Two-step nuclear centring by competing microtubule- and actin-based mechanisms in 2-cell embryos
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ABSTRACT:
SUBMITTER: Ms. Yunan Ye
PROVIDER: S-SCDT-10_15252-EMBR_202255251 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Microtubules typically promote nuclear centring during early embryonic divisions in centrosome-containing vertebrates. In acentrosomal mouse zygotes, microtubules also centre male and female pronuclei prior to the first mitosis, this time in concert with actin. How nuclear centring is brought about in subsequent acentrosomal embryonic divisions has not been studied. Here, using time-lapse imaging in mouse embryos, we find that although nuclei are delivered to the cell centre upon completion of the first mitotic anaphase, the majority do not remain stationary and instead travel all the way to the cortex in a microtubule-dependent manner. High cytoplasmic viscosity in 2-cell embryos is associated with non-diffusive mechanisms involving actin for subsequent nuclear centring when microtubules again exert a negative influence. Thus, following the first mitotic division, pro-centring actin-dependent mechanisms work against microtubule-dependent de-centring forces. Disrupting the equilibrium of this tug-of-war compromises nuclear centring and symmetry of the subsequent division potentially risking embryonic development. This circuitous centring process exposes an embryonic vulnerability imposed by microtubule-dependent de-centring forces.
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Project description:Successful mammalian cloning using somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II (MII)-arrested oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing totipotency in somatic cell nuclei. However, these poorly defined maternal factors presumably decline sharply after fertilization, as the cytoplasm of pronuclear-stage zygotes is reportedly inactive. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase, can also support derivation of embryonic stem (ES) cells after SCNT, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in the metaphase but not in the interphase cytoplasm are 'trapped' inside the nucleus during interphase and effectively removed during enucleation. Here we investigated the presence of reprogramming activity in the cytoplasm of interphase two-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated metaphase and interphase zygotes and two-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Second, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ES cell, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ES cells capable of contributing to traditional germline and tetraploid chimaeras. Last, direct transfer of cloned embryos, reconstructed with ES cell nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming, with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to use interphase cytoplasm in SCNT could aid efforts to generate autologous human ES cells for regenerative applications, as donated or discarded embryos are more accessible than unfertilized MII oocytes.
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Project description:The interaction between actin filaments and microtubules is crucial for many eukaryotic cellular processes, such as, among others, cell polarization, cell motility and cellular wound healing. The importance of this interaction has long been recognized, yet very little is understood about both the underlying mechanisms and the consequences for the spatial (re)organization of the cellular cytoskeleton. At the same time, understanding the causes and the consequences of the interaction between different biomolecular components are key questions for in vitro research involving reconstituted biomolecular systems, especially in the light of current interest in creating minimal synthetic cells. In this light, recent in vitro experiments have shown that the actin-microtubule interaction mediated by the cytolinker TipAct, which binds to actin lattice and microtubule tips, causes the directed transport of actin filaments. We develop an analytical theory of dynamically unstable microtubules, nucleated from the centre of a spherical cell, in interaction with actin filaments. We show that, depending on the balance between the diffusion of unbound actin filaments and propensity to bind microtubules, actin is either concentrated in the centre of the cell, where the density of microtubules is highest, or becomes localized to the cell cortex.
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Project description:BackgroundEntry into mitosis triggers profound changes in cell shape and cytoskeletal organisation. Here, by studying microtubule remodelling in human flat mitotic cells, we identify a two-step process of interphase microtubule disassembly.ResultsFirst, a microtubule-stabilising protein, Ensconsin/MAP7, is inactivated in prophase as a consequence of its phosphorylation downstream of Cdk1/cyclin B. This leads to a reduction in interphase microtubule stability that may help to fuel the growth of centrosomally nucleated microtubules. The peripheral interphase microtubules that remain are then rapidly lost as the concentration of tubulin heterodimers falls following dissolution of the nuclear compartment boundary. Finally, we show that a failure to destabilise microtubules in prophase leads to the formation of microtubule clumps, which interfere with spindle assembly.ConclusionsThis analysis highlights the importance of the step-wise remodelling of the microtubule cytoskeleton and the significance of permeabilisation of the nuclear envelope in coordinating the changes in cellular organisation and biochemistry that accompany mitotic entry.
