Lsc is required to form a single-dominant pseudopod in formyl-peptide–stimulated neutrophils. DIC photomicrographs (25 × objective) of WT and Lsc KO neutrophils stimulated with 10 μM fMLP in a Zigmond chamber were obtained at 15-second intervals for 15 minutes and subjected to quantitative analyses of cell shape (see Supplemental Videos 1-2 and Table 1). (A) Representative sequential photomicrographs of fMLP-stimulated WT and Lsc KO neutrophils. WT neutrophils form and sustain a single-dominant pseudopod (solid arrowhead). In contrast, Lsc KO neutrophils rapidly generate and retract pseudopodia at random locations around the cell perimeter. (B) fMLP-stimulated Lsc KO neutrophils undergo larger cumulative positive and negative changes (Δ) in area, perimeter, and roundness. These differences result from the rapid turnover of supernumerary pseudopodia in Lsc KO neutrophils. Data are the mean ± SEM for 60 cells of each genotype from 3 independent experiments. (C-F) Confocal images (63 ×) of WT and Lsc KO neutrophils adhered to a glass coverslip and incubated with 1 μM fMLP for 5 minutes. The cells were then fixed, permeabilized, and labeled with phalloidin (red) and the indicated antibodies. Bars represent 10 μm. Images are representative of 2 independent experiments. (C-D) fMLP-stimulated PIP3 and Akt accumulation at the leading edge of pseudopodia is similar in WT and Lsc KO cells. PIP3 and Akt are not visible in all pseudopodia of WT or Lsc KO neutrophils, likely reflecting varying stages of pseudopod formation. (E-F) fMLP-stimulated pMLC and RhoA accumulation at the trailing edge of the uropod is similar in WT and Lsc KO cells. Solid arrowhead indicates leading edge; open arrowhead, trailing edge. All photomicrographs are representative of at least 2 independent experiments.
