Abstract
Packaging of DNA into condensed chromosomes during mitosis is essential for the faithful segregation of the genome into daughter nuclei. Although the structure and composition of mitotic chromosomes have been studied for over 30 years, these aspects are yet to be fully elucidated. Here, we used stable isotope labeling with amino acids in cell culture to compare the proteomes of mitotic chromosomes isolated from cell lines harboring conditional knockouts of members of the condensin (SMC2, CAP-H, CAP-D3), cohesin (Scc1/Rad21), and SMC5/6 (SMC5) complexes. Our analysis revealed that these complexes associate with chromosomes independently of each other, with the SMC5/6 complex showing no significant dependence on any other chromosomal proteins during mitosis. To identify subtle relationships between chromosomal proteins, we employed a nano Random Forest (nanoRF) approach to detect protein complexes and the relationships between them. Our nanoRF results suggested that as few as 113 of 5058 detected chromosomal proteins are functionally linked to chromosome structure and segregation. Furthermore, nanoRF data revealed 23 proteins that were not previously suspected to have functional interactions with complexes playing important roles in mitosis. Subsequent small-interfering-RNA-based validation and localization tracking by green fluorescent protein-tagging highlighted novel candidates that might play significant roles in mitotic progression.
Highlights
Mitotic chromosome condensation, regulated sister chromatid cohesion, and chromosome interactions with the spindle are crucial to ensuring appropriate genome segregation during mitosis and meiosis
Dependence of the Mitotic Chromosome Proteome on Condensins I and II—Previous findings revealed that SMC2-depleted chromosomes appeared relatively normal during early mitosis, they lost their organized structure during anaphase [20]
Results presented in this study demonstrated that the condensin, cohesin, and SMC5/6 structural maintenance of chromosomes (SMC) complexes associate with mitotic chromosomes independently of each other
Summary
Proteomics Analysis with a Nano Random Forest Approach Reveals Novel Functional Interactions Regulated by SMC Complexes on Mitotic Chromosomes*□S. Multivariate profiling employing principal component analysis has proven useful [34], as has the multiclassifier combinatorial proteomics approach for integrating data from multiple “classifiers” using Random-Forest analysis [35] This latter approach appeared to be useful in distinguishing cytoplasmic “hitchhikers” associated with isolated mitotic chromosomes from proteins that contribute to chromosomal structure and segregation during mitosis [35, 36]. Condensin depletions appeared to have the most profound effects on the mitotic chromosome proteome, and depletions of the SMC5/6 complex appeared to have the least impact, with the only significant changes occurring exclusively among members of that complex This analysis revealed few structural links between cohesin and condensin, in contrast to suggestions of several published studies [14, 38]. This information was subsequently used to predict and confirm proteins that affect mitotic progression
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have