From lamins to lamina: a structural perspective

Lamin proteins are the major constituents of the nuclear lamina, a proteinaceous network that lines the inner nuclear membrane. Primarily, the nuclear lamina provides structural support for the nucleus and the nuclear envelope; however, lamins and their associated proteins are also involved in most of the nuclear processes, including DNA replication and repair, regulation of gene expression, and signaling. Mutations in human lamin A and associated proteins were found to cause a large number of diseases, termed ‘laminopathies.' These diseases include muscular dystrophies, lipodystrophies, neuropathies, and premature aging syndromes. Despite the growing number of studies on lamins and their associated proteins, the molecular organization of lamins in health and disease is still elusive. Likewise, there is no comprehensive view how mutations in lamins result in a plethora of diseases, selectively affecting different tissues. Here, we discuss some of the structural aspects of lamins and the nuclear lamina organization, in light of recent result

Mapping eGFP Oligomer Mobility in Living Cell Nuclei

Movement of particles in cell nuclei can be affected by viscosity, directed flows, active transport, or the presence of obstacles such as the chromatin network. Here we investigate whether the mobility of small fluorescent proteins is affected by the chromatin density. Diffusion of inert fluorescent proteins was studied in living cell nuclei using fluorescence correlation spectroscopy (FCS) with a two-color confocal scanning detection system. We first present experiments exposing FCS-specific artifacts encountered in live cell studies as well as strategies to prevent them, in particular those arising from the choice of the fluorophore used for calibration of the focal volume, as well as temperature and acquisition conditions used for fluorescence fluctuation measurements. After defining the best acquisition conditions, we show for various human cell lines that the mobility of GFP varies significantly within the cell nucleus, but does not correlate with chromatin density. The intranuclear diffusional mobility strongly depends on protein size: in a series of GFP-oligomers, used as free inert fluorescent tracers, the diffusion coefficient decreased from the monomer to the tetramer much more than expected for molecules free in aqueous solution. Still, the entire intranuclear chromatin network is freely accessible for small proteins up to the size of eGFP-tetramers, regardless of the chromatin density or cell line. Even the densest chromatin regions do not exclude free eGFP-monomers or multimers

Lamin B Receptor

Lamin B Receptor (LBR) is an integral membrane protein of the interphase nuclear envelope (NE). The N-terminal end resides in the nucleoplasm, binding to lamin B and heterochromatin, with the interactions disrupted during mitosis. The C-terminal end resides within the inner nuclear membrane, retreating with the ER away from condensing chromosomes during mitotic NE breakdown. Some of these properties are interpretable in terms of our current structural knowledge of LBR, but many of the structural features remain unknown. LBR apparently has an evolutionary history which brought together at least two ancient conserved structural domains (i.e. Tudor and sterol reductase). This convergence may have occurred with the emergence of the chordates and echinoderms. It is not clear what survival values have maintained LBR structure during evolution. But it seems likely that roles in post-mitotic nuclear reformation, interphase NE growth and compartmentalization of nuclear architecture might have provided some evolutionary advantage to preservation of the LBR gene

Myopathic lamin mutations impair nuclear stability in cells and tissue and disrupt nucleo-cytoskeletal coupling

Lamins are intermediate filament proteins that assemble into a meshwork underneath the inner nuclear membrane, the nuclear lamina. Mutations in the LMNA gene, encoding lamins A and C, cause a variety of diseases collectively called laminopathies. The disease mechanism for these diverse conditions is not well understood. Since lamins A and C are fundamental determinants of nuclear structure and stability, we tested whether defects in nuclear mechanics could contribute to the disease development, especially in laminopathies affecting mechanically stressed tissue such as muscle. Using skin fibroblasts from laminopathy patients and lamin A/C-deficient mouse embryonic fibroblasts stably expressing a broad panel of laminopathic lamin A mutations, we found that several mutations associated with muscular dystrophy and dilated cardiomyopathy resulted in more deformable nuclei; in contrast, lamin mutants responsible for diseases without muscular phenotypes did not alter nuclear deformability. We confirmed our results in intact muscle tissue, demonstrating that nuclei of transgenic Drosophila melanogaster muscle expressing myopathic lamin mutations deformed more under applied strain than controls. In vivo and in vitro studies indicated that the loss of nuclear stiffness resulted from impaired assembly of mutant lamins into the nuclear lamina. Although only a subset of lamin mutations associated with muscular diseases caused increased nuclear deformability, almost all mutations tested had defects in force transmission between the nucleus and cytoskeleton. In conclusion, our results indicate that although defective nuclear stability may play a role in the development of muscle diseases, other factors, such as impaired nucleo-cytoskeletal coupling, likely contribute to the muscle phenotyp

