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

Abstract

Podosomes 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 micrometers of matrix in 24 hours. Podosome-associated 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.

Fig. 1.

Correlative LM and EM of the DC podosomes. (A-C) Low-magnification fluorescent micrographs of a DC podosome region stained with TRITC-phalloidin (A) or paxillin (B); merged image (C). (D-F) The boxed area from C is magnified in (D) (arrows indicate actin-rich foci) and (F) (asterisks indicate interconnected paxillin-rich rings). (E) The correlative EM image (epoxy resin section). Discrete electron lucent regions (arrows) correlate with the actin-rich cores in E (arrows). The electron lucent areas are surrounded by an interconnected electron-dense reticular profiles (asterisks in E) that correlate with paxillin-rich ring (asterisks in F). Arrowhead in E shows irregular border between core and ring regions. (G-I) Micrographs are alternate epoxy resin sections (90 nm thick) through correlated podosome regions (LM not shown). Image G is closest to the membrane and I furthest inside the cell. Dots in G-I indicate positions of actin-rich cores and asterisks electron-dense rings. (J) Reconstruction of podosomes using six successive 90-nm sections from series G-I (see Materials and Methods). Darkest green is closest to the membrane and lightest green furthest inside the cell. The reconstructed podosome cores (dots) are dome shaped and, here, extend at least 450 nm into the cell. Bars (A-C) 2 μm; (D-J) 1 μm.

Fig. 2.

EM and SIM of DC podosomes. (A-D) EM sections of DC podosomes orientated orthogonal to the substratum. (A) Epoxy resin section in which the dome shape is evident (arrows) and the peripheral ring regions (asterisks) lie adjacent to dome-shaped core. (B) HM23 resin section in which gold labelling for actin is most intense towards the core centre. The matrix has slightly greater electron density compared with the less intensely labelled periphery (arrows). (C) Epoxy resin section. The core regions of three dome-shaped podosomes (arrowed) are flanked by adjacent ring regions (bars). The ring regions display puckered membrane profiles, and in HM23 section displayed in D the gold labelling for paxillin is located along striations extending orthogonally from the plasma membrane (arrows). The insert illustrates the striations as they appear in optimally contrasted epoxy resin sections extending from puckered membrane regions of the ring. In these orthogonal sections, podosome height was 685 nm and width at the membrane was 1245 nm (see Materials and Methods; n=96, s.d.=0.22 μm for each, respectively). (E-H) SIM. Image E shows actin (phalloidin labelling; red) concentrated in the core regions that have irregular borders, whereas ring regions contain actin filaments (insert; arrowheads). In F the vinculin ring (using Alexa-Flour 488 secondary antibody; green) is distributed in patches often situated between the actin filaments (arrows in E and F). Image H is a Z-projection through the same podosome region as in E-G, taken along the white line in G. Arrows in G and H indicate corresponding actin-rich podosome cores, and white arrowheads in H indicates vinculin staining in linear arrays extending away from the membrane. Bars (A-D) 200 nm, insert in (D) 100 nm. (E-H) 2 μm and insert in (E) 1 μm.

Fig. 3.

Protrusion and matrix degradation in polycarbonate filters impregnated with crosslinked gelatin. (A) Protrusion from a DC podosome region (*) into pores of a polycarbonate filter in collage of EM micrographs. (B) Detail of protrusion in A. The protrusion contacts gelatin in the pore (pale green) and is immunogold labelled for actin along its length. The protrusion base is continuous with podosomes above and is also labelled for actin. At the pore rim, striations extend into the podosome (arrow). (C) Pore protrusion shows irregular close contact with gelatin. Electron-dense striations (arrows) extend into the podosome close to the pore-aperture and extend from puckered plasma membrane profiles typical of a podosome ring region (line). (D) Representative paxillin distribution (HM23 section). Gold particles label electron-dense areas (arrows) that flank the protrusion base and a striation is indicated (arrowhead). Gelatin is coloured pale green in A-D. Bars in (A) 1 μm, (B-D) 500 nm.

Fig. 4.

Mapping of podosome components using quantitative immuno-EM. (A-H) Immunogold labelling over podosomes. (A-C,F) Gold labelling quantified in standardised fractions of the total podosome profile(s) as described in Materials and Methods. Black outlines represent the average location of the cytoplasmic border and the grey line the plasma membrane. Examples of labelling for actin and paxillin are shown in Fig. 2, and here for Tyr-P in panel D and gelsolin in panel G. Bar charts in panels E (Tyr-P) and H (gelsolin) show the fraction of specific label over different compartments estimated from control experiments. Cyt, cytosol; Nuc, Nucleus; ER, endoplasmic reticulum; End, endosome; Ruff, ruffles; PM, plasma membrane; Pod, podosome. See Materials and Methods for further details. For each protein n=5-28 podosomes, >200 total gold particles from three spleen preparations were analysed. (I-K) Immunogold labelling over protrusions; quantification as described in Materials and Methods. Black dashed line represents the average location of the podosome-cytoplasm junction; grey line represents the average location of plasma membrane. For each protein n=5-32 podosomes, 200 gold particles and three spleen preparations were analysed. (L,M) Localisation of Tyr-P (arrows) in the tip (L) and ring region at the base of a protrusion (M). Bars in density maps (A-C and F; I-K) represent 200 nm, in D and G 200 nm, in L and M 500 nm. (N) Overlay of peak intensities from distributions shown in (A-C,F) and (I-K) in podosomes and protrusions. The actin-rich core is partially obscured by the paxillin and Tyr-P peak intensities.

