Actomyosin-dependent dynamic spatial patterns of cytoskeletal components drive mesoscale podosome organization

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.

Modular actin nano-architecture enables podosome protrusion and mechanosensing

“…Classically, podosomes are characterized by a protrusive actin-rich core with a diameter of 500-700 nm which is surrounded by a 200-300 nm thick adhesive integrin ring enriched for adaptor proteins such as vinculin and talin (16). Neighboring podosomes are interconnected by a network of actin filaments that radiate from the podosome core and facilitate a mesoscale (1.5-10 µm) connectivity (17)(18)(19). While individual podosomes are thought to function as micronsized protrusive machineries (20)(21)(22), their mesoscale connectivity presumably facilitates long-range basement membrane exploration for protrusion-permissive spots (18,23).…”

Strong cytoskeleton activity on millisecond timescales upon particle binding revealed by ROCS microscopy

“…The reliability of those correlation-based methods is often limited by the acquisition speed of the imaging system, making it hardly possible to investigate fast processes that occur on millisecond timescales. A recent example is the study of Meddens et al (Meddens & Meddens, 2016), where spatiotemporal image correlation spectroscopy is used to investigate dynamic spatial patterns and directional movements of cytoskeletal components over the course of 75 min, but only with a very small temporal sampling rate in the range of Δt = 5 -15 s. Recent studies on laser speckle rheology on weakly absorbing fluids were recorded with a fast sCMOS camera at 945 Hz (Hajjarian & Nadkarni, 2014) or even at 1950 Hz (Nader et al, 2016), but without providing any spatial resolution or imaging.…”

“…Studies which are comparable to our approach are fluorescence-based with the aforementioned advantages (protein-specific) and drawbacks (slow, subject to bleaching). A recent example is the study of Meddens et al (Meddens & Meddens, 2016), where spatiotemporal image correlation spectroscopy is used to investigate dynamic spatial patterns and directional movements of cytoskeletal components over the course of 75 min, but only with a very small temporal sampling rate in the range of Δt = 5 -15 s.…”

“…However, these structures are too small to be resolved by conventional light microscopy. A more recent (also fluorescence-based) study (Meddens & Meddens, 2016) investigates the interplay of cytoskeletal components during podosome organization, but is limited in temporal resolution to acquisition times of Δt ≈ 1-10 s. Fluorescence microscopy techniques are generally relatively slow compared to coherent imaging approaches, although there are established approaches based on fluorescence correlation spectroscopy (Levi, Ruan, Kis-Petikova, & Gratton, 2003) or fluorescence fluctuation analysis (Di Rienzo, 2014) that can probe the diffusion speeds of single specifically labelled molecules in the 3D cellular environment on submillisecond timescales.…”

“…A review about fluorescent speckle microscopy of cytoskeleton dynamics can be found in (Danuser & Waterman-Storer, 2006). A more recent (also fluorescence-based) study (Meddens & Meddens, 2016) investigates the interplay of cytoskeletal components during podosome organization, but is limited in temporal resolution to acquisition times of Δt ≈ 1-10 s.…”