The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the inner nuclear membrane and represents an important determinant of interphase nuclear architecture. Its major components are the A- and B-type lamins. Whereas B-type lamins are found in all mammalian cells, A-type lamin expression is developmentally regulated. In the mouse, A-type lamins do not appear until midway through embryonic development, suggesting that these proteins may be involved in the regulation of terminal differentiation. Here we show that mice lacking A-type lamins develop to term with no overt abnormalities. However, their postnatal growth is severely retarded and is characterized by the appearance of muscular dystrophy. This phenotype is associated with ultrastructural perturbations to the nuclear envelope. These include the mislocalization of emerin, an inner nuclear membrane protein, defects in which are implicated in Emery-Dreifuss muscular dystrophy (EDMD), one of the three major X-linked dystrophies. Mice lacking the A-type lamins exhibit tissue-specific alterations to their nuclear envelope integrity and emerin distribution. In skeletal and cardiac muscles, this is manifest as a dystrophic condition related to EDMD.

A critical point during mammalian pregnancy is the implantation of the blastocyst when the embryo attaches to the wall of the uterus. The autonomously developing preimplantation embryo then becomes dependent on the maternal environment for its continued development. Little is known about the regulation of implantation, except that a complex interaction between peptide and steroid hormones synchronizes the preparation of the uterus for implantation with the development of the embryo. Whether the implantation event is under maternal or embryonic control is also unclear (reviewed in refs 1, 2). We have previously shown that a cytokine, leukaemia inhibitory factor (LIF), is expressed in the uterine endometrial glands specifically on the fourth day of pregnancy. This burst of expression is under maternal control and always precedes implantation of the blastocyst. Here we report that transient expression of LIF in mice is essential for implantation. Females lacking a functional LIF gene are fertile, but their blastocysts fail to implant and do not develop. The blastocysts, however, are viable and, when transferred to wild-type pseudopregnant recipients, they can implant and develop to term.

Embryonic stem (ES) cells, the totipotent outgrowths of blastocysts, can be cultured and manipulated in vitro and then returned to the embryonic environment where they develop normally and can contribute to all cell lineages. Maintenance of the stem-cell phenotype in vitro requires the presence of a feeder layer of fibroblasts or of a soluble factor, differentiation inhibitory activity (DIA) produced by a number of sources; in the absence of DIA the ES cells differentiate into a wide variety of cell types. We recently noted several similarities between partially purified DIA and a haemopoietic regulator, myeloid leukaemia inhibitory factor (LIF), a molecule which induces differentiation in M1 myeloid leukaemic cells and which we have recently purified, cloned and characterized. We demonstrate here that purified, recombinant LIF can substitute for DIA in the maintenance of totipotent ES cell lines that retain the potential to form chimaeric mice.

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Eukaryotic cells have a layer of heterochromatin at the nuclear periphery. To investigate mechanisms regulating chromatin distribution, we analyzed heterochromatin organization in different tissues and species, including mice with mutations in the lamin B receptor (Lbr) and lamin A (Lmna) genes that encode nuclear envelope (NE) proteins. We identified LBR- and lamin-A/C-dependent mechanisms tethering heterochromatin to the NE. The two tethers are sequentially used during cellular differentiation and development: first the LBR- and then the lamin-A/C-dependent tether. The absence of both LBR and lamin A/C leads to loss of peripheral heterochromatin and an inverted architecture with heterochromatin localizing to the nuclear interior. Myoblast transcriptome analyses indicated that selective disruption of the LBR- or lamin-A-dependent heterochromatin tethers have opposite effects on muscle gene expression, either increasing or decreasing, respectively. These results show how changes in NE composition contribute to regulating heterochromatin positioning, gene expression, and cellular differentiation during development.

strate that transcriptional activation in response to mechanical stimuli is attenuated in Lmna -/-cells, impairing viability of mechanically strained cells. These data suggest that the structural and gene regulation hypotheses of the laminopathies are in fact closely related, and different mutations may cause specific phenotypes by differentially affecting these processes.

