Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

• EEA1 mobility was compared in serum-starved and EGF-stimulated interphase HeLa cells.
• FRAP analysis revealed fast and slow components of EEA1 recovery in both cases.
• Stimulation of EGFR endocytosis did not affect fast EEA1 turnover.
• EGF stimulation significantly increased half-time of slowly exchanged EEA1 fraction.

Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering.