RESUMEN
Hematopoietic Stem and Progenitor Cells (HSPCs) give rise to all cell types of the hematopoietic system through various processes including asymmetric divisions. However, the contribution of stromal cells of the hematopoietic niches in the control of HSPC asymmetric divisions remains unknown. Using polyacrylamide microwells as minimalist niches, we show that specific heterotypic interaction with osteoblast and endothelial cell promote asymmetric division of human HSPC. Upon interaction, HSPCs polarize in interphase with the centrosome, the Golgi apparatus, and lysosomes positioned close to the site of contact. Subsequently, during mitosis, HSPCs orient their spindle perpendicular to the plane of contact. This division mode gives rise to siblings with unequal amounts of lysosomes and of CD34 differentiation marker. Such asymmetric inheritance generates heterogeneity in the progeny, which is likely to contribute to the plasticity of the early steps of hematopoiesis.
RESUMEN
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.
Asunto(s)
Diferenciación Celular , Expresión Génica , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Microtúbulos , Línea Celular , Linaje de la Célula , Núcleo Celular/genética , Núcleo Celular/fisiología , Citocinas , Células Madre Hematopoyéticas/citología , Histonas , Humanos , TranscriptomaRESUMEN
Invariant natural killer T (iNKT) cells expressing the retinoic acid receptor-related orphan receptor γt (RORγt) and producing IL-17 represent a minor subset of CD1d-restricted iNKT cells (iNKT17) in C57BL/6J (B6) mice. We aimed in this study to define the reasons for their low distribution and the sequence of events accompanying their normal thymic development. We found that RORγt+ iNKT cells have higher proliferation potential and a greater propensity to apoptosis than RORγt- iNKT cells. These cells do not likely reside in the thymus indicating that thymus emigration, and higher apoptosis potential, could contribute to RORγt+ iNKT cell reduced thymic distribution. Ontogeny studies suggest that mature HSAlow RORγt+ iNKT cells might develop through developmental stages defined by a differential expression of CCR6 and CD138 during which RORγt expression and IL-17 production capabilities are progressively acquired. Finally, we found that RORγt+ iNKT cells perceive a strong TCR signal that could contribute to their entry into a specific 'Th17 like' developmental program influencing their survival and migration. Overall, our study proposes a hypothetical thymic developmental sequence for iNKT17 cells, which could be of great use to study molecular mechanisms regulating this developmental program.
Asunto(s)
Células T Asesinas Naturales/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/deficienciaRESUMEN
It is well established that chromosome segregation in female meiosis I (MI) is error-prone. The acentrosomal meiotic spindle poles do not have centrioles and are not anchored to the cortex via astral microtubules. By Cre recombinase-mediated removal in oocytes of the microtubule binding site of nuclear mitotic apparatus protein (NuMA), which is implicated in anchoring microtubules at poles, we determine that without functional NuMA, microtubules lose connection to MI spindle poles, resulting in highly disorganized early spindle assembly. Subsequently, very long spindles form with hyperfocused poles. The kinetochores of homologs make attachments to microtubules in these spindles but with reduced tension between them and accompanied by alignment defects. Despite this, the spindle assembly checkpoint is normally silenced and the advance to anaphase I and first polar body extrusion takes place without delay. Females without functional NuMA in oocytes are sterile, producing aneuploid eggs with altered chromosome number. These findings establish that in mammalian MI, the spindle assembly checkpoint is unable to sustain meiotic arrest in the presence of one or few misaligned and/or misattached kinetochores with reduced interkinetochore tension, thereby offering an explanation for why MI in mammals is so error-prone.
