Pregled bibliografske jedinice broj: 1221219
Comparison of different FYVE domains and anti-PI3P antibody for monitoring the localization of PI3P in BALB 3T3 cells
Comparison of different FYVE domains and anti-PI3P antibody for monitoring the localization of PI3P in BALB 3T3 cells, 2020., diplomski rad, diplomski, Odjel za biotehnologiju, Rijeka
CROSBI ID: 1221219 Za ispravke kontaktirajte CROSBI podršku putem web obrasca
Naslov
Comparison of different FYVE domains and anti-PI3P
antibody for monitoring the localization of PI3P
in BALB 3T3 cells
Autori
Bruketa, Tea
Vrsta, podvrsta i kategorija rada
Ocjenski radovi, diplomski rad, diplomski
Fakultet
Odjel za biotehnologiju
Mjesto
Rijeka
Datum
17.07
Godina
2020
Stranica
75
Mentor
Antonija Jurak Begonja
Neposredni voditelj
Antonija Jurak Begonja
Ključne riječi
PI3P, Vps34, FYVE domain, nucleus
Sažetak
Phosphoinositides are phosphorylated membrane lipids that control the intracellular membrane traffic and have a role in cell signaling. Phosphatidylinositol 3-phosphate (PI3P) is a key regulator of membrane and vesicular trafficking localized primarily on the early and late endosomal membranes where it regulates endosomal maturation. The major source of PI3P is the class III phosphoinositide 3-kinase vacuolar protein sorting 34 (Vps34). In the cells, PI3P can be visualized using antibodies and/or protein domains with a specific PI3P-binding property, such as FYVE or PX. In this thesis. we tested the validity of three FYVE domain-containing probes (GFP-FYVEEEA1, GFP- 2xFYVEEEA1 and GFP-2xFYVEHrs), and anti-PI3P antibody for the detection of PI3P in BALB 3T3 cells. Our results showed that all three probes and the antibody can be used to visualize cytoplasmic PI3P that was mostly confined to the early and late endosomes/lysosomes. In addition to endo-lysosomal PI3P in the cytoplasm, we also detected PI3P in the nucleus and nucleolus. We visualized nuclear PI3P with all of the probes and the antibody, while nucleolar PI3P could be visualized only with GFP-2xFYVEEEA1 and GFP- 2xFYVEHrs which have a higher PI3P-binding affinity. Furthermore, by inhibiting Vps34, we showed that all the probes and the antibody were highly specific for PI3P, and confirmed that Vps34 is a major source of PI3P within the cell. Next, we observed that the simultaneous inhibition of Vps34 and the overexpression of high affinity probes slightly increased heterochromatin levels in the nucleus. Finally, we investigated the expression of nucleolar markers UBF, B23 and fibrillarin in human megakaryoblastic leukemia cell line (Dami) and found that their expression remains mostly unchanged during Dami cells maturation. These data indicate that PI3P may be involved in different nuclear processes. Also, these results open new questions about the role of PI3P in the process of chromatin remodeling, which is a subject of future research.
Izvorni jezik
Engleski
Znanstvena područja
Biologija, Temeljne medicinske znanosti, Biotehnologija u biomedicini (prirodno područje, biomedicina i zdravstvo, biotehničko područje)
POVEZANOST RADA
Projekti:
NadSve-Sveučilište u Rijeci-uniri-biomed-18-188-1343 - Identifikacija novih interakcijskih partnera Vps34 u megakariopoezi (Jurak Begonja, Antonija, NadSve - UNIRI projekti 2018) ( CroRIS)
HRZZ-UIP-2014-09-2400 - Uloga fosfoinozitida u nastanku trombocita (MkPI) (Jurak Begonja, Antonija, HRZZ - 2014-09) ( CroRIS)
Ustanove:
Sveučilište u Rijeci - Odjel za biotehnologiju
Profili:
Antonija Jurak Begonja
(mentor)