SciCombinator

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Concept: Nuclear envelope

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Nuclear pore complexes form a selective filter that allows the rapid passage of transport factors (TFs) and their cargoes across the nuclear envelope, while blocking the passage of other macromolecules. Intrinsically disordered proteins (IDPs) containing phenylalanyl-glycyl (FG) rich repeats line the pore and interact with TFs. However, the reason that transport can be both fast and specific remains undetermined, through lack of atomic-scale information on the behavior of FGs and their interaction with TFs. We used NMR spectroscopy to address these issues. We show that FG repeats are highly dynamic IDPs, stabilized by the cellular environment. Fast transport of TFs is supported because the rapid motion of FG motifs allows them to exchange on and off TFs extremely quickly through transient interactions. Because TFs uniquely carry multiple pockets for FG repeats, only they can form the many frequent interactions needed for specific passage between FG repeats to cross the NPC.

Concepts: Pharmacology, Cell nucleus, Nuclear magnetic resonance, NMR spectroscopy, Interaction, Nuclear pore, Nuclear envelope, The Passage

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Much of life’s essential molecular machinery consists of large protein assemblies that currently pose challenges for structure determination. A prominent example is the nuclear pore complex (NPC), for which the organization of its individual components remains unknown. By combining stochastic superresolution microscopy, to directly resolve the ringlike structure of the NPC, with single-particle averaging, to use information from thousands of pores, we determined the average positions of fluorescent molecular labels in the NPC with a precision well below 1 nanometer. Applying this approach systematically to the largest building block of the NPC, the Nup107-160 subcomplex, we assessed the structure of the NPC scaffold. Thus, light microscopy can be used to study the molecular organization of large protein complexes in situ in whole cells.

Concepts: DNA, Protein, Cell nucleus, Light, Messenger RNA, Nuclear pore, Nuclear envelope, Microscopy

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Much of the structural stability of the nucleus comes from meshworks of intermediate filament proteins known as lamins forming the inner layer of the nuclear envelope called the nuclear lamina. These lamin meshworks additionally play a role in gene expression. Abnormalities in nuclear shape are associated with a variety of pathologies, including some forms of cancer and Hutchinson-Gilford Progeria Syndrome, and often include protruding structures termed nuclear blebs. These nuclear blebs are thought to be related to pathological gene expression; however, little is known about how and why blebs form. We have developed a minimal continuum elastic model of a lamin meshwork that we use to investigate which aspects of the meshwork could be responsible for bleb formation. Mammalian lamin meshworks consist of two types of lamin proteins, A type and B type, and it has been reported that nuclear blebs are enriched in A-type lamins. Our model treats each lamin type separately and thus, can assign them different properties. Nuclear blebs have been reported to be located in regions where the fibers in the lamin meshwork have a greater separation, and we find that this greater separation of fibers is an essential characteristic for generating nuclear blebs. The model produces structures with comparable morphologies and distributions of lamin types as real pathological nuclei. Thus, preventing this opening of the meshwork could be a route to prevent bleb formation, which could be used as a potential therapy for the pathologies associated with nuclear blebs.

Concepts: DNA, Gene, Cell nucleus, Cell, Nuclear envelope, Intermediate filament, Progeria, Lamin

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The lamin proteins are essential components of the nuclear lamina of eukaryotic cells, that are involved in a complex association mechanism to attain a functional supermolecular structure. Mutations of the lamin A/C gene are associated with several different neuromuscular diseases, and the detailed effect of disease-associated amino acid substitutions on the structure and stability of human lamin dimers is yet unknown. Here we present a structural and thermodynamic characterization by means of molecular dynamics simulations of the effect of pathological mutations (S326T, R331P, R331Q, E347K, E358K, M371K, and R377H) on the association of the coil 2B domains that mediate lamin A/C oligomerization. The structures attained during the simulations, along with the quantification of the contribution of each residue to the dimerization energies, support a lamin association mechanism mediated by homophilic intermolecular interactions promoted by dissociative conformational changes at distinct positions in the coiled coil. The pathogenic mutations can both increase or decrease the stability of lamin A/C dimers, and a possible correlation between the effect of the amino acid substitutions and disease onset and severity is presented.

Concepts: DNA, Protein, Cell nucleus, Bacteria, Amino acid, Amine, Structure, Nuclear envelope

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Nuclear export of mRNAs is thought to occur exclusively through nuclear pore complexes. In this issue of Cell, Speese et al. identify an alternate pathway for mRNA export in muscle cells where ribonucleoprotein complexes involved in forming neuromuscular junctions transit the nuclear envelope by fusing with and budding through the nuclear membrane.

Concepts: DNA, Cell nucleus, Nuclear pore, Nuclear envelope

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Mitosis in metazoa requires nuclear envelope (NE) disassembly and reassembly. NE disassembly is driven by multiple phosphorylation events. Mitotic phosphorylation of the protein BAF reduces its affinity for chromatin and the LEM family of inner nuclear membrane proteins; loss of this BAF-mediated chromatin-NE link contributes to NE disassembly. BAF must reassociate with chromatin and LEM proteins at mitotic exit to reform the NE; however, how its dephosphorylation is regulated is unknown. Here, we show that the C. elegans protein LEM-4L and its human ortholog Lem4 (also called ANKLE2) are both required for BAF dephosphorylation. They act in part by inhibiting BAF’s mitotic kinase, VRK-1, in vivo and in vitro. In addition, Lem4/LEM-4L interacts with PP2A and is required for it to dephosphorylate BAF during mitotic exit. By coordinating VRK-1- and PP2A-mediated signaling on BAF, Lem4/LEM-4L controls postmitotic NE formation in a function conserved from worms to humans.