Asunto(s)
Segregación Cromosómica/fisiología , Infertilidad Femenina/fisiopatología , Cinetocoros/fisiología , Meiosis/fisiología , Proteínas Nucleares/genética , Huso Acromático/fisiología , Anafase/fisiología , Aneuploidia , Animales , Proteínas de Ciclo Celular , Células Cultivadas , Femenino , Infertilidad Femenina/genética , Infertilidad Femenina/metabolismo , Mamíferos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microtúbulos/fisiología , Proteínas Nucleares/metabolismo , Oocitos/citología , Oocitos/fisiología , Transducción de Señal/fisiología , Estrés MecánicoRESUMEN
The vast majority of animal cells contain canonical centrosomes as a main microtubule-organizing center defined by a central pair of centrioles. As a rare and striking exception to this rule, vertebrate oocytes loose their centrioles at an early step of oogenesis. At the end of oogenesis, centrosomes are eventually replaced by numerous acentriolar microtubule-organizing centers (MTOCs) that shape the spindle poles during meiotic divisions. The mechanisms involved in centrosome and acentriolar MTOCs metabolism in oocytes have not been elucidated yet. In addition, little is known about microtubule organization and its impact on intracellular architecture during the oocyte growth phase following centrosome disassembly. We have investigated this question in the mouse by coupling immunofluorescence and live-imaging approaches. We show that growing oocytes contain dispersed pericentriolar material, responsible for microtubule assembly and distribution all over the cell. The gradual enlargement of PCM foci eventually leads in competent oocytes to the formation of big perinuclear MTOCs and to the assembly of large microtubule asters emanating from the close vicinity of the nucleus. Upon meiosis resumption, perinuclear MTOCs spread around the nuclear envelope, which in parallel is remodelled before breaking-down, via a MT- and dynein-dependent mechanism. Only fully competent oocytes are able to perform this dramatic reorganization at NEBD. Therefore, the MTOC-MT reorganization that we describe is one of key feature of mouse oocyte competency.
Asunto(s)
Centriolos/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Oogénesis , Animales , Núcleo Celular/metabolismo , Proliferación Celular , Dineínas/metabolismo , Femenino , Meiosis , Ratones , Microtúbulos/metabolismo , Oocitos/citología , Oocitos/metabolismoRESUMEN
The bone marrow (BM) is a complex microenvironment in which hematopoietic stem and progenitor cells (HSPCs) interact with multiple cell types that regulate their quiescence, growth, and differentiation. These cells constitute local niches where HSPCs are confined and subjected to specific set of physical and biochemical cues. Endothelial cells forming the walls of blood capillaries have been shown to establish a vascular niche, whereas osteoblasts lying along the bone matrix organize the endosteal niche with distinct and specific impact on HSPC fate. The observation of the interaction of HSPCs with niche cells, and the investigation of its impact on HSPCs behavior in vivo is hindered by the opacity of the bone matrix. Therefore, various experimental strategies have been devised to reconstitute in vitro the interaction of HSPCs with distinct sets of BM-derived cells. In this chapter, we present a method to manufacture a pseudo BM-on-a-chip with separated compartments mimicking the vascular and the endosteal niches. Such a configuration with connected but distant compartments allowed the investigation of the specific contribution of each niche to the regulation of HSPC behavior. We describe the microfabrication of the chip with a maskless photolithography method that allows the iterative improvement of the geometric design of the chip in order to optimize the adaptation of the multicellular architecture to the specific aim of the study. We also describe the loading and culture of the various cell types in each compartment.
Asunto(s)
Células de la Médula Ósea/fisiología , Células Madre Hematopoyéticas/fisiología , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Nicho de Células Madre , Ingeniería de Tejidos/instrumentación , Diferenciación Celular , Línea Celular , Técnicas de Cocultivo , Células Endoteliales/fisiología , Diseño de Equipo , Humanos , Hidrogeles , Osteoblastos/fisiología , FenotipoRESUMEN
The fate of hematopoietic stem and progenitor cells (HSPCs) is regulated by their interaction with stromal cells in the bone marrow. However, the cellular mechanisms regulating HSPC interaction with these cells and their potential impact on HSPC polarity are still poorly understood. Here we evaluated the impact of cell-cell contacts with osteoblasts or endothelial cells on the polarity of HSPC. We found that an HSPC can form a discrete contact site that leads to the extensive polarization of its cytoskeleton architecture. Notably, the centrosome was located in proximity to the contact site. The capacity of HSPCs to polarize in contact with stromal cells of the bone marrow appeared to be specific, as it was not observed in primary lymphoid or myeloid cells or in HSPCs in contact with skin fibroblasts. The receptors ICAM, VCAM, and SDF1 were identified in the polarizing contact. Only SDF1 was independently capable of inducing the polarization of the centrosome-microtubule network.
