RESUMEN
Cell division requires the wholesale reorganization of cell architecture. At the same time as the microtubule network is remodelled to generate a bipolar spindle, animal cells entering mitosis replace their interphase actin cytoskeleton with a contractile mitotic actomyosin cortex that is tightly coupled to the plasma membrane--driving mitotic cell rounding. Here, we consider how these two processes are coordinated to couple chromosome segregation and cell division. In doing so we explore the relative roles of cell shape and the actin cortex in spindle morphogenesis, orientation and positioning.
Asunto(s)
Citoesqueleto de Actina/metabolismo , División Celular , Microtúbulos/metabolismo , Huso Acromático/metabolismo , Animales , Forma de la Célula , Segregación Cromosómica , Humanos , Mitosis , Multimerización de ProteínaRESUMEN
The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. Here we show that the conserved kinase NHK-1 (Drosophila Vrk-1) is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, we find unrepaired DNA breaks formed during recombination suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates) through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Meiosis/genética , Protamina Quinasa/genética , Animales , Línea Celular , Polaridad Celular , Roturas del ADN de Doble Cadena/efectos de la radiación , Daño del ADN , Reparación del ADN/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Histonas/metabolismo , Immunoblotting , Masculino , Modelos Biológicos , Mutación , Oocitos/metabolismo , Fosforilación , Protamina Quinasa/metabolismoRESUMEN
Proliferating animal cells are able to orient their mitotic spindles along their interphase cell axis, setting up the axis of cell division, despite rounding up as they enter mitosis. This has previously been attributed to molecular memory and, more specifically, to the maintenance of adhesions and retraction fibers in mitosis [1-6], which are thought to act as local cues that pattern cortical Gαi, LGN, and nuclear mitotic apparatus protein (NuMA) [3, 7-18]. This cortical machinery then recruits and activates Dynein motors, which pull on astral microtubules to position the mitotic spindle. Here, we reveal a dynamic two-way crosstalk between the spindle and cortical motor complexes that depends on a Ran-guanosine triphosphate (GTP) signal [12], which is sufficient to drive continuous monopolar spindle motion independently of adhesive cues in flattened human cells in culture. Building on previous work [1, 12, 19-23], we implemented a physical model of the system that recapitulates the observed spindle-cortex interactions. Strikingly, when this model was used to study spindle dynamics in cells entering mitosis, the chromatin-based signal was found to preferentially clear force generators from the short cell axis, so that cortical motors pulling on astral microtubules align bipolar spindles with the interphase long cell axis, without requiring a fixed cue or a physical memory of interphase shape. Thus, our analysis shows that the ability of chromatin to pattern the cortex during the process of mitotic rounding is sufficient to translate interphase shape into a cortical pattern that can be read by the spindle, which then guides the axis of cell division.
Asunto(s)
Dineínas/fisiología , Mecanotransducción Celular , Microtúbulos/fisiología , Mitosis , Huso Acromático/fisiología , Células HeLa , Humanos , Transducción de SeñalRESUMEN
Accurate animal cell division requires precise coordination of changes in the structure of the microtubule-based spindle and the actin-based cell cortex. Here, we use a series of perturbation experiments to dissect the relative roles of actin, cortical mechanics, and cell shape in spindle formation. We find that, whereas the actin cortex is largely dispensable for rounding and timely mitotic progression in isolated cells, it is needed to drive rounding to enable unperturbed spindle morphogenesis under conditions of confinement. Using different methods to limit mitotic cell height, we show that a failure to round up causes defects in spindle assembly, pole splitting, and a delay in mitotic progression. These defects can be rescued by increasing microtubule lengths and therefore appear to be a direct consequence of the limited reach of mitotic centrosome-nucleated microtubules. These findings help to explain why most animal cells round up as they enter mitosis.
Asunto(s)
Actinas/metabolismo , Forma de la Célula , Mitosis , Huso Acromático/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Centrosoma/metabolismo , Cromosomas Humanos/genética , Cromosomas Humanos/metabolismo , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Microtúbulos/genética , Microtúbulos/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Complejo Shelterina , Huso Acromático/genética , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
Both symmetric and asymmetric divisions rely on alignment of the mitotic spindle with cues from the environment. A study now shows that mitotic spindles find their position by reading the map of forces that load-bearing retraction fibres exert on the cell body.
Asunto(s)
Adhesión Celular , Mecanotransducción Celular , Mitosis , Huso Acromático/fisiología , Actinas/metabolismo , Animales , Polaridad Celular , Forma de la Célula , Fibronectinas/metabolismo , Homeostasis , Humanos , Morfogénesis , Rotación , Huso Acromático/metabolismo , Estrés MecánicoRESUMEN
Accurate chromosome segregation in meiosis requires dynamic changes in chromatin organization. In Drosophila melanogaster, upon completion of recombination, meiotic chromosomes form a single, compact cluster called the karyosome in an enlarged oocyte nucleus. This clustering is also found in humans; however, the mechanisms underlying karyosome formation are not understood. In this study, we report that phosphorylation of barrier to autointegration factor (BAF) by the conserved kinase nucleosomal histone kinase-1 (NHK-1; Drosophila Vrk1) has a critical function in karyosome formation. We find that the noncatalytic domain of NHK-1 is crucial for its kinase activity toward BAF, a protein that acts as a linker between chromatin and the nuclear envelope. A reduction of NHK-1 or expression of nonphosphorylatable BAF results in ectopic association of chromosomes with the nuclear envelope in oocytes. We propose that BAF phosphorylation by NHK-1 disrupts anchorage of chromosomes to the nuclear envelope, allowing karyosome formation in oocytes. These data provide the first mechanistic insight into how the karyosome forms.