Figure 4. The cytoplasmic tail of ICAM-1... | Rockefeller University Press

Figure 4.

$Figure 4. The cytoplasmic tail of ICAM-1 mediates clustering and lateral mobility in the plasma membrane of BMDCs. (A) Sequence alignments of the ICAM-1 cytoplasmic tail and ICAM-1 constructs. (B) ICAM-1−/− BMDCs were transduced with ICAM-1 mutants, and cell surface levels were compared by flow cytometry. ICAM-1−/− cells (shaded gray line), WT cells expressing endogenous ICAM-1 (solid black line), ICAM-1−/− cells reconstituted with WT (solid gray line), Δ Tail (broken gray line), or chimera (broken black line) constructs. Results are representative of three individual experiments. (C and D) Mobility of endogenous ICAM-1 and exogenous ICAM-1 mutants expressed in ICAM-1−/− BMDCs was analyzed using FRAP. (C) Mobile fraction; (D) diffusion coefficient. Dots represent individual FRAP measurements (n = 42–523). Data were pooled from five independent experiments except for endogenous, which was pooled from two experiments, and Y518F, which was from a single experiment. (E) Immunofluorescence microscopy showing the distribution of moesin and F-actin with respect to ICAM-1 in LPS matured ICAM-1−/− BMDCs reconstituted with exogenous WT, Δ Tail, or chimeric ICAM-1. Bars, 10 µm. (F) Images collected as in E were analyzed for ICAM-1 clustering by measuring the coefficient of variation (standard deviation/mean) of surface ICAM-1 intensity. Data are from one experiment (n = 50 cells) representative of three independent experiments. (G) Capping of exogenous ICAM-1 in transduced DCs was quantified from midplane images similar to Fig. 3 E. Data are means ± standard deviation (error bars) from four replicate coverslips (∼50 cells each) in one experiment, representative of two independent experiments. **, P < 0.001; ***, P < 0.0001.$

The cytoplasmic tail of ICAM-1 mediates clustering and lateral mobility in the plasma membrane of BMDCs. (A) Sequence alignments of the ICAM-1 cytoplasmic tail and ICAM-1 constructs. (B) ICAM-1^−/− BMDCs were transduced with ICAM-1 mutants, and cell surface levels were compared by flow cytometry. ICAM-1^−/− cells (shaded gray line), WT cells expressing endogenous ICAM-1 (solid black line), ICAM-1^−/− cells reconstituted with WT (solid gray line), Δ Tail (broken gray line), or chimera (broken black line) constructs. Results are representative of three individual experiments. (C and D) Mobility of endogenous ICAM-1 and exogenous ICAM-1 mutants expressed in ICAM-1^−/− BMDCs was analyzed using FRAP. (C) Mobile fraction; (D) diffusion coefficient. Dots represent individual FRAP measurements (n = 42–523). Data were pooled from five independent experiments except for endogenous, which was pooled from two experiments, and Y518F, which was from a single experiment. (E) Immunofluorescence microscopy showing the distribution of moesin and F-actin with respect to ICAM-1 in LPS matured ICAM-1^−/− BMDCs reconstituted with exogenous WT, Δ Tail, or chimeric ICAM-1. Bars, 10 µm. (F) Images collected as in E were analyzed for ICAM-1 clustering by measuring the coefficient of variation (standard deviation/mean) of surface ICAM-1 intensity. Data are from one experiment (n = 50 cells) representative of three independent experiments. (G) Capping of exogenous ICAM-1 in transduced DCs was quantified from midplane images similar to Fig. 3 E. Data are means ± standard deviation (error bars) from four replicate coverslips (∼50 cells each) in one experiment, representative of two independent experiments. **, P < 0.001; ***, P < 0.0001.