, in which at least 100 cells were analyzed.