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Project description:Correct nuclear position is crucial for cellular function and tissue development. Depending on cell context, however, the cytoskeletal elements responsible for nuclear positioning vary. While these cytoskeletal mechanisms have been intensely studied in single cells, how nuclear positioning is linked to tissue morphology is less clear. Here, we compare apical nuclear positioning in zebrafish neuroepithelia. We find that kinetics and actin-dependent mechanisms of nuclear positioning vary in tissues of different morphology. In straight neuroepithelia, nuclear positioning is controlled by Rho-ROCK-dependent myosin contractility. In contrast, in basally constricted neuroepithelia, a novel formin-dependent pushing mechanism is found for which we propose a proof-of-principle force generation theory. Overall, our data suggest that correct nuclear positioning is ensured by the adaptability of the cytoskeleton to cell and tissue shape. This in turn leads to robust epithelial maturation across geometries. The conclusion that different nuclear positioning mechanisms are favored in tissues of different morphology highlights the importance of developmental context for the execution of intracellular processes.
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Project description:BackgroundDendrite morphogenesis plays an essential role in establishing the connectivity and receptive fields of neurons during the development of the nervous system. To generate the diverse morphologies of branched dendrites, neurons use external cues and cell surface receptors to coordinate intracellular cytoskeletal organization; however, the molecular mechanisms of how this signaling forms branched dendrites are not fully understood.MethodsWe performed in vivo time-lapse imaging of the PVD neuron in C. elegans in several mutants of actin regulatory proteins, such as the WAVE Regulatory Complex (WRC) and UNC-34 (homolog of Enabled/Vasodilator-stimulated phosphoprotein (Ena/VASP)). We examined the direct interaction between the WRC and UNC-34 and analyzed the localization of UNC-34 in vivo using transgenic worms expressing UNC-34 fused to GFP.ResultsWe identify a stereotyped sequence of morphological events during dendrite outgrowth in the PVD neuron in C. elegans. Specifically, local increases in width ("swellings") give rise to filopodia to facilitate a "rapid growth and pause" mode of growth. In unc-34 mutants, filopodia fail to form but swellings are intact. In WRC mutants, dendrite growth is largely absent, resulting from a lack of both swelling and filopodia formation. We also found that UNC-34 can directly bind to the WRC. Disrupting this binding by deleting the UNC-34 EVH1 domain prevented UNC-34 from localizing to swellings and dendrite tips, resulting in a stunted dendritic arbor and reduced filopodia outgrowth.ConclusionsWe propose that regulators of branched and linear F-actin cooperate to establish dendritic branches. By combining our work with existing literature, we propose that the dendrite guidance receptor DMA-1 recruits the WRC, which polymerizes branched F-actin to generate "swellings" on a mother dendrite. Then, WRC recruits the actin elongation factor UNC-34/Ena/VASP to initiate growth of a new dendritic branch from the swelling, with the help of the actin-binding protein UNC-115/abLIM. Extension of existing dendrites also proceeds via swelling formation at the dendrite tip followed by UNC-34-mediated outgrowth. Following dendrite initiation and extension, the stabilization of branches by guidance receptors further recruits WRC, resulting in an iterative process to build a complex dendritic arbor.
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Project description:The lamellipodium of migrating animal cells protrudes by directed polymerization of a branched actin network. The underlying mechanisms of filament growth, branching, and capping can be studied in in vitro assays. However, conflicting results have been reported for the force-velocity relation of such actin networks, namely both convex and concave shapes as well as history dependencies. Here we model branching as a reaction that is independent of the number of existing filaments, in contrast to capping, which is assumed to be proportional to the number of existing filaments. Using both stochastic network simulations and deterministic rate equations, we show that such a description naturally leads to the stability of two qualitatively different stationary states of the system, namely a +/- 35 degrees and a +70/0/-70 degrees orientation pattern. Changes in network growth velocity induce a transition between these two patterns. For sufficiently different protrusion efficiency of the two network architectures, this leads to hysteresis in the growth velocity of actin networks under force. Dependent on the history of the system, convex and concave regimes are obtained for the force-velocity relation. Thus a simple generic model can explain the experimentally observed anomalies, with far reaching consequences for cell migration.
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