Cytoskeletal prestress regulates nuclear shape and stiffness in cardiac myocytes

Mechanical stresses on the myocyte nucleus have been associated with several diseases and potentially transduce mechanical stimuli into cellular responses. Although a number of physical links between the nuclear envelope and cytoplasmic filaments have been identified, previous studies have focused on the mechanical properties of individual components of the nucleus, such as the nuclear envelope and lamin network. The mechanical interaction between the cytoskeleton and chromatin on nuclear deformability remains elusive. Here, we investigated how cytoskeletal and chromatin structures influence nuclear mechanics in cardiac myocytes. Rapid decondensation of chromatin and rupture of the nuclear membrane caused a sudden expansion of DNA, a consequence of prestress exerted on the nucleus. To characterize the prestress exerted on the nucleus, we measured the shape and the stiffness of isolated nuclei and nuclei in living myocytes during disruption of cytoskeletal, myofibrillar, and chromatin structure. We found that the nucleus in myocytes is subject to both tensional and compressional prestress and its deformability is determined by a balance of those opposing forces. By developing a computational model of the prestressed nucleus, we showed that cytoskeletal and chromatin prestresses create vulnerability in the nuclear envelope. Our studies suggest the cytoskeletal–nuclear–chromatin interconnectivity may play an important role in mechanics of myocyte contraction and in the development of laminopathies by lamin mutations.     

Preblastoderm Nuclear Division in the Embryo of the Large Milkweed Bug, Oncopeltus fasciatus (Dallas)

The preblastoderm nuclei of the large milkweed bug, Oncopeltus fasciatus (Dallas), divide in the absence of centrioles and a spindle fibre apparatus. The preblastoderm nuclei occur in cytoplasmic islands that are not bound by cytoplasmic membranes. The chromatin condenses in association with the nuclear envelope and intranuclear lamellae into two compact masses. The chromatin of the sister nuclei disperse as the nuclei increase in volume and move away from each other in a common cytoplasm. A model is proposed for the orderly separation of the chromatin of the indicated eukaryotic system that is patterned after the site and site territory concept for the control of prokaryotic DNA replication by the membrane. In the case of the milkweed bug preblastoderm nuclei, the nuclear envelope would play a distributive role by providing only one site and site territory in each half of the nucleus for the attachment of a homologue. The presence of a site on the membrane for the attachment of a given chromosome would prevent the formation of a new site for the chromosome homologue within a certain distance.     

Remodeling the sperm nucleus into a male pronucleus at fertilization

After fertilization, the dormant sperm nucleus undergoes morphological and biochemical transformations leading to the development of a functional nucleus, the male pronucleus. We have investigated the formation of the male pronucleus in a cell-free system consisting of permeabilized sea urchin sperm nuclei incubated in fertilized sea urchin egg extract containing membrane vesicles. The first sperm nuclear alteration in vitro is the disassembly of the sperm nuclear lamina as a result of lamin phosphorylation mediated by egg protein kinase C. The conical sperm nucleus decondenses into a spherical pronucleus in an ATP-dependent manner. The new nuclear envelope (NE) forms by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of vesicles to each other. Three cytoplasmic membrane vesicle fractions with distinct biochemical, chromatin-binding and fusion properties, are required for pronuclear envelope assembly. Binding of each fraction to chromatin requires two detergent-resistant lipophilic structures at each pole of the sperm nucleus, which are incorporated into the NE by membrane fusion. Targeting of the bulk of NE vesicles to chromatin is mediated by a lamin B receptor (LBR)-like integral membrane protein. The last step of male pronuclear formation involves nuclear swelling. Nuclear swelling is associated with import of soluble lamin B into the nucleus and growth of the nuclear envelope by fusion of additional vesicles. In the nucleus, lamin B associates with LBR, which apparently tethers the NE to the lamina. Thus male pronuclear envelope assembly in vitro involves a highly ordered series of events. These events are similar to those characterizing the remodeling of somatic and embryonic nuclei transplanted into oocytes. The relationship between sperm nuclear remodeling at fertilization and nuclear remodeling after nuclear transplantation is discussed.     

Interaction of the activated cytoplasmic glucocorticoid hormone receptor complex with the nuclear envelope

Highly purified activated cytoplasmic glucocorticoid hormone receptor binds with high affinity to sites in the nuclear envelope. Nuclear envelope fragments can be isolated from purified chromatin. They bind activated cytoplasmic glucocorticoid receptor with the same equilibrium constant as nuclear envelopes. The presence of envelope components in chromatin is confirmed by the virtual identity of the gel electrophoretic glycoprotein pattern of nuclear envelope, chromatin nonhistones, and nuclear envelope fragments from chromatin.     

