Abstract
Podosomes are cytoskeletal structures crucial for cell protrusion and matrix remodelling in osteoclasts, activated endothelial cells, macrophages and dendritic cells. In these cells, hundreds of podosomes are spatially organized in diversely shaped clusters. Although we and others established individual podosomes as micron-sized mechanosensing protrusive units, the exact scope and spatiotemporal organization of podosome clustering remain elusive. By integrating a newly developed extension of Spatiotemporal Image Correlation Spectroscopy with novel image analysis, we demonstrate that F-actin, vinculin and talin exhibit directional and correlated flow patterns throughout podosome clusters. Pattern formation and magnitude depend on the cluster actomyosin machinery. Indeed, nanoscopy reveals myosin IIA-decorated actin filaments interconnecting multiple proximal podosomes. Extending well-beyond podosome nearest neighbours, the actomyosin-dependent dynamic spatial patterns reveal a previously unappreciated mesoscale connectivity throughout the podosome clusters. This directional transport and continuous redistribution of podosome components provides a mechanistic explanation of how podosome clusters function as coordinated mechanosensory area.
Highlights
Podosomes are cytoskeletal structures crucial for cell protrusion and matrix remodelling in osteoclasts, activated endothelial cells, macrophages and dendritic cells
The coordination shown by the podosome clusters in both spatial arrangements is supported by images of actin and vinculin labelled in fixed dendritic cells (DCs) that were analysed by super-resolution microscopy using structured illumination microscopy (SIM) (Supplementary Fig. 1c,d)
By integrating nanoscopy of multiple podosome components with measurements of their spatiotemporal molecular dynamics using an extension of Spatiotemporal Image Correlation Spectroscopy (STICS) and with the application of dedicated image analysis algorithms, we here revealed the dynamic and structural mechanisms driving mesoscale coordination of podosome clusters in human DCs
Summary
Podosomes are cytoskeletal structures crucial for cell protrusion and matrix remodelling in osteoclasts, activated endothelial cells, macrophages and dendritic cells. Depending on cell type and substrate physicochemical properties, different cellular structures and molecular mechanisms have been investigated that allow cells to anchor to, remodel and migrate through the extracellular matrix[2,3,4,5,6]. Common to all these processes is the capacity of actin filaments to organize in different assemblies by interacting with actin-binding proteins such as talin, vinculin and non-muscle myosin IIA. Individual podosomes exhibit stiffness oscillations and density fluctuations of actin and mechanosensitive components such as vinculin and zyxin that depend on myosin IIA contractility[23,25]
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