RESUMO
Lysosomes are membrane-surrounded cytoplasmic organelles filled with a powerful cocktail of hydrolases. Besides degrading cellular constituents inside the lysosomal lumen, lysosomal hydrolases promote tissue remodeling when delivered to the extracellular space and cell death when released to the cytosol. Here, we show that spatially and temporally controlled lysosomal leakage contributes to the accurate chromosome segregation in normal mammalian cell division. One or more chromatin-proximal lysosomes leak in the majority of prometaphases, after which active cathepsin B (CTSB) localizes to the metaphase chromatin and cleaves a small subset of histone H3. Stabilization of lysosomal membranes or inhibition of CTSB activity during mitotic entry results in a significant increase in telomere-related chromosome segregation defects, whereas cells and tissues lacking CTSB and cells expressing CTSB-resistant histone H3 accumulate micronuclei and other nuclear defects. These data suggest that lysosomal leakage and chromatin-associated CTSB contribute to proper chromosome segregation and maintenance of genomic integrity.
Assuntos
Catepsina B/metabolismo , Cromatina/metabolismo , Segregação de Cromossomos , Lisossomos/metabolismo , Mitose , Animais , Catepsina B/antagonistas & inibidores , Catepsina B/genética , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/patologia , Segregação de Cromossomos/genética , Feminino , Inativação Gênica , Histonas/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Lisossomos/enzimologia , Metáfase , Camundongos , Mitose/genética , Permeabilidade , Telômero/metabolismoRESUMO
As microRNAs (miRs) are gaining increasing attention as key regulators of cellular processes, expressional quantification is widely applied. However, in the processing of relatively quantified data, the importance of testing the stability of several reference mRNAs and/or miRs and choosing among these for normalization is often overlooked, potentially leading to biased results. Here, we have optimized the purification of miR-enriched total RNA from pancreatic insulin-producing INS-1 cells. Additionally, we optimized and analyzed miR expression by a qPCR-based microarray and by specific qPCR and tested the stability of candidate reference mRNAs and miRs. Hence, this study gives a widely applicable example on how to easily and systematically test and decide how to normalize miR quantification. We suggest that caution in the interpretation of miR quantification studies that do not comprise stability analysis should be exerted.
