RESUMEN
The nucleus is the stiffest organelle in the cell. Several morphogenetic processes depend on its deformation such as cell migration, cell differentiation, or senescence. Recent studies have revealed various mechanisms involved in the regulation of nucleus stiffness and deformation. The implication of chromatin swelling, lamin density, actin filament, and microtubule network revealed that nucleus shape is the outcome of a fine balance between various sources of external forces and numerous means of internal resistance. In adherent cells, the actin network is the dominant player in external force production, whereas in nonadherent cells microtubules seem to take over. It is therefore important to set up reconstitution assays in order to decipher the exact contribution of each player in this mechanical balance. In this method, we describe a nucleus purification protocol that is suitable for nonadherent cells. We also show that purified nuclei can interact with microtubules and that nuclei purified from distinct cell types get differentially wrapped into the array of microtubules. A combination with a microtubule gliding assay offers the possibility to counterbalance the binding to the nucleus membrane by active motor-based forces pulling on microtubules. So this protocol allows an in-depth study of microtubule-nucleus interactions in vitro.
Asunto(s)
Núcleo Celular , Microtúbulos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Núcleo Celular/metabolismo , Fenómenos Mecánicos , Microtúbulos/metabolismoRESUMEN
Hematopoietic stem and progenitor cells (HSPC) can differentiate into all hematopoietic lineages to support hematopoiesis. Cells from the myeloid and lymphoid lineages fulfill distinct functions with specific shapes and intra-cellular architectures. The role of cytokines in the regulation of HSPC differentiation has been intensively studied but our understanding of the potential contribution of inner cell architecture is relatively poor. Here, we show that large invaginations are generated by microtubule constraints on the swelling nucleus of human HSPC during early commitment toward the myeloid lineage. These invaginations are associated with a local reduction of lamin B density, local loss of heterochromatin H3K9me3 and H3K27me3 marks, and changes in expression of specific hematopoietic genes. This establishes the role of microtubules in defining the unique lobulated nuclear shape observed in myeloid progenitor cells and suggests that this shape is important to establish the gene expression profile specific to this hematopoietic lineage. It opens new perspectives on the implications of microtubule-generated forces, in the early commitment to the myeloid lineage.