Kanser hücre bölünmesinde keratin 8'in düzenlenmesi

Keratins are cytoskeletal proteins classified as Type I and Type II intermediate filaments. These filamentous structures provide mechanical strength and stability to the cells, but it is not known well how these robust structures are regulated and function during dynamic processes, such as cell division. To better understand the biochemistry of cell division and the regulation of keratins, I focused on keratin 8 in cancer cell division. Keratin 8 is a good candidate with a maintained expression during tumorigenesis and has diverse expression patterns in different cell types. During cell division, these filamentous structures split into non-filamentous clusters with mechanisms that are not well understood. To uncover the phosphorylation-dependent disassembly, I analyzed the cell cycle-dependent phosphoproteome of keratin 8 in different cell lines and investigated its commonly regulated phosphorylation sites. I took label-free and dimethyl labeling-based quantitative approaches combined with TiO2 phosphopeptide enrichment methodology. I applied DDA, DIA, and PRM-based mass spectrometry methods and identified and quantified commonly regulated or cell-type specific phosphorylation sites of keratin 8 during cell division. To understand the functional role of keratins, interaction partners of keratin 8 were investigated by immunoprecipitation. I focused on the relation of keratin 8 with other cytoskeletal elements and further searched for its role in mitosis by analyzing its relation with microtubules. I performed microtubule pelleting assays to determine keratin 8-dependent microtubule interactors. I focused on the NDC80 complex during metaphase and further investigated the role of keratin 8 at the kinetochore-microtubule attachment by super-resolution imaging. Our study brought a better understanding of the regulation and functioning of keratins during cell division.