Dendritic cell podosomes are protrusive and invade the extracellular matrix using metalloproteinase MMP-14

Abstract

SummaryPodosomes are spot-like actin-rich structures formed at the ventral surface of monocytic and haematopoietic cells. Podosomes degrade extracellular matrix and are proposed to be involved in cell migration. A key question is whether podosomes form protrusions similar to the invadopodia of cancer cells. We characterised podosomes of immature dendritic cells using electron microscopy combined with both conventional and novel high-resolution structured illumination light microscopy. Dendritic cell podosomes are composed of actin foci surrounded by a specialised ring region that is rich in material containing paxillin. We found that podosomes were preferential sites for protrusion into polycarbonate filters impregnated with crosslinked gelatin, degrading up to 2 mm of matrix in 24 hours. Podosomeassociated uptake of colloidal gold-labelled gelatin matrix appeared to occur via large phagosome-like structures or narrow tubular invaginations. The motor protein myosin-II was excluded from ring or core regions but was concentrated around them and the myosin-II inhibitor Blebbistatin reduced the length of podosome protrusions. Finally, we found that degradation, protrusion and endocytosis in this system are dependent on the matrix metalloproteinase MMP-14. We propose that podosomes mediate migration of dendritic cells through tissues by means of myosin-II-dependent protrusion coupled to MMP-14-dependent degradation and endocytosis.

Three‐dimensional invasion of macrophages is mediated by cysteine cathepsins in protrusive podosomes

“…MRC 1 was enriched in the protrusive podosomes and it was colocalized with CtsB in spherical or ring-shaped areas that might correspond to the nascent endocytic vesicles. Previous studies proposed that the degraded ECM is internalized by endocytosis at podosomes [16,17]. This is in line with our results showing that the inhibitor of endocytosis Dynasore reduces the intracellular degradation of DQ-collagen IV by Mfs in 3D environment (Fig.…”

“…Our results on ECM degradation, together with the real-time studies of 3D Mf migration and the detailed dynamics of CtsB activity during protrusion formation and function, suggest that the protrusive podosomes that form during mesenchymal migration are characterized by at least two functionally different regions: the protruding tip, rich in actin and adhesive molecules, and a proteolytic posterior region (the base of the protrusion). In previous studies the tips of protrusive podosomes were regarded as the main ECM-degradation sites of the invading immune cells [16,17,57]. Conversely, the highest pericellular collagenolytic activity was observed to be at the base, which is more similar to what was observed in tumor cells in 3D, where the invading leading edge develops an adhesive anterior and a proteolytic posterior [58].…”

“…Although, U937 cells originate from a human lymphoma, the role of cysteine cathepsins in ECM degradation by PMA-differentiated U937 Mfs was extremely comparable to that of M-CSF differentiated primary human Mfs, excluding the potential influence of the cancer-related invasive phenotype. We propose that the overall process of ECM remodeling mediated by cysteine cathepsins plays a central role in facilitating Mf progression through the dense 3D matrices that trigger the protease-dependent migration mode.It was recently suggested that protrusive podosomes of immune cells promote cell invasion, as they have been recognized as structures that degrade ECM matrix [16,17,57]. However, the mechanism by which the interplay between protrusive podosomes and ECM degradation mediates cell migration was not investigated.…”

Dynamics of podosome stiffness revealed by atomic force microscopy

“…Altogether this work leads us to propose that F-actin treadmilling and actomyosin complex activities generate periodic forces at podosomes. Because these processes have been proposed to participate in the protrusion of podosomes into the ECM (10,11), the involvement of their periodicity during macrophage migration remains to be elucidated. Podosome biophysical properties are likely to be coupled with their mechanosensing activity.…”

Extracellular proteolysis in macrophage migration: Losing grip for a breakthrough

“…In migrating macrophages, podosomes are concentrated at the leading edge. As for invadopodia, the tumor cell counterpart of podosomes, a link between matrix degradation and secretion of proteases at podosomes has been described [22][23][24]. Degradation of gelatin by macrophage podosomes is MMP-dependent [5] and degradation of fibronectin or collagen I correlates with the presence of MT1-MMP (also known as MMP14) at podosomes [24,25].…”