Asunto(s)
Médula Ósea/metabolismo , Médula Ósea/fisiología , Quimiocina CXCL12/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/fisiología , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/fisiología , HumanosRESUMEN
TPX2 has multiple functions during mitosis, including microtubule nucleation around the chromosomes and the targeting of Xklp2 and Aurora A to the spindle. We have performed a detailed domain functional analysis of TPX2 and found that a large N-terminal domain containing the Aurora A binding peptide interacts directly with and nucleates microtubules in pure tubulin solutions. However, it cannot substitute the endogenous TPX2 to support microtubule nucleation in response to Ran guanosine triphosphate (GTP) and spindle assembly in egg extracts. By contrast, a large C-terminal domain of TPX2 that does not bind directly to pure microtubules and does not bind Aurora A kinase rescues microtubule nucleation in response to RanGTP and spindle assembly in TPX2-depleted extract. These and previous results suggest that under physiological conditions, TPX2 is essential for microtubule nucleation around chromatin and functions in a network of other molecules, some of which also are regulated by RanGTP.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Extractos Celulares/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Oocitos/citología , Fosfoproteínas/metabolismo , Huso Acromático/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , Aurora Quinasas , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Polaridad Celular , Activación Enzimática , Cinesinas/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Oocitos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Estructura Terciaria de Proteína , Huso Acromático/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
The postnatal mammalian ovary contains the primary follicles, each comprising an immature oocyte surrounded by a layer of somatic granulosa cells. Oocytes reach meiotic and developmental competence via folliculogenesis. During this process, the granulosa cells proliferate massively around the oocyte, form an extensive extracellular matrix (ECM) and differentiate into cumulus cells. As the ECM component hyaluronic acid (HA) is thought to form the backbone of the oocyte-granulosa cell complex, we deleted the relevant domain of the Receptor for HA Mediated Motility (RHAMM) gene in the mouse. This resulted in folliculogenesis defects and female hypofertility, although HA-induced signalling was not affected. We report that wild-type RHAMM localises at the mitotic spindle of granulosa cells, surrounding the oocyte. Deletion of the RHAMM C-terminus in vivo abolishes its spindle association, resulting in impaired spindle orientation in the dividing granulosa cells, folliculogenesis defects and subsequent female hypofertility. These data reveal the first identified physiological function for RHAMM, during oogenesis, and the importance of this spindle-associated function for female fertility.
RESUMEN
BACKGROUND: During meiosis, mammalian oocytes undergo two successive cell divisions without an intermediate replicative phase. This brief period, called 'meiotic maturation', is crucial for the formation of an egg capable of being fertilized and of generating viable and euploid offspring. METHODS: We review our current knowledge of the cellular and molecular mechanisms that control asymmetry and appear to be shared between mammalian species, as well as the associated misfunctions that impair the formation of functional female gametes. RESULTS AND CONCLUSIONS: The two successive divisions that comprise mammalian oogenesis are asymmetric. They lead to the formation of small polar bodies and the large and polarized egg. This asymmetry depends upon the dynamic organization of the oocyte cytoskeleton during both divisions. During meiosis I, microfilaments and associated molecules ensure the targeting of the microtubule spindle at the oocyte periphery. During meiosis II, they anchor the spindle under the plasma membrane. In parallel, the cortex overhanging the spindle is dramatically reorganized. Establishment and maintenance of this cortical domain are crucial for the completion of fertilization. Loss of this differentiated area is characteristic of ageing or low-quality gametes and associated with increased maternal age or post-ovulatory ageing.
