Institutional creator:

Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN.

Contributor:

Joachimiak, Ewa (1973- ) : Supervisor

Publisher:

Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN

Place of publishing:

Warszawa

Description:

165 pages : illustrations ; 30 cm ; Bibliography ; Summary in English

Degree name:

PhD in Biological Sciences

Degree discipline :

Biological Sciences

Degree grantor:

Nencki Institute of Experimental Biology PAS ; degree obtained in 2024

Type of object:

Thesis

Abstract:

Cilia and flagella are evolutionary conserved structures which protrude from the apical surface of the eukaryotic cells in both unicellular organisms and humans. There are two major types of cilia – motile and non-motile cilia (also called primary cilia). In mammals, motile cilia are formed on the apical surface of epithelial cells lining e.g. respiratory tract. Sperm cells possess special type of cilium called flagellum. Lack or defects in motile cilia lead to primary ciliary dyskinesia that involves e.g. chronic airway diseases and defects in fertility. Motile cilia axoneme consists of nine outer microtubule doublets which are accompanied by protein complexes responsible for generation of cilia movement. In the central part of the axoneme localizes structure called central apparatus which comprises two single microtubules surrounded by multiprotein complexes called projections. Up to now, exact protein composition of these protein complexes remains unknown, therefore the function of central apparatus and its role in generation of cilia movement is only partially resolved. One of the hypotheses suggests that the signal needed for cilia beating is initiated in central apparatus. However, it cannot be excluded that functions of central apparatus are regulated by enzymatic proteins. Nevertheless, the available data are only fragmentary. This thesis focuses on so-far uncharacterized central apparatus proteins, potentially being part of ciliary MAP kinases-regulatory pathway. Experiments presented in this thesis were conducted with use of well established, unicellular model organism – Tetrahymena thermophila. Based on analysis of previously obtained ciliomes from mutants that lack C1b projection (SPEF2A-CoDel), three kinases– Mapk3, Map2k7, Nek6, two phosphatases: Pp2c and Dusp5 and kinesin Kif9 which build C2c projection were selected for further research. All of these proteins were downregulated in Tetrahymena mutants lacking C1b projection. Amino acid sequence analysis of studied proteins revealed that all of them are evolutionary conserved. Conducted research allowed to confirm that all examined proteins localize in Tetrahymena cilia and when overexpressed are present in both cilia and cell body. BioID and coimmunoprecipitation experiments showed that Dusp5 not only localizes in close proximity but also forms stable complex with C1b projection proteins in Tetrhymena - that is with Spef2A, Cfap69, Androglobin and Cfap246/Lrguk. BioID and pull-down experiments results showed also that both Dusp5 and kinesin Kif9 directly interact with Mapk3, Map2k7 and Nek6 – that is with all examined kinases. None of them interacted with Pp2c phosphatase. These results allowed to propose a model of interaction network between these proteins. Moreover, functional analysis of Dusp5 and Kif9 was also performed. For this, Tetrahymena knock-out mutants lacking Dusp5 or Kif9 were prepared. Lack of Dusp5 or Kif9 resulted in reduced cell swimming speed. Interestingly, both mutants had slower cilia regeneration rate after experimental deciliation which suggest that both of them can participate in ciliary intraflagellar transport. Additionally, no changes in cell proliferation, phagocytosis or cilia length have been observed. To sum up, conducted experiments allowed to identify and at least partly characterize novel potential regulators of the central apparatus functions.

Detailed Resource Type:

PhD Dissertations

Resource Identifier:

oai:rcin.org.pl:242821

Source:

IBD PAN, call no. 20570

Language:

pol

Language of abstract:

eng

Rights:

Rights Reserved - Free Access

Terms of use:

Copyright-protected material. May be used within the limits of statutory user freedoms

Copyright holder:

Publication made available with the written permission of the author

Digitizing institution:

Nencki Institute of Experimental Biology of the Polish Academy of Sciences

Original in:

Library of the Nencki Institute of Experimental Biology PAS

Access:

Open