Influence of Lamin A on the Mechanical Properties of Amphibian Oocyte Nuclei Measured by Atomic Force Microscopy

The nuclear lamina is part of the nuclear envelope (NE). Lamin filaments provide the nucleus with mechanical stability and are involved in many nuclear activities. The functional importance of these proteins is highlighted by mutations in lamin genes, which cause a variety of human diseases (laminopathies). Here we describe a method that allows one to quantify the contribution of lamin A protein to the mechanical properties of the NE. Lamin A is ectopically expressed in Xenopus oocytes, where it is incorporated into the NE of the oocyte nucleus, giving rise to a prominent lamina layer at the inner nuclear membrane. Nuclei are then isolated and probed by atomic force microscopy. From the resulting force curves, stiffness values are calculated and compared with those of control nuclei. Expression of lamin A significantly increases the stiffness of oocyte nuclei in a concentration-dependent manner. Since chromatin adds negligibly to nuclear mechanics in these giant nuclei, this method allows one to measure the contribution of individual NE components to nuclear mechanics.     

Mechanical properties of the cell nucleus and the effect of emerin deficiency

Nuclear structure and mechanics are gaining recognition as important factors that affect gene expression, development, and differentiation in normal function and disease, yet the physical mechanisms that govern nuclear mechanical stability remain unclear. Here we examined the physical properties of the cell nucleus by imaging fluorescently labeled components of the inner nucleus (chromatin and nucleoli) and the nuclear envelope (lamins and membranes) in nuclei deformed by micropipette aspiration (confocal imaged microdeformation). We investigated nuclei, both isolated and in intact, living cells, and found that nuclear volume significantly decreased by 60-70% during aspiration. While nuclear membranes exhibited blebbing and fluid characteristics during aspiration, the nuclear lamina exhibited behavior of a solid-elastic shell. Under large deformations of GFP-lamin A-labeled nuclei, we observed a decay of fluorescence intensity into the tip of the deformed tongue that we interpreted in terms of nonlinear, two-dimensional elasticity theory. Here we applied this method to study nuclear envelope stability in disease and found that mouse embryo fibroblasts lacking the inner nuclear membrane protein, emerin, had a significantly decreased ratio of the area expansion to shear moduli (K/mu) compared to wild-type cells (2.1 +/- 0.2 versus 5.1 +/- 1.3). These data suggest that altered nuclear envelope elasticity caused by loss of emerin could contribute to increased nuclear fragility in Emery-Dreifuss muscular dystrophy patients with mutations in the emerin gene. Based on our experimental results and theoretical considerations, we present a model describing how the nucleus is stabilized in the pipette. Such a model is essential for interpreting the results of any micropipette study of the nucleus and porous materials in general.     

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.     

Power-law rheology of isolated nuclei with deformation mapping of nuclear substructures

Force-induced changes in genome expression as well as remodeling of nuclear architecture in development and disease motivate a deeper understanding of nuclear mechanics. Chromatin and green fluorescent protein-lamin B dynamics were visualized in a micropipette aspiration of isolated nuclei, and both were shown to contribute to viscoelastic properties of the somatic cell nucleus. Reversible swelling by almost 200% in volume, with changes in salt, demonstrates the resilience and large dilational capacity of the nuclear envelope, nucleoli, and chromatin. Swelling also proves an effective way to separate the mechanical contributions of nuclear elements. In unswollen nuclei, chromatin is a primary force-bearing element, whereas swollen nuclei are an order of magnitude softer, with the lamina sustaining much of the load. In both cases, nuclear deformability increases with time, scaling as a power law-thus lacking any characteristic timescale-when nuclei are either aspirated or indented by atomic force microscopy. The nucleus is stiff and resists distortion at short times, but it softens and deforms more readily at longer times. Such results indicate an essentially infinite spectrum of timescales for structural reorganization, with implications for regulating genome expression kinetics.     

The integration of tissue structure and nuclear function.

Living cells can filter the same set of biochemical signals to produce different functional outcomes depending on the deformation of the cell. It has been suggested that the cell may be "hard-wired" such that external forces can mediate internal nuclear changes through the modification of established, balanced, internal cytoskeletal tensions. This review will discuss the potential of subnuclear structures and nuclear chromatin to participate in or respond to transduction of mechanical signals originating outside the nucleus. The mechanical interactions of intranuclear structure with the nuclear lamina will be examined. The nuclear lamina, in turn, provides a structural link between the nucleus and the cytoplasmic and cortical cytoskeleton. These mechanical couplings may provide a basis for regulating gene expression through changes in cell shape.     

Nuclear lamina at the crossroads of the cytoplasm and nucleus

The nuclear lamina is a protein meshwork that lines the nuclear envelope in metazoan cells. It is composed largely of a polymeric assembly of lamins, which comprise a distinct sequence homology class of the intermediate filament protein family. On the basis of its structural properties, the lamina originally was proposed to provide scaffolding for the nuclear envelope and to promote anchoring of chromatin and nuclear pore complexes at the nuclear surface. This viewpoint has expanded greatly during the past 25 years, with a host of surprising new insights on lamina structure, molecular composition and functional attributes. It has been established that the self-assembly properties of lamins are very similar to those of cytoplasmic intermediate filament proteins, and that the lamin polymer is physically associated with components of the cytoplasmic cytoskeleton and with a multitude of chromatin and inner nuclear membrane proteins. Cumulative evidence points to an important role for the lamina in regulating signaling and gene activity, and in mechanically coupling the cytoplasmic cytoskeleton to the nucleus. The significance of the lamina has been vaulted to the forefront by the discovery that mutations in lamins and lamina-associated polypeptides lead to an array of human diseases. A key future challenge is to understand how the lamina integrates pathways for mechanics and signaling at the molecular level. Understanding the structure of the lamina from the atomic to supramolecular levels will be essential for achieving this goal.     

