Imaging podosome dynamics and matrix degradation

Abstract

Invasive cell migration is critical for leukocyte trafficking into tissues. Podosomes are matrix-degrading adhesive structures that are formed by macrophages and are necessary for macrophage migration and invasion. Here, we describe methods for imaging and quantifying podosomes in primary human macrophages and in THP-1 cells, a monocyte cell line that can be differentiated to a macrophage-like state. Moreover, we outline detailed methods for live imaging of podosomes, which are highly dynamic, and for the quantification of rates of podosome turnover. Finally, we discuss methods for the quantitative analysis of matrix degradation on fluorescent-gelatin-coated cover slips.

Figure 1. Comparison of podosomes, invadopodia, and focal adhesions

(a) Primary human macrophages differentiated with MCSF for 7 days and (b) THP-1 cells differentiated for 48 hours after PMA treatment were stained with rhodamine phalloidin and an anti-vinculin antibody. Note the prominent ring structures in the vinculin images that are characteristic of podosomes. (c) MTLn3 rat mammary carcinoma cells stained with rhodamine phalloidin and an anti-vinculin antibody. Note the localization of vinculin to focal adhesions, which are formed at the termini of actin stress fibers (arrows), and the absence of vinculin rings in areas corresponding to the punctate invadopodia (arrowheads). Scale bar = 10 μm for all panels.

Figure 2. Schematic of Podosome Structure

Pictured on the top right is a cell forming a cluster of podosomes as viewed from above. The gross podosome structure is depicted as black dots and grey rings. Below is a magnified cross section of an individual podosome. The colors of the proteins in the cross-section correspond to their location in the podosome core (black) or ring (grey). Matrix metalloproteinase 9 (MMP-9) is secreted from podosome cores and is capable of degrading the underlying extracellular matrix (ECM). Degraded matrix is depicted as dashed lines.

Figure 3. Live fluorescent imaging of podosomes in primary human macrophages

Primary human macrophages were differentiated for 7 days with MCSF. On day 7, the macrophages were nucleofected using 0.5 μg of pRuby-Lifeact and 0.5 μg of pEGFP-vinculin and plated on fibrinogen-coated glass bottom dishes. Time-lapse images were acquired using the following settings: binning = 3 × 3, gain = 2, Lifeact exposure = 15 ms, and vinculin exposure = 15 ms. Images were taken at 1 minute intervals for 20 minutes. Lifeact and vinculin images are shown at 2 minute intervals. Arrows indicate assembling or disassembling podosomes. Scale bar = 10 μm.

Figure 4. Gelatin degradation by primary macrophages

Cover slips were coated with 200 μg/mL Oregon Green-488 gelatin. 3 × 10⁴ primary human macrophages, which had been differentiated for 7 days with MCSF, were added to the cover slips. Podosomes were stained with rhodamine phalloidin. To determine the percentage of cell area degraded, a degradation coefficient of 0.3 was used for each image. The white shading in the right-most column indicates the area that was thresholded as degradation. The percentage of cell area degraded is indicated. Images were selected to demonstrate various degrees of degradation. Scale bar = 10 μm.