Fig. 7.

Mutation of Ser756 to Asp rescues acute LPS-driven podosome loss. (A) Itgb2-null BMDCs were infected with retroviruses containing either the WT-Itgb2-EGFP or the S756D-Itgb2-EGFP mutant constructs (green EGFP staining) and treated with (+LPS) or without (–LPS) 50 ng/ml LPS for 30 minutes before fixation and then stained to visualize F-actin (red) and α-actinin 4 (grey). Images were acquired using a Zeiss LSM700, as described for Fig. 1. The images show that podosomes formed normally in S756D-Itgb2-EGFP expressing cells and that LPS induced podosome dissolution. Scale bars: 5 µm. (B) Percentages of infected (GFP positive) cells showing podosomes when reconstituted with empty vector (pBMN-I-GFP), or constructs for WT β2 integrin or the S745D and S756D mutants, with or without LPS treatment were quantitated. Podosomes in cells expressing the S745D and S756D mutants were responsive to LPS (**P = 0.001 and *P = 0.014, unpaired t-tests). (C) Itgb2-null BMDCs expressing EGFP fusion proteins of WT, S745A, S745D, S756A or S756D-Itgb2 were cultured in glass bottom dishes for FRAP analysis, as in Fig. 4. Cells expressing Itgb2-EGFP in a typical honeycomb shaped podosome ring pattern in the ventral plasma membrane were selected for photobleaching and the half-life for fluorescence recovery measured for each construct as in Materials and Methods (ten cells per experiment). S745D and S756D mutants show reduced half-life compared to corresponding Ala mutants (**P = 0.001 and **P = 0.006, respectively, paired t-test). Data were from three independent experiments from different bone marrow and virus preparations.