Asunto(s)
Meiosis/fisiología , Oocitos/citología , Citoesqueleto de Actina/fisiología , Citoesqueleto de Actina/ultraestructura , Animales , Polaridad Celular , Femenino , Fertilización/fisiología , Humanos , Mamíferos , Microtúbulos/fisiología , Microtúbulos/ultraestructura , Oocitos/ultraestructura , Huso Acromático/fisiología , Huso Acromático/ultraestructuraRESUMEN
In contrast to somatic cells, formation of acentriolar meiotic spindles relies on the organization of microtubules (MTs) and MT-organizing centers (MTOCs) into a stable bipolar structure. The underlying mechanisms are still unknown. We show that this process is impaired in hepatoma up-regulated protein (Hurp) knockout mice, which are viable but female sterile, showing defective oocyte divisions. HURP accumulates on interpolar MTs in the vicinity of chromosomes via Kinesin-5 activity. By promoting MT stability in the spindle central domain, HURP allows efficient MTOC sorting into distinct poles, providing bipolarity establishment and maintenance. Our results support a new model for meiotic spindle assembly in which HURP ensures assembly of a central MT array, which serves as a scaffold for the genesis of a robust bipolar structure supporting efficient chromosome congression. Furthermore, HURP is also required for the clustering of extra centrosomes before division, arguing for a shared molecular requirement of MTOC sorting in mammalian meiosis and cancer cell division.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centrosoma/metabolismo , Meiosis/fisiología , Centro Organizador de los Microtúbulos/metabolismo , Mitosis/fisiología , Neoplasias/patología , Huso Acromático/metabolismo , Anafase/genética , Animales , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Segregación Cromosómica/genética , Femenino , Expresión Génica/genética , Humanos , Cinesinas/antagonistas & inhibidores , Cinesinas/metabolismo , Cinetocoros/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neoplasias/genética , Oocitos/metabolismo , ARN Interferente Pequeño/genética , Huso Acromático/genéticaRESUMEN
Formation of female gametes requires acentriolar spindle assembly during meiosis. Mitotic spindles organize from centrosomes and via local activation of the RanGTPase on chromosomes. Vertebrate oocytes present a RanGTP gradient centred on chromatin at all stages of meiotic maturation. However, this gradient is dispensable for assembly of the first meiotic spindle. To understand this meiosis I peculiarity, we studied TPX2, a Ran target, in mouse oocytes. Strikingly, TPX2 activity is controlled at the protein level through its accumulation from meiosis I to II. By RNAi depletion and live imaging, we show that TPX2 is required for spindle assembly via two distinct functions. It controls microtubule assembly and spindle pole integrity via the phosphorylation of TACC3, a regulator of MTOCs activity. We show that meiotic spindle formation in vivo depends on the regulation of at least a target of Ran, TPX2, rather than on the regulation of the RanGTP gradient itself.
Asunto(s)
Ciclo Celular , Meiosis , Oocitos/citología , Animales , Secuencia de Bases , Femenino , Técnica del Anticuerpo Fluorescente , Ratones , Oocitos/metabolismo , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño , Huso AcromáticoRESUMEN
During meiotic maturation, mammalian oocytes undergo an asymmetric division which is crucial for the formation of a functional gamete. In various organisms, accurate positioning of the nucleus before M-phase plays a major role in asymmetric cell divisions. However, the role of the position of the nucleus (or germinal vesicle, GV) during the prophase I arrest has not been investigated in mammalian oocytes. Here, we show that incompetent mouse oocytes possess a peripheral GV, while competent oocytes mainly exhibit a central position of the GV. At that time, the position of the GV correlates with the ability of the oocyte to complete meiotic maturation. Moreover, a lower efficiency in GV centering and a reduced ability to progress through meiosis are observed in oocytes from old mice. Thus, the position of the GV could be used as a simple morphological marker of oocyte quality.
Asunto(s)
Núcleo Celular/ultraestructura , Mamíferos/fisiología , Profase Meiótica I , Oocitos/ultraestructura , Oogénesis/fisiología , Animales , Supervivencia Celular , Células Cultivadas , Femenino , Edad Materna , RatonesRESUMEN
We present an in vitro system to analyze quantitatively the interactions of green fluorescent protein (GFP)-tagged recombinant proteins with microtubules. This method relies on photoconversion of GFP and time-lapse microscopy. Specific interactions can be detected and binding kinetics can be determined rapidly and accurately. This method provides an alternative to classical in vitro microtubule-binding assays to analyze microtubule-associated proteins binding to microtubules. It has the potential to be extended to study interactions of proteins or multi-protein complexes with different biopolymers like actin microfilaments or organelle membranes.
Asunto(s)
Proteínas Fluorescentes Verdes , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Coloración y Etiquetado , HumanosRESUMEN
During meiotic maturation of mammalian oocytes, two successive divisions occur without an intermediate phase of DNA replication, so that haploid gametes are produced. Moreover, these two divisions are asymmetric, to ensure that most of the maternal stores are retained within the oocyte. This leads to the formation of daughter cells with different sizes: the large oocyte and the small polar bodies. All these events are dependent upon the dynamic changes in the organization of the oocyte cytoskeleton (microtubules and microfilaments) and are highly regulated in time and space. We review here the current knowledge of the interplay between the cytoskeleton and the cell cycle machinery in mouse oocytes, with an emphasis on the two major activities that control meiotic maturation in vertebrates, MPF (Maturation promoting factor) and CSF (Cytostatic factor).