Fig. 5.

Quantitative analysis of podosome protrusion and degradation in wild-type and MMP-14^−/− DCs. (A) Fractional area of pore occupied by protrusions, quantified using intersection counting of podosome and non-podosome regions (see Materials and Methods); Wt, wild-type; MMP-14^−/−, MMP-14 knockout in (A,C-E). (B) Representative diagram of the measurements and terminology used in text and (C-E). (C) Measurement of processes extending into pores beneath podosome and non-podosome regions (Non-podo). (D) The pore-aperture to gelatin distance (Pore depth) was measured at podosome regions, non-podosome areas (Non-podo) and cell free areas of the filter (Non-cell). (E) The fraction of protrusions contacting gelatin directly (defined in Materials and Methods) as counted in podosome and non-podosome regions. Data in A,C-E were obtained from three independent experiments. Error bars represent ± s.e.m.

Fig. 6.

Myosin-IIa distribution and quantitative effects of Blebbistatin on DC podosomes. (A) Myosin-IIa distribution in podosome regions. Upper panels show overviews of a podosome region stained for vinculin, myosin-IIa and actin. Lower panels show Z-reconstructions from the same cell. Podosomes in lower panels are from the white line of the merged image in the upper panel. Myosin-IIa is distributed to the periphery of the actin staining of the podosome (see upper panel) but does not overlap with actin (core) or vinculin staining (ring) (B) Migration of SDCs through a 3-μm filter pore in the absence (Control) and presence of 10 μM Blebbistatin over 8 hours. (C) Podosome protrusion length in the control after 7.5 hours or 0.5 hours of treatment with 10 μM Blebbistatin. (D) Frequency distribution of podosome protrusion lengths. For clarity, categories (0.39 μm) are labelled alternately with only the lower size limit included. Data in B and C were obtained from from three independent experiments; data in D were obtained from 300 measurements per experimental condition. Error bars represent ± s.e.m. Bars (A) upper panels 5 μm; lower panels 1 μm.

Fig. 7.

Gold-particle uptake is associated with podosomes. DCs grown on crosslinked gelatin-containing colloidal gold-gelatin complexes were processed for epoxy resin TEM. (A,B) Low-power micrographs. Non-podosome containing cells (A) lie on a smooth-surfaced unlabelled gelatin layer that is superficial to a gold-labelled zone lying on the filter surface (arrowhead). In podosome-containing (B, asterisk) cells, the smooth unlabelled gelatin surface is absent and the podosome displays an irregular plasma membrane inside the gold-labelled gelatin. Insets show lysosome-like profiles that are label free in A (non-podosome cell profiles) and gold-labelled in B (podosome cell profiles). (C,D) Higher magnification views of podosome regions (C) and podosome processes (D) show invagination of cell surface (arrowheads) and compaction of gold-gelatin complexes close to the membrane (arrows). (E) Canaliculus (arrowhead) in a podosome region that contains gold particles (arrows). (F) Canaliculi (arrows) extend into a podosome ring region from groups of compacted gelatin-gold particles that were surrounded by plasma membrane invaginations. Bars, (A,B) 1 μm, inserts 200 nm; (C,D) 500 nm; (E,F) 300 nm.

Fig. 8.

Models of protrusive podosomes from quantitative EM data. (A) Representative model drawn to scale to illustrate the data shown in Fig. 5. The podosome region is 50 μm² and pores measure 1 μm across. The panel illustrates the increased frequency of pore occupancy and increased length of protrusions below podosomes. (B) Model of protruding podosome proposed from the data shown in Figs 1, 2, 3, 4, 5, 6 and 7. In the periphery, recruitment of paxillin and vinculin at sites of integrin binding (not shown) promotes assembly of the ring structure, which has a fibrillar composition. The inner surfaces of the ring provides an interaction domain for actin filaments, vinculin and paxillin, allowing for stabilisation of the interaction, possible force generation and membrane puckering. Centrally, close to the plasma membrane, podosome assembly occurs via phosphotyrosine signalling and the phosphorylation of actin regulatory proteins. Protrusion at the membrane is driven by actin treadmilling activity that is associated with gelsolin capping and cleavage. Without progressive protrusion the podosome rapidly disassembles. Actin assembly, therefore, occurs centrally in the developing protrusion while mechanical tension is generated in the more stable periphery. (C) Protrusion is coupled to degradation by membrane located MMP-14 and protrusion extends into space generated by the degradation. Degraded matrix is removed at the periphery of the protrusion by tubules or larger endocytic carriers (not shown).