Involvement of a Triton-insoluble floating fraction in Dictyostelium cell-cell adhesion

We have isolated and characterized a Triton-insoluble floating fraction (TI FF) from Dictyostelium, Ten major proteins were consistently detected in TI FF, and six species were identified by mass spectrometry as actin, porin, c omitin, regulatory myosin light chain, a novel member of the CD36 family, a nd the phospholipid-anchored cell adhesion molecule gp80, TIFF was enriched with many acylated proteins. Also, the sterol phospholipid ratio of TIFF w as 10-fold higher than that of the bulk plasma membrane. Immunoelectron mic roscopy showed that TIFF has vesicular morphology and confirmed the associa tion of gp80 and comitin with TIFF membranes. Several TIFF properties were similar to those of Dictyostelium contact regions, which were isolated as a cytoskeleton-associated membrane fraction. Mass spectrometry demonstrated that TIFF and contact regions shared the same major proteins. During develo pment, gp80 colocalized with F-actin, porin, and comitin at cell-cell conta cts. These proteins were also recruited to gp80 caps induced by antibody cr osslinking. Filipin staining revealed high sterol levels in both gp80-enric hed cell-cell contacts and gp80 caps. Moreover, sterol sequestration by fil ipin and digitonin inhibited gp80-mediated cell-cell adhesion. This study r eveals that Dictyostelium TIFF has structural properties previously attribu ted to vertebrate TIFF and establishes a role for Dictyostelium TIFF in cel l-cell adhesion during development.