Concepts: Cell nucleus, Cell, Signal transduction, Enzyme, Eukaryote, Nuclear envelope, Mitosis, Inner nuclear membrane proteins

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The serine/threonine kinase Akt/PKB is a major signaling hub integrating metabolic, survival, growth, and cell cycle regulatory signals. The definition of the phospho-motif cipher driving phosphorylation by Akt led to the identification of hundreds of putative substrates, and it is therefore pivotal to identify those whose phosphorylation by Akt is of consequence to biological processes. The Lmna gene products lamin A/C and the lamin A precursor prelamin A are type V intermediate filament proteins forming a filamentous meshwork, the lamina, underneath the inner nuclear membrane, for nuclear envelope structures organization and interphase chromatin anchoring. In our previous work, we reported that A-type lamins are phosphorylated by Akt at S301 and S404 in physiological conditions and are therefore bona fide substrates of Akt. We report here that Akt phosphorylation at S404 targets the precursor prelamin A for degradation. We further demonstrate that Akt also regulates Lmna transcription. Our study unveils a previously unknown function of Akt in the control of prelamin A stability and expression. Moreover, given the large number of diseases related to prelamin A, our findings represent a further important step bridging basic A-type lamin physiology to therapeutic approaches for lamin A-linked disorders.-Bertacchini, J., Beretti, F., Cenni, V., Guida, M., Gibellini, F., Mediani, L., Marin, O., Maraldi, N. M., de Pol, A., Lattanzi, G., Cocco, L., Marmiroli, S. The protein kinase Akt/PKB regulates both prelamin A degradation and Lmna gene expression.

Concepts: DNA, Gene, Cell nucleus, Cell, Transcription, Adenosine triphosphate, Nuclear envelope, Lamin

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Nuclear pore complexes (NPCs) perforate the nuclear envelope and allow the exchange of macromolecules between the nucleus and the cytoplasm. To acquire a deeper understanding of this transport mechanism, we analyse the structure of the NPC scaffold and permeability barrier, by reconstructing the Xenopus laevis oocyte NPC from native nuclear envelopes up to 20 Å resolution by cryo-electron tomography in conjunction with subtomogram averaging. In addition to resolving individual protein domains of the NPC constituents, we propose a model for the architecture of the molecular gate at its central channel. Furthermore, we compare and contrast this native NPC structure to one that exhibits reduced transport activity and unveil the spatial properties of the NPC gate.

Concepts: Protein, Cell nucleus, Cell, Messenger RNA, Nuclear pore, Nuclear envelope, Cell anatomy, Postage stamp

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The nuclear lamina represents a multifunctional platform involved in such diverse yet interconnected processes as spatial organization of the genome, maintenance of mechanical stability of the nucleus, regulation of transcription and replication. Most of lamina activities are exerted through tethering of lamina-associated chromatin domains (LADs) to the nuclear periphery. Yet, the lamina is a dynamic structure demonstrating considerable expansion during the cell cycle to accommodate increased number of LADs formed during DNA replication. We analyzed dynamics of nuclear growth during interphase and changes in lamina structure as a function of cell cycle progression. The nuclear lamina demonstrates steady growth from G1 till G2, while quantitative analysis of lamina meshwork by super-resolution microscopy revealed that microdomain organization of the lamina is maintained, with lamin A and lamin B microdomain periodicity and interdomain gap sizes unchanged. FRAP analysis, in contrast, demonstrated differences in lamin A and B1 exchange rates; the latter showing higher recovery rate in S-phase cells. In order to further analyze the mechanism of lamina growth in interphase, we generated a lamina-free nuclear envelope in living interphase cells by reversible hypotonic shock. The nuclear envelope in nuclear buds formed after such a treatment initially lacked lamins, and analysis of lamina formation revealed striking difference in lamin A and B1 assembly: lamin A reassembled within 30 min post-treatment, whereas lamin B1 did not incorporate into the newly formed lamina at all. We suggest that in somatic cells lamin B1 meshwork growth is coordinated with replication of LADs, and lamin A meshwork assembly seems to be chromatin-independent process.

Concepts: DNA, Cell nucleus, Chromosome, Cell cycle, Nuclear envelope, DNA replication, Interphase, Lamin

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Lamina-associated polypeptide 2α (LAP2α) localizes throughout the nucleoplasm and interacts with the fraction of lamins A/C that is not associated with the peripheral nuclear lamina. The LAP2α-lamin A/C complex negatively affects cell proliferation. Lamins A/C are encoded by LMNA, a single heterozygous mutation of which causes Hutchinson-Gilford progeria syndrome (HGPS). This mutation generates the lamin A variant progerin, which we show here leads to loss of LAP2α and nucleoplasmic lamins A/C, impaired proliferation, and down-regulation of extracellular matrix components. Surprisingly, contrary to wild-type cells, ectopic expression of LAP2α in cells expressing progerin restores proliferation and extracellular matrix expression but not the levels of nucleoplasmic lamins A/C. We conclude that, in addition to its cell cycle-inhibiting function with lamins A/C, LAP2α can also regulate extracellular matrix components independently of lamins A/C, which may help explain the proliferation-promoting function of LAP2α in cells expressing progerin.

Concepts: DNA, Gene, Cell nucleus, Extracellular matrix, DNA repair, Nuclear envelope, Progeria, LMNA