This study has revealed the existence of a new organization of invadosomes, with a specific linear architecture, selectively and specifically induced by the physiological fibrillar organization of collagen I, in a β1- and β3-independent manner. These structures, which we named “linear invadosomes,” were observed for the first time in primary LSECs in which the original organization of the actin cytoskeleton, and more precisely the low abundance of stress fibers, helped us to detect them. We were then able to find them in other cell types, even in the presence of a high level of stress fibers, thanks to their accumulation of the scaffold protein Tks5, which has been proven essential for their formation. Indeed, linear invadosomes were observed in all cell types examined, including, as described here, endothelial cells from various vascular beds, macrophages, fibroblasts, and tumor cells. However, the percentage of cell-forming linear invadosomes and the level of associated degradation vary from cell type to cell type. As linear invadosomes may result from the association of multiple protein partners and integration of different molecular events, we assume that this difference reflects the variation of their contributions according to cell type.
Linear invadosomes are inducible structures strictly dependent on the presence of collagen I fibrils. They were assembled within minutes upon contact with collagen I fibers. This is notably faster than the induction delay of invadosomes by soluble agents, which is variable, being 6 h with transforming growth factor β for BAECs (Varon et al., 2006
) or 30 min to 1 h with phorbol-12 myristate-13-acetate (PMA), phorbol-12,13 dibotyrate (PDBu), and sodium fluoride (NaF) for different cell types, such as endothelial and smooth muscle cells (Hai et al., 2002
; Tatin et al., 2010
). In addition, following induction, the number of podosomes starts to decrease after 30 min to 1 h upon PMA or PDBu treatment (Hai et al., 2002
; Tatin et al., 2010
), whereas linear invadosomes appear stable for up to several hours. Altogether, this suggests that the contact with collagen I fibers is sufficient to assemble and stably maintain the invadosome machinery, F-actin and associated proteins, such as Tks5 and metalloproteinases.
Dynamic observations clearly demonstrated movements of fibrils associated with those of linear invadosomes, suggesting a strong link between both structures. However, whereas every linear invadosome is associated with a type I collagen fibril, it is important to notice that every collagen I fibril does not induce linear invadosome formation. This observation suggests that the nature, density, diameter, and, potentially, the cross-linking level of the fibril may be involved in linear invadosome formation. Whereas it has already been shown that collagen matrix architecture dictates three-dimensional migration modes of human macrophages (Van Goethem et al., 2010
), we show that collagen I architecture also induces the linear invadosome formation.
As integrins are the major receptors for collagen and are found associated to podosomes and invadopodia, we took great care to examine the presence and function of integrins in linear invadosomes. Using immunocytochemistry, integrin-deficient cells, and RGD peptide, we show that β1 and β3 integrins are not localized within linear invadosomes, nor are they necessary for linear invadosome formation and activity. The lack of β1 and β3 integrins in linear invadosomes thus reflects a major difference between linear invadosomes and other invadosomes and raises the question of the identity of the collagen receptor responsible for linear invadosome formation.
At this time, four major classes of vertebrate transmembrane receptors are known to interact directly with the native collagen triple helix: collagen-binding β1 integrins, discoidin domain receptors (DDRs), glycoprotein VI (GPVI), and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1; Leitinger and Hohenester, 2007
). Our data eliminated β1 and β3 integrins. Since GPVI is present only on platelets and LAIR-1 on leukocytes, we turned our attention to DDRs, which are ubiquitously expressed. Our preliminary data seem to exclude a role for DDRs since: 1) LSECs isolated from DDR2−/−
mice (Labrador et al., 2001
) were still able to form linear invadosomes to the same extent as WT LSECs (unpublished data); and 2) nilotinib and imatinib, which potently inhibit the kinase activity of both DDR1 and DDR2 receptors (Day et al., 2008
), did not affect the formation of linear invadosomes in cells exposed to type I collagen fibrils (unpublished data). We also ruled out the role of CD44, another type of collagen I receptor (Jalkanen and Jalkanen, 1992
) known to play an important role in podosome formation in osteoclasts (Chabadel et al., 2007
), since CD44−/−
MEFs (Shi et al., 2006
) did not show any defect in linear invadosome formation (unpublished data). Further work is required to identify the collagen I receptor involved in the formation of linear invadosomes. In addition, redundancy and association between receptors, including with other classes of integrins, need to be considered to fully investigate the molecular mechanism allowing formation of linear invadosomes.
Like integrins themselves, integrin-associated proteins, such as vinculin or paxillin, are also not localized within linear invadosomes. Thus, linear invadosomes may be viewed as simplified but functional invadosomes containing only core elements, such as Tks5, which is necessary for linear invadosome formation. Recently it was shown that podosome cores can be formed in osteoclasts lacking the integrin adaptor kindlin-3−/−
(Schmidt et al., 2011
). The presence of podosome cores in β1−/−
, and αv−/−
triple-null integrin knockout osteoclasts suggests that podosomes cores are integrin-independent structures (Schmidt et al., 2011
). Linear invadosomes retain their capacity to degrade ECM elements. The absence of adhesion proteins in linear invadosomes suggests that collagen I fibrils could bypass the role of podosome adhesion rings in terms of organization.
One of the major results of this study is the finding of a strong matrix degradation activity associated with linear invadosomes. In the case of Src-activated fibroblasts, this activity is even higher than the activity of invadosome rosettes observed on gelatin alone. These observations can be reconciled with the long-known finding that pro-MMP2 can be activated by the culture of cells on fibrillar collagen I, but not on any other type of ECM component (Azzam and Thompson, 1992
; Ruangpanit et al., 2001
). Activation of the latent pro-MMP-2 zymogen is mainly due to membrane type MT1-MMP. Accordingly, we show here that linear invadosomes induced by collagen I fibrils can promote a concentration of MT1-MMP to linear invadosomes and an increase of gelatinolytic activity in a MT1-MMP–dependent process, and that the degradation activity is strictly limited to the vicinity of the collagen I fibrils. Our data also strongly suggest that linear invadosomes can degrade the underlying collagen I fibrils as well. Altogether, the results suggest that linear invadosomes can act as collagen I fibril sensors and are able to remodel the ECM.
Linear invadosomes were found in all cells tested. Their presence in normal cells (endothelial cells, fibroblasts, and macrophages) as well as in tumor cell lines suggests that they may have a major impact on physiological and pathological conditions. Since they are formed along the physiological form of collagen I and are found in a three-dimensional environment, this is compatible with a presence in vivo. We believe that they may represent one of the “physiological” configurations of invadosomes. The discovery of collagen I as a physiological inducer of invadosomes will probably help to better characterize their roles in vivo. Owing to their capacity to localize the degradation machinery along fibrils, linear invadosomes could be implicated in matrix remodeling and cell migration, either in physiological conditions, such as angiogenesis, or conditions in which collagen I is accumulated, such as fibrosis, atherosclerosis, and cancer.