The nuclear lamins and the nuclear envelope

The cell nucleus is separated from the rest of the cell by the nuclear envelope. The nuclear envelope, nuclear envelope proteins and nuclear lamina organise the structure of the entire nucleus and the chromatin via a myriad of interactions. These interactions are dynamic, change with the change (progress) of the cell cycle, with cell differentiation and with changes in cell physiology.     

Chromatin organization in relation to the nuclear periphery

In the limited space of the nucleus, chromatin is organized in a dynamic and non-random manner. Three ways of chromatin organization are compaction, formation of loops and localization within the nucleus. To study chromatin localization it is most convenient to use the nuclear envelope as a fixed viewpoint. Peripheral chromatin has both been described as silent chromatin, interacting with the nuclear lamina, and active chromatin, interacting with nuclear pore proteins. Current data indicate that the nuclear envelope is a reader as well as a writer of chromatin state, and that its influence is not limited to the nuclear periphery.     

应用HeLa细胞染色体(簇)和蛙卵提取物进行细胞核体外重建试验

将HeLa细胞中期染色体(簇)、非洲爪蟾卵提取物和ATP再生体系混合温育,能够促使细胞核自发重建。在此非细胞体系中重建的细胞核处于一般细胞核大小范围,具有典型的双层核膜,核孔复合体、染色质、核纤层、核骨架等结构,核重建具有一个明显的过程;发现环形片层通过与核膜融合方式参与核膜和核孔复合体组装。     

Identification and functional evaluation of cellular and viral factors involved in the alteration of nuclear architecture during herpes simplex virus 1 infection

Herpes simplex virus 1 (HSV-1) replicates in the nucleus of host cells and radically alters nuclear architecture as part of its replication process. Replication compartments (RCs) form, and host chromatin is marginalized. Chromatin is later dispersed, and RCs spread past it to reach the nuclear edge. Using a lamin A-green fluorescent protein fusion, we provide direct evidence that the nuclear lamina is disrupted during HSV-1 infection and that the UL31 and UL34 proteins are required for this. We show nuclear expansion from 8 h to 24 h postinfection and place chromatin rearrangement and disruption of the lamina in the context of this global change in nuclear architecture. We show HSV-1-induced disruption of the localization of Cdc14B, a cellular protein and component of a putative nucleoskeleton. We also show that UL31 and UL34 are required for nuclear expansion. Studies with inhibitors of globular actin (G-actin) indicate that G-actin plays an essential role in nuclear expansion and chromatin dispersal but not in lamina alterations induced by HSV-1 infection. From analyses of HSV infections under various conditions, we conclude that nuclear expansion and chromatin dispersal are dispensable for optimal replication, while lamina rearrangement is associated with efficient replication.     

Involvement of nuclear lamins in postmitotic reorganization of chromatin as demonstrated by microinjection of lamin antibodies

The nuclear lamins are major components of a proteinaceous polymer that is located at the interface of the nuclear membrane and chromatin; these lamins are solubilized and dispersed throughout the cytoplasm during mitosis. It has been postulated that these proteins, assembled into the lamina, provide an architectural framework for the organization of the cell nucleus. To test this hypothesis we microinjected lamin antibodies into cultured PtK2 cells during mitosis, thereby decreasing the soluble pool of lamins. The antibody injected was identified, together with the lamins, in cytoplasmic aggregates by immunoelectron microscopy. We show that microinjected cells are not able to form normal daughter nuclei, in contrast to cells injected with other immunoglobulins. Although cells injected with lamin antibodies are able to complete cytokinesis, the chromatin of their daughter nuclei remains arrested in a telophase-like configuration, and the telophase-like chromatin remains inactive as judged from its condensed state and by the absence of nucleoli. These results indicate that lamins and the nuclear lamina structure are involved in the functional organization of the interphase chromatin.     

高等动物细胞核膜和核纤层结构、功能及动态变化调控机制

细胞核是真核细胞中最大的细胞器．高等动物细胞核主要由双层核膜、核孔复合体、核纤层、染色质和核仁等组成．在细胞有丝分裂期,细胞核呈现去装配和再装配等动态变化．在细胞分裂间期,核膜、核孔复合体和核纤层构成细胞核的外周结构,为遗传物质在染色质和核仁中的代谢提供了一个相对稳定的环境,同时调控细胞核内外的物质转运,在细胞增殖、分化、个体发育和细胞衰老等许多方面发挥着重要作用．本文主要对高等动物细胞核膜和核纤层结构、功能及动态变化调控机制等方面的研究进展进行简要综述．     

Cancer cell migration in 3D tissue: Negotiating space by proteolysis and nuclear deformability

Efficient tumor cell invasion into the surrounding desmoplastic stroma is a hallmark of cancer progression and involves the navigation through available small tissue spaces existent within the dense stromal network. Such navigation includes the reciprocal adaptation of the moving tumor cell, including the nucleus as largest and stiffest organelle, to pre-existent or de-novo generated extracellular matrix (ECM) gaps, pores and trails within stromal compartments. Within the context of migration, we briefly summarize physiological and tumor-related changes in ECM geometries as well as tissue proteolysis. We then focus on mechanisms that ensure the successful translocation of a nucleus through a confining pore by cytoskeleton-mediated coupling, as well as regulators of cell and nuclear deformability such as chromatin organization and nuclear lamina expression. In summary, understanding dynamic nuclear mechanics during migration in response to confined space will add to a better conceptual appreciation of cancer invasion and progression.