Supervisors info:
Γαλανοπούλου Ντία Αναπλ. Καθηγήτρια ΕΚΠΑ (επιβλέπουσα), Παπασιδέρη Ι. Αναπλ. Καθηγήτρια ΕΚΠΑ, Μπολέτη Χ. Ερευνήτρια Β΄ Ελληνικό Ινστιτούτο Παστέρ
Summary:
Phosphoinositides, membrane lipids present in all eukaryotic cells, serve as
regulators of important cellular functions including signal transduction and
membrane trafficking. Regulation is carried out through conserved protein
domains which recognize and specifically bind certain phosphoinositides
resulting either to the protein activation or to the protein translocation and
binding to a specific membrane of the cell. Four members of the
phosphoinositide group are present in the unicellular eukaryotic organism T.
thermophila and there is evidence that they are involved in different stages of
the phagosome formation/enzyme secretion pathway of the organism. Moreover,
signalling PI-PLCs as well as phosphoinositide kinases are also present,
supporting the idea that T. thermophila is an excellent model for the study of
phosphoinositides. The goal of this work was to investigate subcellular
localization of phosphoinositides in T. thermophila cells by fluorescent
microscopy using phosphoinositide recognizing protein domains of both
Tetrahymena and mammalian origin. Using bioinformatics, we found all T.
thermophila proteins containing PH, FYVE, PX, ENTH, GRAM and C2 domains. Based
on the conservation of certain amino acid residues during protein alignment, we
cloned the PH domains of protein kinases TtPDK1 (TTHERM_00188930) and PHK5
(TTHERM_00666380), as well as the FYVE domain of the lipid kinase TtPIPK3
(TTHERM_01005090), which possibly recognize PtdInsP3 and PtdIns3P,
respectively. After total RNA isolation and cDNA production, the three domains
were amplified by PCR. The cDNA was inserted in the pTZ57R/T vector for
sequencing and, then, PHTtPDK1 was ligated in frame to the Tetrahymena-specific
pVGF vector, which contains also the GFP sequence for expression of a
GFP-fusion protein. The cDNA of the mammalian PHPLCδ1, which specifically
recognize PtdIns(4,5)P2, was then used to construct pVGF-GFP-PHPLCδ1. Induction
of protein expression by CdCl2 showed that fluorescence was localized at the
cytostome area, confirming previous biochemical evidence for the involvement of
PtdIns(4,5)P2 in the initiation of phagocytosis. Furthermore, the presence of
AKT (PHK5) and PDK1-type kinases in T. thermophila, together with the
bioinformatic data showing the presence of three PI3K-1 and, also, three
PTEN-type proteins, suggest the possible function of a PtdInsP3-based
signalling pathway in Tetrahymena cells.
Keywords:
Phosphoinositides, Phosphoinositide binding domains, PH, FYVE, Tetrahymena
