Funkcionalno istraživanje fosforilacije proteina CENP-E kinazama Aurora A i B

master's thesis

• Title (english): Functional study of CENP-E phosphorylation by Aurora kinases A and B
• Author: Mirela Kubat
• Mentor: Marin Barišić (mentor)
• Mentor: Inga Marijanović (sumentor)
• Committee member: Inga Marijanović (predsjednik povjerenstva)
• Committee member: Sven Jelaska (član povjerenstva)
• Committee member: Goran Kovačević (član povjerenstva)
• Committee member: Nenad Malenica (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2020-02-28, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Ispravan prostorni raspored kromosoma omogućava ravnopravnu raspodjelu genetičkog materijala prilikom stanične diobe. Kongresija kromosoma koji se nalaze na polovima mitotskog vretena je posredovana proteinom kinezinom-7/CENP-E. Trenutno postoje dva modela koji objašnjavaju kako CENP-E usmjerava kromosome isključivo prema staničnom ekvatoru. U prvom modelu, CENP-E pogoni kretanje kromosoma od visoke koncentracije kinaze Aurora A na polovima prema staničnom ekvatoru, zatim veže protein fosfatazu 1, koja ga defosforilira i omogućuje stabilno vezanje kinetohora na mikrotubule. Drugi model se bazira na različitim preferencijama dvaju motornih protein suprotnih usmjerenja, proteina CENP-E i dineina za detirozinirane i tirozinirane mikrotubule, koji kontroliraju smjer putovanja kromosoma tijekom stanične diobe. Odnos pojedinačnih doprinosa regulacije aktivnosti proteina CENP-E direktnom fosforilacijom i detirozinacijom mikrotubula ostaje za razjasniti. Ovaj set eksperimenata je fokusiran na raščišćavanje funkcije fosforilacije proteina CENP-E kinazama Aurora A i B, kao i na utjecaj fosforilacije na prekidač aktivnosti između proteina CENP-E i dineina. Metode koje su korištene su molekularno kloniranje, lentivirusna infekcija, imunofluorescencija nakon tretmana inhibitorima proteina i siRNA knock down, konfokalna mikroskopija, Western blot te imunoprecipitacija.

Abstract (english)

Spatial layout of chromosomes enables equal allocation of genetic material during cell division. Congression of chromosomes that are positioned on the poles of the mitotic spindle is mediated by kinesin-7/CENP-E, a microtubule plus-end directed motor protein. Currently, there are two models that explain how CENP-E directs chromosomes exclusively towards cell equator. In the first model, CENP-E drives chromosome movements from high Aurora A kinase concentration on the poles, towards the cell equator, then binds protein phosphatase 1 which dephosphorylates it and enables stable binding of kinetochores to microtubules. The second model is based on different preferences of two motor proteins with opposite directionalities, namely CENP-E and dynein, for detyrosinated and tyrosinated microtubules, respectively, which control the directionality of chromosome movements during cell division. However, the relationship of individual contributions of activity regulation of CENP-E by phosphorylation and microtubule detyrosination remains to be elucidated. This set of experiments focused on clarifying the role of CENP-E phosphorylation by Aurora A and B and its impact on CENP-E-dynein activity switch and potential interaction. The hypothesis is that T422 phosphorylation of CENP-E prevents its premature stripping by Dyenin from bioriented chromosomes’ kinetochores, thus facilitating the accurate cell division. The methods used were molecular cloning, lentiviral infection, immunofluorescence following protein inhibition and siRNA knock down, confocal microscopy, Western blot and immunoprecipitation.

• Keywords: CENP-E; Aurora A; Aurora B; dinein; uklanjanje posredovano dineinom; mitoza; kongresija kromosoma.
• Keywords (english): CENP-E; Aurora A; Aurora B; kinase; dynein-mediated stripping; mitosis; chromosome congression
• Language: croatian
• URN:NBN: urn:nbn:hr:217:125685
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2022-02-28
• Created on: 2020-07-13 06:43:40