Single‐Molecule Tracking and Super‐Resolution Microscopy Unveil Actin‐Driven Membrane Nanotopography Shaping Stable Integrin Adhesions in Developing Tissue

ABSTRACT

Single molecule tracking and super‐resolution microscopy of integrin adhesion proteins and actin in developing Drosophila muscle attachment sites reveals that nanotopography triggered by Arp2/3‐dependent actin protrusions promotes stable adhesion formation. The nanodomains formed during this process confine the diffusion of integrins and promote their immobilization. Spatial confinement is also applied to the motion of actin filaments, resulting in enhanced mechanical connection with the integrin adhesion complex. Fabricated nano‐structured surfaces mimicking the nanotopography observed in living tissue are able to recapitulate the formation of these adhesions in isolated muscle cells and the confinement of integrin diffusion. These results emphasize the importance of geometrical regulation of tissue morphogenesis at a single molecule level.

Integrin adhesions mediate the attachment of cells to the extracellular matrix and drive cell locomotion. The most studied type of integrin adhesion is its mature form, called a focal adhesion, which is bound to actomyosin stress fibers and forms on a flat surface, usually between motile cells and a glass coverslip (Kanchanawong and Calderwood 2023). Focal adhesions present a flat but layered organization with an integrin signaling layer at the bottom and an actin layer on the top, sandwiching a force transduction layer involving proteins like talin and vinculin (Kanchanawong et al. 2010). The molecular dynamics of proteins in these adhesion macromolecular complexes have been studied by single particle tracking photo‐activation localization microscopy (sptPALM), revealing that individual molecules can enter the adhesion and engage in cycles of diffusion and immobilization (Orré et al. 2021; Rossier et al. 2012; Tsunoyama et al. 2018).

However, integrin adhesions exhibit morphological and functional diversity in different cell types and tissues where the nanoscale organization and protein dynamics need to adapt to changing environments (Vicente et al. 2023). For example, in the developing Drosophila embryo, the linkage between muscle cells and tendon cells is established by integrin adhesions (Maartens and Brown 2015). These adhesions, once formed, need to last many days and withstand a great deal of mechanical forces exerted by muscle contractions in the larvea. This is in stark contrast to integrin adhesions in motile cells where dynamic assembly and disassembly happens within minutes. When considering that the integrin adhesion complex in fly muscle comprises very similar protein components, this disparity becomes particularly intriguing (Green et al. 2018).

In a recent study, we applied advanced single molecule imaging techniques, including super‐resolution imaging and tracking, to investigate the nanoscale organization and dynamics of integrin adhesion proteins inside developing Drosophila muscle attachment sites (MASs) (Chen et al. 2024). We found that the stability of the adhesion is significantly affected by its nanoscale geometry, which is driven by Arp2/3‐dependent actin protrusions that in turn produces nanodomains inside the adhesion where integrins experience increased immobilization and confinement.

In order to image the drosophila embryo tissue with high spatial and temporal resolutions, we produced ex vivo embryo fillets by removing the chorion as well as the vitelline membrane and dissected the embryo along the dorsal midline to flatten it onto a coverslip. This also stabilized the movement of the tissue and allowed us to image the same adhesions for hours after the dissection. We employed multiple super‐resolution microscopy techniques to decipher the nanoscale organization of MASs. Structured illumination microscopy (SIM) showed that the mature MAS is not a homogeneous structure but presents intricate fingerprint‐like patterns (Figure 1a). Two‐color super‐resolution imaging, using demixing STORM, further illustrated that integrins organize into rings and ridges wrapping around actin‐enriched cores. This organization is reminiscent of invadosomes including invadopodia and podosomes (Linder et al. 2022). Indeed, 3D super‐resolution imaging further revealed that these patterns are protrusive structures that extend up to ~1 μm in depth. These protrusions rely on Arp2/3‐dependent actin polymerization as treatment with an Arp2/3 inhibitor (CK666) abolishes the formation of the mature adhesion pattern. The deformable tissue environment thus allows the development of 3D adhesion structures, membrane nanotopographies, from a flat 2D membrane surface.

FIGURE 1.

Nanoscale organization and dynamics of Drosophila muscle attachment site adhesions. (a) Representative image of a muscle attachment site between a muscle and tendon cell, exhibiting fingerprint‐like patterns of integrin adhesion organization around actin filaments. (b) Endogenously labelled integrin with photoactivatable mEos3.2 enables single molecular tracking of individual integrin proteins, and their diffusion and immobilization can be visualized. (c) Schematic showing one protrusive nanodomain inside a developing muscle attachment site. (d) The muscle attachment site is segmented into nanodomains where integrin diffusion is confined, and immobilization is promoted.

Integrins can undergo cycles of diffusion and immobilization within integrin adhesions, and immobilization events signify integrin binding to extracellular ligand and intracellular actin‐associated proteins, thus reflecting integrin activation states (Rossier et al. 2012). Using CRISPR knock‐in integrins tagged with a photoactivatable mEos3.2 protein, we can directly visualize the molecular movement of endogenous proteins inside a developing attachment site (Figure 1b,c). We found that the immobilization of integrin increases with the maturation of adhesions, accompanied by a reduction of its diffusion coefficient and increased confinement inside the adhesions. The mature adhesion site is thus segmented into different nanodomains with varying diffusion coefficients (Figure 1d), reflecting the membrane nanotopographies of the adhesions observed with super‐resolution imaging.

The confinement of molecular dynamics applies not only to integrins, but also to the actin cytoskeleton. Actin networks at the leading edge of motile cells (Giannone et al. 2004; Ponti et al. 2004) or within integrin adhesions (Hu et al. 2007; Rossier et al. 2012) readily undergoes retrograde flow and the velocity of the flow anti‐correlates with actin‐adhesion engagement. The dynamic interactions between integrin associated proteins (e.g., talin, vinculin) form a molecular clutch (Hu et al. 2007) driving the initiation of integrin adhesions (Giannone et al. 2004, 2007), but also stabilizing mature integrin adhesions (Oria et al. 2017). However, inside the MAS, actin movement is slow and highly confined, with only a small fraction (< 20%) exhibiting directed motion. The nanodomains thus promote strong engagement of integrin adhesion proteins with the actin cytoskeleton and lead to increased integrin activation. By inhibiting dynamic actin polymerization with CK666 or severing actin bundles with Swinholide A, we show that two distinct organizations of actin are important in driving the formation of these nanostructures. While the dynamic actin protrusions are important in initiating and maintaining the nanotopography, actin bundles help stabilize the mature adhesion structure. The confined movement of actin filaments and restricted diffusion of integrin adhesion proteins potentially create more possibilities for interaction and may lead to a stronger molecular clutch in vivo.

To further validate these observations in live tissue, we took a reductionist approach by using fabricated substrates with nanotopographies that mimic the ones found in developing embryos. When individual muscle cells from embryos were isolated and put on the nanostructured substrates, they were able to develop actin protrusions into the nanostructures and develop stable integrin adhesions, compared to flat substrates, where only nascent adhesions can form. By varying the size of these structures, we show that only sub‐micron topographies can promote actin protrusion and adhesion development. This range corresponds well with the nanotopographies observed in living tissue where the width of the nanodomains vary from approximately 200 to 900 nm. With single molecule tracking, we also observed higher confinement and immobilization of integrin on substrates with nanotopographies, especially on the bottom surface within the protrusive nanodomains.

In summary, these results show that nanotopography can promote the formation of stable integrin adhesions by regulating the diffusion of integrin and dynamics of the actin cytoskeleton. The same set of integrin adhesion proteins can produce drastically different structures in morphology and stability in different cell types and tissue contexts (Vicente et al. 2023), in particular physical properties such as rigidity. Our results highlight the role of the geometrical properties of the cellular environment in controlling biological processes (Chen et al. 2024).

Conflicts of Interest

The authors declare no conflicts of interest.

Chen, T. , Fernández‐Espartero C. H., and Giannone G.. 2025. “Single‐Molecule Tracking and Super‐Resolution Microscopy Unveil Actin‐Driven Membrane Nanotopography Shaping Stable Integrin Adhesions in Developing Tissue.” Cytoskeleton 82, no. 7: 471–474. 10.1002/cm.21970.