A network of nuclear envelope membrane proteins linking centromeres to microtubules

In the fission yeast S. pombe, nuclei are actively positioned at the cell center by microtubules. Here, we show that cytoplasmic microtubules are mechanically coupled to the nuclear heterochromatin through proteins embedded in the nuclear envelope. This includes an integral outer nuclear membrane protein of the KASH family (Kms2) and two integral inner nuclear membrane proteins, the SUN-domain protein Sad1 and the previously uncharacterized protein Ima1. Ima1 specifically binds to heterochromatic regions and promotes the tethering of centromeric DNA to the SUN-KASH complex. In the absence of Ima1, or in cells harboring mutations in the centromeric Ndc80 complex, inefficient coupling of centromeric heterochromatin to Sad1 leads to striking defects in the ability of the nucleus to tolerate microtubule-dependent forces, leading to changes in nuclear shape, loss of spindle pole body components from the nuclear envelope, and partial dissociation of SUN-KASH complexes. This work highlights a framework for communication between cytoplasmic microtubules and chromatin.     

Dynamics of chromatin decondensation reveals the structural integrity of a mechanically prestressed nucleus

Genome organization within the cell nucleus is a result of chromatin condensation achieved by histone tail-tail interactions and other nuclear proteins that counter the outward entropic pressure of the polymeric DNA. We probed the entropic swelling of chromatin driven by enzymatic disruption of these interactions in isolated mammalian cell nuclei. The large-scale decondensation of chromatin and the eventual rupture of the nuclear membrane and lamin network due to this entropic pressure were observed by fluorescence imaging. This swelling was accompanied by nuclear softening, an effect that we quantified by measuring the fluctuations of an optically trapped bead adhered onto the nucleus. We also measured the pressure at which the nuclear scaffold ruptured using an atomic force microscope cantilever. A simple theory based on a balance of forces in a swelling porous gel quantitatively explains the diffusive dynamics of swelling. Our experiments on decondensation of chromatin in nuclei suggest that its compaction is a critical parameter in controlling nuclear stability.     

Nucleus size and its effect on nucleosome stability in living cells

DNA architectural proteins play a major role in organization of chromosomal DNA in living cells by packaging it into chromatin, whose spatial conformation is determined by an intricate interplay between the DNA-binding properties of architectural proteins and physical constraints applied to the DNA by a tight nuclear space. Yet, the exact effects of the nucleus size on DNA-protein interactions and chromatin structure currently remain obscure. Furthermore, there is even no clear understanding of molecular mechanisms responsible for the nucleus size regulation in living cells. To find answers to these questions, we developed a general theoretical framework based on a combination of polymer field theory and transfer-matrix calculations, which showed that the nucleus size is mainly determined by the difference between the surface tensions of the nuclear envelope and the endoplasmic reticulum membrane as well as the osmotic pressure exerted by cytosolic macromolecules on the nucleus. In addition, the model demonstrated that the cell nucleus functions as a piezoelectric element, changing its electrostatic potential in a size-dependent manner. This effect has been found to have a profound impact on stability of nucleosomes, revealing a previously unknown link between the nucleus size and chromatin structure. Overall, our study provides new insights into the molecular mechanisms responsible for regulation of the nucleus size, as well as the potential role of nuclear organization in shaping the cell response to environmental cues.     

CRUCIFORM NUCLEAR DIVISION IN WORONINA PYTHII (PLASMODIOPHOROMYCETES)

Somatic nuclear divisions in sporangiogenous plasmodia of Woronina pythii Goldie-Smith were studied with transmission electron microscopy. During metaphase, each nucleus formed a cruciform configuration as chromatin became aligned at the equatorial plate perpendicular to the persistent nucleolus. Except for polar fenestrations, the original nuclear envelope remained intact throughout the mitotic division. Intranuclear membranous vesicles appeared to bleb off the inner membrane of the original nuclear envelope, adhered to the surfaces of the separating chromatin, and eventually formed new daughter nuclear envelope within the original nuclear envelope. During the first 24 hr of vegetative plasmodial growth, each telophase nucleus exhibited an obvious constriction of the original nuclear envelope in the interzonal region. Similar constrictions were not evident in telophase nuclei found in 24–36-hr-old plasmodia. This variation in the ultrastructural morphology of cruciform division appears to be related to the age and size of each sporangiogenous plasmodium, and is the first to be documented within this group of fungal pathogens.     