Mutations in the nuclear envelope proteins lamins A and C cause a broad variety of human diseases, including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. Cells lacking lamins A and C have reduced nuclear stiffness and increased nuclear fragility, leading to increased cell death under mechanical strain and suggesting a potential mechanism for disease. Here, we investigated the contribution of major lamin subtypes (lamins A, C, and B1) to nuclear mechanics by analyzing nuclear shape, nuclear dynamics over time, nuclear deformations under strain, and cell viability under prolonged mechanical stimulation in cells lacking both lamins A and C, cells lacking only lamin A (i.e."lamin C-only" cells), cells lacking wild-type lamin B1, and wild-type cells. Lamin A/C-deficient cells exhibited increased numbers of misshapen nuclei and had severely reduced nuclear stiffness and decreased cell viability under strain. Lamin C-only cells had slightly abnormal nuclear shape and mildly reduced nuclear stiffness but no decrease in cell viability under strain. Interestingly, lamin B1-deficient cells exhibited normal nuclear mechanics despite having a significantly increased frequency of nuclear blebs. Our study indicates that lamins A and C are important contributors to the mechanical stiffness of nuclei, whereas lamin B1 contributes to nuclear integrity but not stiffness.Lamins are type V intermediate filament proteins that form the nuclear lamina, a filamentous network underlying the inner nuclear membrane of eukaryotic cells. Lamins form stable structures in the nuclear lamina and the nucleoplasm, determine nuclear shape and size, resist nuclear deformation, and position nuclear pore complexes (reviewed in Refs. 1-3). In addition, lamins recruit and anchor, either directly or indirectly, several nuclear envelope proteins (e.g. nesprin-1␣, emerin, and the lamin B receptor) to the inner nuclear membrane (3).Mammalian cells express two types of lamins, the A and B types. Both share a common structural organization: a globular N-terminal domain separated from a larger C-terminal globular domain by a central helical rod domain that allows lamins to form parallel coiled-coil dimers, which in turn assemble into stable strings and higher order networks. Lamins A and C, the major A-type lamins, are alternatively spliced isoforms of a single gene, LMNA. The expression of A-type lamins is developmentally regulated, beginning midway through embryonic development (4). A-type lamins are expressed in most but not all differentiated cells (5). B-type lamins (lamins B1 and B2) are encoded by separate genes, LMNB1 and LMNB2, respectively (6). Unlike A-type lamins, B-type lamins are expressed in all cells and throughout development (7,8), although it is not clear if they are always coexpressed at equivalent levels in the same cell.

Defects in nuclear morphology often correlate with the onset of disease, including cancer, progeria, cardiomyopathy, and muscular dystrophy. However, the mechanism by which a cell controls its nuclear shape is unknown. Here, we use adhesive micropatterned surfaces to control the overall shape of fibroblasts and find that the shape of the nucleus is tightly regulated by the underlying cell adhesion geometry. We found that this regulation occurs through a dome-like actin cap that covers the top of the nucleus. This cap is composed of contractile actin filament bundles containing phosphorylated myosin, which form a highly organized, dynamic, and oriented structure in a wide variety of cells. The perinuclear actin cap is specifically disorganized or eliminated by inhibition of actomyosin contractility and rupture of the LINC complexes, which connect the nucleus to the actin cap. The organization of this actin cap and its nuclear shape-determining function are disrupted in cells from mouse models of accelerated aging (progeria) and muscular dystrophy with distorted nuclei caused by alterations of A-type lamins. These results highlight the interplay between cell shape, nuclear shape, and cell adhesion mediated by the perinuclear actin cap.LINC complexes ͉ nucleus I n 1921, Champy and Carleton suggested an apparent correlation between the shape of various types of animal cells and the shape of their respective nuclei (1). Moreover, defects in nuclear shape are routinely used in the lab and in clinical settings as markers of disease and differentiation in human cells and tissues (2). However, remarkably little is known about the factors that determine nuclear morphology in living cells. In particular, the molecular mechanisms that govern the shape of the interphase nucleus are unknown. Here we show that an actin filament structure that forms a cap or dome located above the apical surface of the nucleus tightly controls nuclear shape and identify key associated cytoskeletal regulators of its organization and nuclear shape-determining function. The organization and function of the perinuclear actin cap are deregulated in diseased cells with distorted nuclei. Results and DiscussionTo test the hypothesis of a correlation between the shape of the nucleus and the overall cell shape, mouse embryonic fibroblasts were dispersed on fibronectin (FN)-coated glass substrates. Using morphometric analysis, we found that nuclear shape and cellular shape correlated ( Fig. 1 A and B). Shape factor, defined as 4 A/P 2 (where A and P are the nuclear area and perimeter), approaches 1 for a rounded nucleus and approaches 0 for an elongated nucleus. Elongated cells typically showed an elongated nucleus of low shape factor; rounded cells showed a rounded nucleus of high shape factor (Fig. 1 A). To control cell shape and, therefore, be able to quantify nuclear shape as a function of cell shape, we developed adhesive FN-coated micropatterned stripes of width ranging between 10 and 50 m, which alternated with stripes covered with nonadhesive poly...