Dual-color superresolution microscopy reveals nanoscale organization of mechanosensory podosomes.

Podosomes are multimolecular mechanosensory assemblies that coordinate mesenchymal migration of tissue-resident dendritic cells. They have a protrusive actin core and an adhesive ring of integrins and adaptor proteins, such as talin and vinculin. We recently demonstrated that core actin oscillations correlate with intensity fluctuations of vinculin but not talin, suggesting different molecular rearrangements for these components. Detailed information on the mutual localization of core and ring components at the nanoscale is lacking. By dual-color direct stochastic optical reconstruction microscopy, we for the first time determined the nanoscale organization of individual podosomes and their spatial arrangement within large clusters formed at the cell-substrate interface. Superresolution imaging of three ring components with respect to actin revealed that the cores are interconnected and linked to the ventral membrane by radiating actin filaments. In core-free areas, αMß2 integrin and talin islets are homogeneously distributed, whereas vinculin preferentially localizes proximal to the core and along the radiating actin filaments. Podosome clusters appear as self-organized contact areas, where mechanical cues might be efficiently transduced and redistributed. Our findings call for a reevaluation of the current "core-ring" model and provide a novel structural framework for further understanding the collective behavior of podosome clusters.

Interplay between myosin IIA-mediated contractility and actin network integrity orchestrates podosome composition and oscillations.

Tissue-resident dendritic cells patrol for foreign antigens while undergoing slow mesenchymal migration. Using actomyosin-based structures called podosomes, dendritic cells probe and remodel extracellular matrix topographical cues. Podosomes comprise an actin-rich protrusive core surrounded by an adhesive ring of integrins, cytoskeletal adaptor proteins and actin network filaments. Here we reveal how the integrity and dynamics of protrusive cores and adhesive rings are coordinated by the actomyosin apparatus. Core growth by actin polymerization induces podosome protrusion and provides tension within the actin network filaments. The tension transmitted to the ring recruits vinculin and zyxin and preserves overall podosome integrity. Conversely, myosin IIA contracts the actin network filaments and applies tension to the vinculin molecules bound, counterbalancing core growth and eventually reducing podosome size and protrusion. We demonstrate a previously unrecognized interplay between actin and myosin IIA in podosomes, providing novel mechanistic insights into how actomyosin-based structures allow dendritic cells to sense the extracellular environment.

Relationship between inhomogeneity phenomena and granule growth mechanisms in a high-shear mixer.

A poorly understood phenomenon observed during high-shear granulation is the poor distribution of a drug in the granulate. To investigate the causes of this inhomogeneity, lactose of three different particle sizes was granulated with 0.1% micronized estradiol (5 microm) in a 10 l high-shear mixer. An aqueous solution of HPC was used as binder. Granulation with the largest lactose particles (141 microm) yielded a homogeneous granulate. However, at a prolonged process time demixing was observed. Contrary to the largest particles, granulation with the smaller lactose particles (50 and 23 microm) already leads to demixing in the first minute, although to a lesser extent. It was concluded that granulation with the largest particles resulted in breakage behavior of the granulate, thereby preventing demixing. However, once granules are strong enough (smaller particle size and prolonged process time) to survive the shear forces demixing is observed. Theoretical calculations of dynamic and static granule strength were used to explain the influence of lactose particle size and process time on breakage behavior. It was argued that once granules survive, preferential growth of the small estradiol particles in favor of the larger lactose particles causes the demixing. The extent of demixing depends on the particle size difference.