RESUMO
We report here the functional analysis of human Regulator of Ribosome Synthesis 1 (RRS1) protein during mitosis. We demonstrate that RRS1 localizes in the nucleolus during interphase and is distributed at the chromosome periphery during mitosis. RNA interference experiments revealed that RRS1-depleted cells show abnormalities in chromosome alignment and spindle organization, which result in mitotic delay. RRS1 knockdown also perturbs the centromeric localization of Shugoshin 1 and results in premature separation of sister chromatids. Our results suggest that a nucleolar protein RRS1 contributes to chromosome congression.
Assuntos
Cromossomos Humanos/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Nucléolo Celular/metabolismo , Nucléolo Celular/ultraestrutura , Pareamento Cromossômico , Segregação de Cromossomos , Cromossomos Humanos/ultraestrutura , Células HeLa , Humanos , Microscopia de Fluorescência , Mitose , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fuso Acromático/metabolismo , Fuso Acromático/ultraestruturaRESUMO
BACKGROUND: Cell-based assays utilizing digital image cytometry yield multivariate sets of information measuring the efficacy of medicines/chemicals. The use of a HeLa cell line that expresses a GFP-Histone-H1 fusion protein further enhances the performance of these systems, avoiding the use of dyes that may have detrimental influence on cells. Aside from the mitotic index, the distribution of the cell-cycle phases during mitosis can be used as measures of drug/treatment efficacy. Quantification of these parameters, however, requires skill and is time consuming. The purpose of this research was therefore to create a classifier to be incorporated into a system that can automatically identify the cell-cycle phases in a given image. METHODS: Features based on the shape and texture of the chromosomal regions in images of live HeLa cells were measured and analyzed. Linear discriminant functions were calculated for the eight cell-cycle phases: interphase, prophase, prometaphase, metaphase, early anaphase, anaphase, telophase and cytokinesis. RESULTS: The multistage linear discriminant classifier developed had an average classification efficiency of 87.30%. CONCLUSION: We demonstrated the possibility of creating a classifier to discriminate between cell-cycle phases using shape and texture features of chromosomal regions. The classifier can be fused to an algorithm for image segmentation, forming a system to automatically and rapidly measure the aforementioned parameters. The results can then be collated to constitute an assay assessing the effects of a drug or treatment on mammalian cells.