Nuclear cytoplasmic shuttling by thyroid hormone receptors. multiple protein interactions are required for nuclear retention

In this report, we have studied the intracellular dynamics and distribution of the thyroid hormone receptor-beta (TRbeta) in living cells, utilizing fusions to the green fluorescent protein. Wild-type TRbeta was mostly nuclear in both the absence and presence of triiodothyronine; however, triiodothyronine induced a nuclear reorganization of TRbeta. By mutating defined regions of TRbeta, we found that both nuclear corepressor and retinoid X receptor are involved in maintaining the unliganded receptor within the nucleus. A TRbeta mutant defective in DNA binding had only a slightly altered nuclear/cytoplasmic distribution compared with wild-type TRbeta; thus, site-specific DNA binding is not essential for maintaining TRbeta within the nucleus. Both ATP depletion studies and heterokaryon analysis demonstrated that TRbeta rapidly shuttles between the nuclear and the cytoplasmic compartments. Cotransfection of nuclear corepressor and retinoid X receptor markedly decreased the shuttling by maintaining unliganded TRbeta within the nucleus. In summary, our findings demonstrate that TRbeta rapidly shuttles between the nucleus and the cytoplasm and that protein-protein interactions of TRbeta with various cofactors, rather than specific DNA interactions, play the predominant role in determining the intracellular distribution of the receptor.     

MFP1, a novel plant filament-like protein with affinity for matrix attachment region DNA.

The interaction of chromatin with the nuclear matrix via matrix attachment regions (MARs) on the DNA is considered to be of fundamental importance for higher order chromatin organization and regulation of gene expression. Here, we report a novel nuclear matrix-localized MAR DNA binding protein, designated MAR binding filament-like protein 1 (MFP1), from tomato. In contrast to the few animal MAR DNA binding proteins thus far identified, MFP1 contains a predicted N-terminal transmembrane domain and a long filament-like alpha-helical domain that is similar to diverse nuclear and cytoplasmic filament proteins from animals and yeast. DNA binding assays established that MFP1 can discriminate between animal and plant MAR DNAs and non-MAR DNA fragments of similar size and AT content. Deletion mutants of MFP1 revealed a novel, discrete DNA binding domain near the C terminus of the protein. MFP1 is an in vitro substrate for casein kinase II, a nuclear matrix-associated protein kinase. Its structure, MAR DNA binding activity, and nuclear matrix localization suggest that MFP1 is likely to participate in nuclear architecture by connecting chromatin with the nuclear matrix and potentially with the nuclear envelope.     

Generation of micronuclei during interphase by coupling between cytoplasmic membrane blebbing and nuclear budding

Micronucleation, mediated by interphase nuclear budding, has been repeatedly suggested, but the process is still enigmatic. In the present study, we confirmed the previous observation that there are lamin B1-negative micronuclei in addition to the positive ones. A large cytoplasmic bleb was found to frequently entrap lamin B1-negative micronuclei, which were connected to the nucleus by a thin chromatin stalk. At the bottom of the stalk, the nuclear lamin B1 structure appeared broken. Chromatin extrusion through lamina breaks has been referred to as herniation or a blister of the nucleus, and has been observed after the expression of viral proteins. A cell line in which extrachromosomal double minutes and lamin B1 protein were simultaneously visualized in different colors in live cells was established. By using these cells, time-lapse microscopy revealed that cytoplasmic membrane blebbing occurred simultaneously with the extrusion of nuclear content, which generated lamin B1-negative micronuclei during interphase. Furthermore, activation of cytoplasmic membrane blebbing by the addition of fresh serum or camptothecin induced nuclear budding within 1 to 10 minutes, which suggested that blebbing might be the cause of the budding. After the induction of blebbing, the frequency of lamin-negative micronuclei increased. The budding was most frequent during S phase and more efficiently entrapped small extrachromosomal chromatin than the large chromosome arm. Based on these results, we suggest a novel mechanism in which cytoplasmic membrane dynamics pulls the chromatin out of the nucleus through the lamina break. Evidence for such a mechanism was obtained in certain cancer cell lines including human COLO 320 and HeLa. The mechanism could significantly perturb the genome and influence cancer cell phenotypes.     

A cytological study of micronuclear elongation during conjugation in Tetrahymena

Micronuclear elongation is the first major event in a series of nuclear changes occurring during the sexual stage of the life cycle of Tetrahymena. Beginning at about one hour after cells of complementary mating types have conjugated, the micronucleus leaves its recess in the macronucleus and swells slightly. This is accompanied by a reorganization of its chromatin from a reticular to a solid body. In the next stage the micronucleus assumes an egg shape, a development concomitant with the appearance of microtubules. While the chromatin spins out from the dense body, and microtubules increase in number, the nucleus assumes a spindle shape. During the elongation, which increases the length of the nucleus some fifty fold, microtubules are prominent in clusters just internal to the nuclear membrane, and parallel to the longitudinal axis of the nucleus. When elongation is completed the nucleus is curved around the macronucleus. Internally, partially condensed strands of chromatin are located off-center, towards the macronuclear side, and the density of the microtubules is diminished. At all the stages, DNA is located throughout the nucleus; neither discrete chromosomes nor synaptonemal complexes are seen. Occasionally cytoplasmic membrane systems are seen fused to the nuclear envelope which retains the typical appearance of a double membrane with pores.     

