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.
Nuclear Mechanics in Disease Zwerger, Monika; Ho, Chin Yee; Lammerding, Jan
Annual review of biomedical engineering,
08/2011, Volume:
13, Issue:
1
Journal Article
Peer reviewed
Open access
Over the past two decades, the biomechanical properties of cells have emerged as key players in a broad range of cellular functions, including migration, proliferation, and differentiation. Although ...much of the attention has focused on the cytoskeletal networks and the cell's microenvironment, relatively little is known about the contribution of the cell nucleus. Here, we present an overview of the structural elements that determine the physical properties of the nucleus and discuss how changes in the expression of nuclear components or mutations in nuclear proteins can not only affect nuclear mechanics but also modulate cytoskeletal organization and diverse cellular functions. These findings illustrate that the nucleus is tightly integrated into the surrounding cellular structure. Consequently, changes in nuclear structure and composition are highly relevant to normal development and physiology and can contribute to many human diseases, such as muscular dystrophy, dilated cardiomyopathy, (premature) aging, and cancer.
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.
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► LBR- and lamin-A/C-dependent tethers maintain peripheral heterochromatin ► In their absence, all heterochromatin clusters in the nuclear interior ► During cellular differentiation, the LBR tether precedes the lamin A/C tether ► Lamin A/C promotes, whereas LBR delays myogenic differentiation
The lamin B receptor and lamin A/C have sequential roles in the tethering of peripheral heterochromatin during cellular differentiation, corresponding to their differential effects on heterochromatin positioning and transcription of tissue-specific genes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
Background: The unusual nuclear shape of neutrophils has been speculated to facilitate their passage through confined spaces.
Results: Levels of nuclear protein lamin A modulate cell passage through micron-scale pores.
Conclusion: The unique protein composition of neutrophil nuclei facilitates their deformation; lobulated nuclear shape is not essential.
Significance: Altered nuclear envelope composition, as reported in cancer cells, could impact cell passage through physiological gaps.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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 phenotype.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mutations in the human LMNA gene cause muscular dystrophy by mechanisms that are incompletely understood. The LMNA gene encodes A-type lamins, intermediate filaments that form a network underlying ...the inner nuclear membrane, providing structural support for the nucleus and organizing the genome. To better understand the pathogenesis caused by mutant lamins, we performed a structural and functional analysis on LMNA missense mutations identified in muscular dystrophy patients. These mutations perturb the tertiary structure of the conserved A-type lamin Ig-fold domain. To identify the effects of these structural perturbations on lamin function, we modeled these mutations in Drosophila Lamin C and expressed the mutant lamins in muscle. We found that the structural perturbations had minimal dominant effects on nuclear stiffness, suggesting that the muscle pathology was not accompanied by major structural disruption of the peripheral nuclear lamina. However, subtle alterations in the lamina network and subnuclear reorganization of lamins remain possible. Affected muscles had cytoplasmic aggregation of lamins and additional nuclear envelope proteins. Transcription profiling revealed upregulation of many Nrf2 target genes. Nrf2 is normally sequestered in the cytoplasm by Keap-1. Under oxidative stress Nrf2 dissociates from Keap-1, translocates into the nucleus, and activates gene expression. Unexpectedly, biochemical analyses revealed high levels of reducing agents, indicative of reductive stress. The accumulation of cytoplasmic lamin aggregates correlated with elevated levels of the autophagy adaptor p62/SQSTM1, which also binds Keap-1, abrogating Nrf2 cytoplasmic sequestration, allowing Nrf2 nuclear translocation and target gene activation. Elevated p62/SQSTM1 and nuclear enrichment of Nrf2 were identified in muscle biopsies from the corresponding muscular dystrophy patients, validating the disease relevance of our Drosophila model. Thus, novel connections were made between mutant lamins and the Nrf2 signaling pathway, suggesting new avenues of therapeutic intervention that include regulation of protein folding and metabolism, as well as maintenance of redox homoeostasis.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Lamin B receptor Olins, Ada L.; Rhodes, Gale; Welch, David B. Mark ...
Nucleus (Austin, Tex.),
20/1/1/, Volume:
1, Issue:
1
Journal Article
Peer reviewed
Open access
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.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Lamins are intermediate filament proteins forming a fibrous meshwork, called nuclear lamina, between the inner nuclear membrane and peripheral heterochromatin of metazoan cells. The assembly and ...incorporation of lamin A/C into the lamina as well as their various functions are still not well understood. Here, we employed designed ankyrin repeat proteins (DARPins) as novel experimental tools for lamin research. We screened for DARPins that specifically bind lamin A/C, interfere with lamin assembly in vitro, and with incorporation of lamin A/C into the native lamina in living cells. Selected DARPins inhibit lamin assembly and delocalize A-type lamins to the nucleoplasm without modifying lamin expression levels or amino acid sequence. Using these lamin binders we demonstrate the importance of proper integration of lamin A/C into the lamina for nuclear mechanical properties and nuclear envelope integrity. Finally, our study provides evidence for cell-type specific differences in lamin functions.
Objective Lamin B receptor (LBR) is an integral protein of the inner nuclear membrane. Recent studies have demonstrated that genetic deficiency of LBR during granulopoiesis results in hypolobulation ...of the mature neutrophil nucleus, as observed in human Pelger-Huët anomaly and mouse ichthyosis ( ic ). In this study, we utilized differentiated early promyelocytes (EPRO cells) that were derived from the bone marrow of homozygous and heterozygous ichthyosis mice to examine changes to the expression of nuclear envelope proteins and heterochromatin structure that result from deficient LBR expression. Materials and Methods Wild-type (+/+), heterozygous (+/ ic ), and homozygous ( ic / ic ) granulocytic forms of EPRO cells were analyzed for the expression of multiple lamins and inner nuclear envelope proteins by immunostaining and immunoblotting techniques. The heterochromatin architecture was also examined by immunostaining for histone lysine methylation. Results Wild-type (+/+) and heterozygous (+/ ic ) granulocytic forms revealed ring-shaped nuclei and contained LBR within the nuclear envelope; ic / ic granulocytes exhibited smaller ovoid nuclei devoid of LBR. The pericentric heterochromatin of undifferentiated and granulocytic ic / ic cells was condensed into larger spots and shifted away from the nuclear envelope, compared to +/+ and +/ ic cell forms. Lamin A/C, which is normally not present in mature granulocytes, was significantly elevated in LBR-deficient EPRO cells. Conclusions Our observations suggest roles for LBR during granulopoiesis, which can involve augmenting nuclear membrane growth, facilitating compartmentalization of heterochromatin, and promoting downregulation of lamin A/C expression.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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