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Lokalizacja i funkcja miozyny VI w jąderku komórek neurosekrecyjnych PC12 : praca doktorska
Creator: Institutional creator:Instytut Biologii Doświadczalnej im. Marcelego Nenckiego PAN
Contributor:Rędowicz, Maria Jolanta : Supervisor ; Lenartowski, Robert : Assistant supervisor
Publisher:Instytut Biologii Doświadczalnej im. M. Nenckiego PAN
Place of publishing: Date issued/created: Description:157 pages : illustrations ; 30 cm ; Affiliation of an assistant supervisor: Uniwersytet Mikołaja Kopernika w Toruniu ; Bibliography ; Summary in English
Degree name: Degree discipline : Degree grantor:Nencki Institute of Experimental Biology PAS ; degree obtained: 24.02.2023
Type of object: Subject and Keywords:Actinomycin D (ActD) ; Fibrillarin ; B23 protein ; Myosin VI ; Nucleolin ; Nucleolus
Abstract:Myosin VI (MVI) is a unique motor protein that, unlike other myosins, moves toward the "minus" end of actin filaments. MVI is involved in migration, adhesion, exo- and endocytosis, stabilization of the Golgi apparatus structure, cytokinesis and gene expression. Its involvement in these processes involves cargo transport and/or cargo anchoring with actin filaments. The presence of MVI in the nucleus has been known since 2006, when it was shown that in transcriptionally active HeLa cells, MVI colocalizes with the RNA polymerase II complex (Pol II) and with newly formed mRNA transcripts. Subsequent studies conducted in the Laboratory of Molecular Basis of Cell Motility, Nencki Institute of Experimental Biology of the Polish Academy of Sciences, on PC12 neurosecretory cells derived from rat adrenal medulla tumor confirmed these observations. Moreover, it was shown that after stimulation of PC12 cells with KCl the level of MVI in the nucleus increases, and this is accompanied by an increase in its colocalization with the active form of Pol II. In addition, mass spectrometry analysis allowed for identification of a number of new potential nuclear partners of MVI, which may determine its role in the cell nucleus. Among them was nucleolin, a nucleolar protein involved in ribosome biogenesis as well as S6, a structural protein of the small ribosomal subunit (40S). The lack of literature data on the function of MVI in the nucleolus encouraged me to investigate the potential role of this motor protein in this region of the nucleus.In my study, I confirmed the interaction of MVI with nucleolin [a protein characteristic of the granular component (GC) of the nucleus, where the assembly of ribosome subunits takes place] and the ribosomal protein S6 (involved in the export of the small subunit of the ribosome from the nucleus to the cytoplasm). I also showed that other nucleolar proteins also interact with MVI. These included upstream binding factor (UBF) [characteristic of the fibrillar center (FC) of the nucleolus, where rDNA transcription takes place], fibrillarin [characteristic of the dense fibrillar component (DFC) of the nucleolus, where pre-rRNA maturation takes place] and B23 protein [characteristic of the GC]. Thus, the interaction of MVI with nucleolar proteins and S6 suggests the involvement of MVI in various steps of ribosome biogenesis. Using electron microscopy, I determined the subnuclear localization of MVI; MVI is mainly found in the DFC and the GC region. Further analysis using electron microscopy showed that under actinomycin D (ActD)-induced nucleolar stress, the nucleolus disintegrates. In addition, I observed that in MVI-knockdown cells the ultrastructure of the nucleolus was less compact compared to control cells, which might indicate that MVI is involved in the maintaining the normal organization of the nucleolus. Moreover, confocal microscopy analysis showed that MVI is involved in the localization of the nucleolar protein B23 under control and nucleolar stress conditions, which may suggest the functionality of these interactions. In addition, I observed that reduced level of MVI is accompanied by disruption of the organization of the endoplasmic reticulum (ER). Also, I observed that in cells with reduced levels of MVI, staining against the ribosomal protein S6 was significantly less intense compared to control cells, which may suggest the involvement of MVI in the organization and function of ribosomes. In contrast, I did not show an effect of reduced MVI levels on 45S pre-rRNA level.In summary, my results indicate that MVI is involved in the organization of the nucleolus and ER but does not play a significant role in rDNA transcription. Since the maintenance of proper nucleolar organization is important for ribosome biogenesis (the disruption of which leads to many serious diseases), the results presented here open the possibility for further research into the mechanisms of MVI function in the nucleolus
Resource type: Detailed Resource Type: Source: Language: Language of abstract: Rights: 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
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