Actin nano-architecture of phagocytic podosomes

Abstract

Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterized the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy (iPALM), a super-resolution microscopy technique capable of 15-20 nm resolution, together with structured illumination microscopy (SIM) and localization-based superresolution microscopy. Phagocytic podosomes were observed during frustrated phagocytosis, a model in which cells attempt to engulf micro-patterned IgG antibodies. For circular patterns, this resulted in regular arrays of podosomes with well-defined geometry. Using persistent homology, we developed a pipeline for semi-automatic identification and measurement of podosome features. These studies revealed an “hourglass” shape of the podosome actin core, a protruding “knob” at the bottom of the core, and two actin networks extending from the core. Additionally, the distributions of paxillin, talin, myosin II, α-actinin, cortactin, and microtubules relative to actin were characterized.

Spatial Models of Pattern Formation During Phagocytosis

“…S2C). Radii of podosomes have been observed to be anywhere from ∼0.15 – 0.6 microns [18], [41]–[43]. Therefore, for our simulations we used D u = 0.004 μm 2 s -1 , which resulted in spots with radius 0.31 ± 0.02 µm (Fig.…”

“…The IgG patterns on glass coverslips were made using the microcontact printing of Polydimethylsiloxane (PDMS) as previously described [53]. The silicon master with an array of 3.5 μm holes spaced 8 μm apart or 10 μm holes spaced 20 μm apart was made using photoresist lithography, and PDMS stamping on glass coverslips was carried out as described previously [43].…”

“…For experimental results, the number of podosomes per site were calculated using a pipeline we developed previously [43] (https://github.com/elstonlab/PodosomeImageAnalysis). In essence, this pipeline uses persistent homology, a type of topological data analysis, to identify significantly persistent features (connected components, holes) within images followed by post-processing.…”

“…S2C). Radii of podosomes have been observed to be anywhere from ~0.15 -0.6 microns [18], [41]- [43].…”

“…For experimental results, the number of podosomes per site were calculated using a pipeline we developed previously [43] (https://github.com/elstonlab/PodosomeImageAnalysis).…”