4D). molecular level, the actin network localized to the immunological synapse exhibited reduced stability, in particular, of the actin-related protein-2/3-dependent, short-filament network. This was associated with decreased polarization of dendritic cell-associated ICAM-1 and MHC class II, which was partially dependent on Wiskott-Aldrich syndrome protein phosphorylation. With the Slco2a1 use of supported planar lipid bilayers incorporating anti-ICAM-1 and ICI 211965 anti-MHC class II antibodies, the dendritic cell actin cytoskeleton organized into recognizable synaptic structures but interestingly, created Wiskott-Aldrich syndrome protein-dependent podosomes within this area. These findings demonstrate that intrinsic dendritic cell cytoskeletal remodeling is a key regulatory component of normal immunological synapse formation, likely through consolidation of adhesive conversation and modulation of immunological synapse stability. test was used to test significance among DC types; ***test was used to test significance among DC types; **values. The protein abundances (proportion of each network), values showed a second-order exponential fit, confirming the presence of 2 unique actin networks (Table 2 and Supplemental Fig. 2); 1 explains a short-filament, fast-recovery network, and the other corresponds to a long-filament, slow-recovery network. Thus, by separating the components of the recovery curve, the rates and proportions of the individual actin networks contributing to recovery can be calculated (Fig. 2). TABLE 2. Fitted parameters R2values = 0.0232; **= 0.0092; ns, = 0.0572. (D) WT, WASKO, and Y293F DCs interacting with the -MHC II-Cy5 (reddish) bilayer were fixed and stained with phalloidin (blue). Initial scale bars, 5 m. (E) Three parameters were measured by use of ImageJ Measure and Analyze particles functions in cells interacting with an -MHC II-Cy5 bilayer: common actin intensity across the contact; MHC II area as a percentage of the total contact area (actin); and quantity of peripheral microclusters (MC) per cell (size 600 nm2). Means and sem are shown for a minimum of 25 cells per condition analyzed in 2 experiments. ***= 0.0241, **= 0.0073 (F) WT DC contacting an -MHC II-Cy5 and -ICAM-1 bilayer, showing actin-rich podosomes (blue) and immunofluorescent staining (yellow): capping protein (F-actin capping protein, subunit; upper), vinculin (lower). Colocalization of F-actin capping protein and actin produces a white overlay; 36 WT DCs were analyzed to determine colocalization. Pearson correlation coefficient = 0.442 0.14; Manders overlap coefficient = 0.777 0.04. Initial scale bars, 5 m. A 3 zoom is shown to the right. (G) DCs were seeded on 2 different bilayers and on fibronectin (50 g/ml) and fixed at set ICI 211965 intervals. Diameter of the podosome actin cores was measured in ImageJ; 100 podosomes were measured for each condition. ICI 211965 Synapse podosomes did not change significantly over time and showed a similar size to those formed around the ventral side of cells adhering to fibronectin. Over time, podosomes assembled into a unique ring surrounding the central MHC II cluster, and crucially, this business was dependent on engagement of ICAM-1 and MHC II (Fig. 4E). Contact with anti-ICAM-1-only bilayers induced podosome-like structures that created clusters or rosettes rather than rings (Fig. 4D). In the absence of ICAM-1 ligation, podosomes-like structures did not form at any time point (Fig. 3D). These podosome-like structures were completely absent in WASKO DCs (Fig. 4A). To characterize these actin-rich structures further, we used immunostaining for vinculin (Fig. 4F), which was present in rings surrounding the individual actin structures, much like canonical podosomes explained elsewhere [48, 49], suggesting that ICI 211965 these actin-rich structures represent true podosome cores. Staining for F-actin capping protein, subunit 1 localized to the actin-rich podosome cores (Manders overlap coefficient, 0.777 0.04), highlighting that Arp2/3 nucleation, polymerization, and filament capping are important for these podosome structures. Furthermore, the podosome diameter was similar to that of classic podosomes created on fibronectin (Fig..