Nuclear Envelope Composition Determines The Ability Of Neutrophil-Type Cells To Passage Through Micron-Scale Constrictions

Neutrophils are characterized by their distinct nuclear shape, which is thought to facilitate the transit of these cells through pore spaces less than one-fifth of their diameter. We used human promyelocytic leukemia (HL-60) cells as a model system to investigate the effect of nuclear shape in whole cell deformability. We probed neutrophil-differentiated HL-60 cells lacking expression of lamin B receptor, which fail to develop lobulated nuclei during granulopoiesis and present an in vitro model for Pelger-Huët anomaly; despite the circular morphology of their nuclei, the cells passed through micron-scale constrictions on similar timescales as scrambled controls. We then investigated the unique nuclear envelope composition of neutrophil-differentiated HL-60 cells, which may also impact their deformability; although lamin A is typically down-regulated during granulopoiesis, we genetically modified HL-60 cells to generate a subpopulation of cells with well defined levels of ectopic lamin A. The lamin A-overexpressing neutrophil-type cells showed similar functional characteristics as the mock controls, but they had an impaired ability to pass through micron-scale constrictions. Our results suggest that levels of lamin A have a marked effect on the ability of neutrophils to passage through micron-scale constrictions, whereas the unusual multilobed shape of the neutrophil nucleus is less essential

From lamins to lamina: a structural perspective

Lamin proteins are the major constituents of the nuclear lamina, a proteinaceous network that lines the inner nuclear membrane. Primarily, the nuclear lamina provides structural support for the nucleus and the nuclear envelope; however, lamins and their associated proteins are also involved in most of the nuclear processes, including DNA replication and repair, regulation of gene expression, and signaling. Mutations in human lamin A and associated proteins were found to cause a large number of diseases, termed 'laminopathies.' These diseases include muscular dystrophies, lipodystrophies, neuropathies, and premature aging syndromes. Despite the growing number of studies on lamins and their associated proteins, the molecular organization of lamins in health and disease is still elusive. Likewise, there is no comprehensive view how mutations in lamins result in a plethora of diseases, selectively affecting different tissues. Here, we discuss some of the structural aspects of lamins and the nuclear lamina organization, in light of recent results

Impaired mechanical response of an EDMD mutation leads to motility phenotypes that are repaired by loss of prenylation

There are roughly 14 distinct heritable autosomal dominant diseases associated with mutations in lamins A/C, including Emery-Dreifuss muscular dystrophy (EDMD). The mechanical model proposes that the lamin mutations change the mechanical properties of muscle nuclei, leading to cell death and tissue deterioration. Here, we developed an experimental protocol that analyzes the effect of disease-linked lamin mutations on the response of nuclei to mechanical strain in living Caenorhabditis elegans We found that the EDMD mutation L535P disrupts the nuclear mechanical response specifically in muscle nuclei. Inhibiting lamin prenylation rescued the mechanical response of the EDMD nuclei, reversed the muscle phenotypes and led to normal motility. The LINC complex and emerin were also required to regulate the mechanical response of C. elegans nuclei. This study provides evidence to support the mechanical model and offers a potential future therapeutic approach towards curing EDMD

Functional architecture of the nuclear pore complex

The nuclear pore complex (NPC) is the sole gateway between the nucleus and the cytoplasm. NPCs fuse the inner and outer nuclear membranes to form aqueous translocation channels that allow the free diffusion of small molecules and ions, as well as receptor-mediated transport of large macromolecules. The NPC regulates nucleocytoplasmic transport of macromolecules, utilizing soluble receptors that identify and present cargo to the NPC, in a highly selective manner to maintain cellular functions. The NPC is composed of multiple copies of approximately 30 different proteins, termed nucleoporins, which assemble to form one of the largest multiprotein assemblies in the cell. In this review, we address structural and functional aspects of this fundamental cellular machinery

Insights into the gate of the nuclear pore complex

Nuclear pore complexes (NPCs) serve as the gateway of the cell nucleus. These macromolecular assemblies form selective aqueous translocation channels permitting the free diffusion of small molecules, as well as receptor-mediated transport of large cargoes. Over the past decade, major progress has been made in both the structural determination of individual nucleoporins and subcomplexes by X-ray crystallography and in the structural analysis of the entire NPC by cryo-electron tomography (cryo-ET). The metazoan NPC structure from Xenopus laevis oocytes was recently resolved up to 20 Å by combining cryo-ET with advanced image processing techniques, revealing for the first time the architecture of the central channel. Here, we discuss the structure of the Xenopus laevis NPC and consider future perspectives that will eventually allow reconstructing the scaffold and gate of the NPC with higher resolution and identifying its transport-relevant regions. This will eventually allow us to describe the structure of the NPC 'in action'