Time-resolved ultrastructural detection of phosphatidylinositol 3-phosphate.
Phosphatidylinositol 3-phosphate [PtdIns(3)P] plays an important role in recruitment of various effector proteins in the endocytic and autophagic pathways. In an attempt to follow the distribution of PtdIns(3)P at the ultrastructural level, we are using the Fab1, YOTB, Vac1, and EEA1 (FYVE) domain, which is a zinc finger motif specifically binding to PtdIns(3)P. To follow PtdIns(3)P trafficking during a defined time window, here we have used a monomeric dimerizable FYVE probe, which binds with high avidity to PtdIns(3)P only after rapalog-induced dimerization. The probe localized to early and late endocytic compartments according to the time period of dimerization, which indicates that PtdIns(3)P is turned over via the endocytic machinery. In the functional context of epidermal growth factor (EGF) stimulation, we observed that dimerization of the probe led to clustering of mainly early endocytic structures, leaving most of the probe localized to the limiting membrane of endosomes. Interestingly, these clustered endosomes contained coats positive for the PtdIns(3)P-binding protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), indicating that the probe did not displace Hrs binding. We conclude that the dimerizer-inducible probe is useful for the time-resolved detection of PtdIns(3)P at the ultrastructural level, but its effects on endosome morphology after EGF stimulation need to be taken into account.
Stuffers
Susanne
S
Dept. of Biochemistry, Institute for Cancer Research, the Norwegian Radium Hospital and Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Montebello, Oslo, Norway.
Malerød
Lene
L
Schink
Kay Oliver
KO
Corvera
Silvia
S
Stenmark
Harald
H
Brech
Andreas
A
D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D055503:Protein Multimerization; D013997:Time Factors; D014162:Transfection; D016335:Zinc Fingers
D018129:Phosphatidylinositol Phosphates; C055525:phosphatidylinositol 3-phosphate; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor