Dataset Information

Super-resolution microscopy reveals nanoscale architecture and regulation of podosome clusters in primary macrophages.

ABSTRACT: Podosomes, an important actin-based adhesive architecture, play critical roles in cell migration and matrix invasiveness. Here, we elucidate the ultrastructural organization and regulation of podosome clusters in primary macrophages. With three-dimensional stochastic optical reconstruction microscopy (3D-STORM), we achieve ∼20/50 nm (lateral/axial) spatial resolution to resolve the mutual localization of podosome core and ring components, and further show that microtubules pass through podosomes at the layer of myosin IIA. The microtubule disruption-caused podosome dissolution is previously ascribed to Rho/ROCK-myosin signaling, yet inhibiting this pathway with Y27632 or blebbistatin only partially recovers podosome assembly, thus suggesting the contribution of the physical supporting of microtubules in stabilizing podosome structures. Through improved substrate-coating technique, we further corroborate that the matrix-degrading capability of macrophages depends on the formation of podosome clusters. Together, 3D-STORM super-resolution microscopy reveals the nanoscale spatial arrangement and the microtubule-dependent regulation of the matrix-degrading podosome clusters in macrophages.

SUBMITTER: Hu F

PROVIDER: S-EPMC9678738 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Super-resolution microscopy reveals nanoscale architecture and regulation of podosome clusters in primary macrophages.

Hu Fen F Zhu Donglan D Dong Hao H Zhang Ping P Xing Fulin F Li Wan W Yan Rui R Zhou Jun J Xu Ke K Pan Leiting L Xu Jingjun J

iScience 20221105 12

Podosomes, an important actin-based adhesive architecture, play critical roles in cell migration and matrix invasiveness. Here, we elucidate the ultrastructural organization and regulation of podosome clusters in primary macrophages. With three-dimensional stochastic optical reconstruction microscopy (3D-STORM), we achieve ∼20/50 nm (lateral/axial) spatial resolution to resolve the mutual localization of podosome core and ring components, and further show that microtubules pass through podosomes ...[more]

PMID: 36425766

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Project description:Super-resolution (SR) microscopy has been used to observe structural details beyond the diffraction limit of ∼250 nm in a variety of biological and materials systems. By combining this imaging technique with both computer-vision algorithms and topological methods, we reveal and quantify the nanoscale morphology of the primary cilium, a tiny tubular cellular structure (∼2-6 μm long and 200-300 nm in diameter). The cilium in mammalian cells protrudes out of the plasma membrane and is important in many signaling processes related to cellular differentiation and disease. After tagging individual ciliary transmembrane proteins, specifically Smoothened, with single fluorescent labels in fixed cells, we use three-dimensional (3D) single-molecule SR microscopy to determine their positions with a precision of 10-25 nm. We gain a dense, pointillistic reconstruction of the surfaces of many cilia, revealing large heterogeneity in membrane shape. A Poisson surface reconstruction algorithm generates a fine surface mesh, allowing us to characterize the presence of deformations by quantifying the surface curvature. Upon impairment of intracellular cargo transport machinery by genetic knockout or small-molecule treatment of cells, our quantitative curvature analysis shows significant morphological differences not visible by conventional fluorescence microscopy techniques. Furthermore, using a complementary SR technique, two-color, two-dimensional stimulated emission depletion microscopy, we find that the cytoskeleton in the cilium, the axoneme, also exhibits abnormal morphology in the mutant cells, similar to our 3D results on the Smoothened-measured ciliary surface. Our work combines 3D SR microscopy and computational tools to quantitatively characterize morphological changes of the primary cilium under different treatments and uses stimulated emission depletion to discover correlated changes in the underlying structure. This approach can be useful for studying other biological or nanoscale structures of interest.

Dataset Information

Super-resolution microscopy reveals nanoscale architecture and regulation of podosome clusters in primary macrophages.

Publications

Super-resolution microscopy reveals nanoscale architecture and regulation of podosome clusters in primary macrophages.

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