Reducing background fluorescence reveals adhesions in 3D matrices

To the editor: Adhesion complexes in cells growing on planar substrates have been studied for over three decades. From these studies, several classes of adhesions have been described based on size, location, morphology, dynamics or molecular composition, and their dual role as signalling centres and linkages that connect the extracellular matrix (ECM) with the cytoskeleton has also emerged1. In contrast to this large and growing understanding of adhesion on two-dimensional (2D) substrates, little is known about the adhesions formed during three-dimensional (3D) growth of cells, including whether they even exist. Immunostaining and microscopy of fixed samples2–8 and dynamic imaging at low spatiotemporal resolution9 have shown the presence of large, elongated adhesions on cells in various 3D model systems. However, attempts to visualize adhesions in living cells growing in 3D with resolution similar to that routinely used in 2D have not been successful10. This has led to the recent conclusion that the adhesions defined and characterized in 2D cultures either do not exist in cells in 3D, or are too small or short-lived to be observed11. One major limitation of imaging live cells in 3D culture is background fluorescence caused by the overexpression of genetically encoded, fluorescently tagged proteins. Having saturated all available association sites, excess fusion proteins accumulate in the cytoplasm resulting in diffuse background fluorescence. In cells on 2D substrates, this cytoplasmic background is less detrimental because of the thinness of the lamellae in migrating cells. Moreover, background fluorescence can be reduced in 2D studies by using TIRF (total internal reflection fluorescence) microscopy, which excludes fluorescence above approximately 100 nm from the substrate. In contrast, protrusions formed on 3D matrices may be thicker12 and must be visualized using wide-field, confocal or multi-photon microscopy, which section a minimum thickness of 500–800 nm (ref. 13). Therefore, overexpression of an adhesion-specific fluorescently tagged protein can be more detrimental to imaging in 3D than in 2D and result in diffuse cytoplasmic fluorescence that masks the signal of molecules localized to adhesions. To address this issue, we used an EGFP (enhanced green fluorescent protein)–paxillin construct under the control of a truncated CMV (cytomegalovirus) promoter that was originally developed to express GFP–β-actin at very low levels14. The truncated CMV promoter reduces expression from the plasmid and therefore results in lower protein levels than those achievable by simply minimizing plasmid copy number. U2OS osteosarcoma cells were transfected with EGFP–paxillin in this vector, seeded within fibrillar collagen gels, and imaged with a laser scanning confocal microscope between 3–5 h after seeding. The collagen fibres were simultaneously imaged in reflectance mode. All images were taken several millimetres from the lateral edges of the gels. The cells were mostly multi-polar with three or more protrusions of variable size and shape that extended out from the cell body, spanned multiple focal planes, and exhibited numerous adhesions (Fig. 1a and Supplementary Information, Fig. S1a–f). Adhesions were rarely observed in areas more proximal to the cell body; however, as these areas of the cell were generally much thicker than the distal ends of the protrusion, and thus had higher cytoplasmic background fluorescence, we cannot exclude their existence. We observed adhesions in cells at depths up to the limit of the working distance of our objective (approximately 350 µm). Regardless of depth, at least 50% of the cells with a detectable level of the low-expressing EGFP–paxillin exhibited adhesions (Supplementary Information, Fig. S1g, m). Cells in which adhesions were not visible tended to have fluorescence intensities that were either relatively high (high background) or relatively low (low overall signal; Supplementary Information, Fig. S1h, n). Figure 1 Cell-matrix adhesions are detected in 3D collagen gels. (a) Z-projection of a multi-polar U2OS cell expressing EGFP–paxillin under the control of a truncated promoter, with adhesions on the distal sections of the protrusions (arrows). See Supplementary ... High-spatial-resolution imaging of the protrusions revealed distinct adhesions (Fig. 1b, lower panels) with a median length of 1 µm (0.8–1.45 µm, 25th–75th percentiles; n = 72 adhesions). Adhesions were also observed in similar short-term experiments with HT-1080 cells (Fig. 1b; median 1.2 µm; n = 36), with both cell types expressing EGFP–vinculin from the same vector (Fig. 1c), and in overnight cultures of U2OS cells (data not shown). In contrast, U2OS cells transfected with low levels of EGFP–paxillin expressed under the full-length CMV promoter, or even with high levels of the truncated promoter plasmid, had a diffuse cytoplasmic fluorescence similar to that of a fluorescent protein expressed alone (Fig. 1b, upper panels). Finally, we observed adhesions in HT-1080 cells cultured overnight in rat-tail collagen gels (Supplementary Information, Fig. S1i–n). To image adhesion formation, growth, and disassembly, we acquired time-lapse Z-stacks of protrusions from U2OS cells cultured 3–5 h as in Figure 1. As highly dynamic nascent adhesions form and disassemble rapidly with an average lifetime of about 1 min15, we imaged with a 10 s time-interval to be sure of capturing short-lived adhesions. Figure 2 shows frames from a representative time-lapse movie of the protrusion of a U2OS cell transfected with a plasmid encoding the promoter-truncated EGFP–paxillin (Supplementary Information, Video S1). The protrusion undergoes cycles of extension and adhesion formation followed by rearward adhesion movement and matrix fibre deformation. Similar observations were made in six independent experiments and with HT-1080 cells (Supplementary Information, Video S2). Figure 2 Dynamics of cell-matrix adhesions in 3D culture. Frames are taken from Supplementary Information, Video S1. The top row shows Z-projections of a protrusion end from a U2OS cell that was transfected with a plasmid encoding promoter-truncated EGFP–paxillin. ... In general, new adhesions formed along collagen fibres at the leading edge of protrusions and travelled rearward, causing fibre deformation. The adhesions had an initial diameter of 0.4 ± 0.07 µm (mean ± s.d.), placing them in the range of focal complexes, but larger than diffraction-limited nascent adhesions1,15. Over the course of the movies, most translocating adhesions grew in intensity and length; adhesions that did not translocate were stable, apparently maintaining isometric force on the matrix. Treatment with a combination of the Rho kinase (ROCK) inhibitor Y-27632 and the myosin light chain kinase (MLCK) inhibitor ML-7 decreased adhesion size similar to their effect in 2D (ref. 1). Our observations demonstrate that cell-matrix adhesions can form in 3D collagen matrices, show that their dynamics can be studied in living cells, and support previous observations by immunostaining and microscopy in collagen2,4,5,8, fibrin7,8, and cell-derived3,8 3D ECMs. In addition to reducing background fluorescence, factors that affect adhesion size (and therefore signal intensity) may affect adhesion detectability. For example, microenvironment stiffness will affect cell contractility and thus adhesion size and composition2,3,16–18. Therefore, more-pliable regions of collagen gels may have smaller and consequently less readily detected adhesions; if so, it would point to pliability, in addition to dimensionality, as the determining factor. In addition, adhesions are difficult to visualize in certain cell types19. For example, leukocytes do not show prominent adhesions on ICAM-1 coated substrates20 and can migrate in an integrin-independent manner21. The effects of cell contractility on adhesion phenotype have been well-studied in 2D systems; future work will need to determine how these interactions occur in 3D. Reducing background fluorescence overcomes an initial barrier to adhesion visualization in 3D and allows studies of the influence of 3D topography, pliability, matrix protein composition, and cell contractility on adhesion phenotype.