Breakdown of the sperm nuclear envelope is a prerequisite for male pronucleus formation: direct evidence from the gynogenetic crucian carp Carassius auratus langsdorfii

The gynogenetic fish, Carassius auratus langsdorfii (the ginbuna, a crucian carp), provides an interesting model for the study of the mechanisms controlling male pronucleus formation. When the sperm nucleus of a different subspecies (C. a. cuvieri) is incorporated into the gynogenetic egg, the nuclear envelope of the spermatozoon is not broken down, and the pronucleus fails to develop, although dispersion of the sperm chromatin occurs to some extent within the space limited by the nuclear envelope. When spermatozoa without plasma membranes and nuclear envelopes were microinjected into mature activated eggs, the sperm nuclei underwent chromatin dispersion, nuclear envelope formation, DNA synthesis, and transformation into male pronuclei. These results indicate that the failure of the male pronucleus to form in ginbuna is primarily due to the failure of sperm nuclear envelope breakdown. We conclude that sperm nuclear envelope breakdown is an indispensable step for the development of the male pronucleus.     

细胞器向核凹入与核液泡的形成和降解

利用电镜观察紫竹梅雄蕊行超薄切片,发现细胞器,如线粒体有向核凹入现象,其线粒体仍处在与细胞质相勾通的核沟槽里,但核沟槽能扩大核被膜表面积,以利用于胞核与胞质的物质交换和信息传递,当核沟槽颈部变窄时,核膜融合,核沟槽脱离核被膜,形成核液泡;随后,核液泡的内外两层膜相继降解,核液泡被消化溶入核基质中。     

An unconventional diacylglycerol kinase that regulates phospholipid synthesis and nuclear membrane growth

Changes in nuclear size and shape during the cell cycle or during development require coordinated nuclear membrane remodeling, but the underlying molecular events are largely unknown. We have shown previously that the activity of the conserved phosphatidate phosphatase Pah1p/Smp2p regulates nuclear structure in yeast by controlling phospholipid synthesis and membrane biogenesis at the nuclear envelope. Two screens for novel regulators of phosphatidate led to the identification of DGK1. We show that Dgk1p is a unique diacylglycerol kinase that uses CTP, instead of ATP, to generate phosphatidate. DGK1 counteracts the activity of PAH1 at the nuclear envelope by controlling phosphatidate levels. Overexpression of DGK1 causes the appearance of phosphatidate-enriched membranes around the nucleus and leads to its expansion, without proliferating the cortical endoplasmic reticulum membrane. Mutations that decrease phosphatidate levels decrease nuclear membrane growth in pah1Delta cells. We propose that phosphatidate metabolism is a critical factor determining nuclear structure by regulating nuclear membrane biogenesis.     

Formation of the sea urchin male pronucleus in vitro: Membrane-independent chromatin decondensation and nuclear envelope-dependent nuclear swelling

We demonstrate that complete sea urchin male pronuclear development in vitro is a two-step process involving membrane-independent chromatin decondensation and nuclear envelope-dependent pronuclear swelling. In the absence of cytoplasmic membrane vesicles (MVs), permeabilized sperm chromatin decondenses into a spherical nucleus of ≈4 μm in diameter. Pronuclear swelling to ≈7 μm requires an intact nuclear envelope, and the degree of swelling is limited by the amount of MVs assembled on the chromatin. Furthermore, after a nuclear envelope is formed, swelling can occur in the absence of additional cytoplasmic MVs. Nuclear swelling also requires ATP hydrolysis, Ca²⁺ and cytosolic factors, some of which are sensitive to heat and to the sulfhy-dryl alkylating agent, N-ethylmaleimide. The requirement for a nuclear envelope and the rate of pronuclear swelling are consistent with previous in vivo observations. © 1995 wiley-Liss, Inc.     

Gold-nanoparticle-assisted laser perturbation of chromatin assembly reveals unusual aspects of nuclear architecture within living cells

Chromatin organization within the nucleus is a vital regulator of genome function, yet its mechanical coupling to the nuclear architecture has remained elusive. To directly investigate this coupling, we locally modulated chromatin structure in living cells using nanoparticle-based laser perturbation. Unusual differences in the response of the cell nucleus were observed depending on the nuclear region that was perturbed--the heterochromatin, the euchromatin, and the nuclear envelope. This response varied under different conditions of cellular perturbations such as ATP depletion, apoptosis, and inhibition of histone deacetylases. Our studies implicate heterochromatin organization in imparting mechanical stability to the cell nucleus and suggest that nuclear size and shape are the result of interplay between nuclear and cytoplasmic anchors.