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 characterize the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy, a super-resolution microscopy technique capable of 15–20 nm resolution, together with structured illumination microscopy and localization-based super-resolution microscopy. Phagocytic podosomes are observed during frustrated phagocytosis, a model in which cells attempt to engulf micropatterned IgG antibodies. For circular patterns, this results in regular arrays of podosomes with well-defined geometry. Using persistent homology, we develop a pipeline for semi-automatic identification and measurement of podosome features. These studies reveal 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 are characterized.

Spatial models of pattern formation during phagocytosis

“…Evidence that supports our current choice of parameters is that the model matches the size and spatial organization of podosomes, uses a membrane-bound diffusion rate for Cdc42 that is estimated from experimental data, and uses a reasonable estimate for the number of Cdc42 molecules expected within podosomes. Finally, we note that the time scale predicted by the model for Cdc42 to form rosettes (~20-40s, S4 Fig ) appears to be faster than our experimental observation for podosome rosette formation (~200 s) [ 18 ]. However, we believe the model’s prediction is consistent with that of podosome formation because the recruitment of actin and other podosome-related molecules would occur following GTPase activation.…”

“…Using this information, we fixed D u = 0.04 μm 2 s -1 and we let D v = D g = D G = 100 D u . Radii of podosomes have been observed to be anywhere from ~0.15–0.6 microns [ 17 , 18 , 48 , 49 ]. Therefore, we tuned the rate constants ( Table 1 ) so that the model produced spots with radius 0.31 ± 0.02 μm.…”

“…The IgG patterns on glass coverslips were made using the microcontact printing of Polydimethylsiloxane (PDMS) as previously described [ 67 ]. 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 [ 18 ].…”

“…From single particle tracking during frustrated phagocytosis (Fig 1D -1G), we estimated the diffusivity for the membrane-bound form of Cdc42 as ~0.04 μm 2 s -1 (S1C Fig) . Using this information, we fixed D u = 0.04 μm 2 s -1 and we let D v = D g = D G = 100 D u . Radii of podosomes have been observed to be anywhere from ~0.15-0.6 microns [17,18,48,49]. Therefore, we tuned the rate constants (Table 1) so that the model produced spots with radius 0.31 ± 0.02 μm.…”

“…Podosomes classically have been considered rod or cone-like structures of dense actin [ 16 , 17 ]. However, we recently demonstrated an hourglass-like shape [ 18 ]. Podosomes recruit many additional molecules and are thought to coordinate interactions between the actin cytoskeleton and the extracellular matrix [ 16 , 17 , 19 ].…”