scholarly journals Supramolecular Structures of the Dictyostelium Lamin NE81

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 162
Author(s):  
Marianne Grafe ◽  
Petros Batsios ◽  
Irene Meyer ◽  
Daria Lisin ◽  
Otto Baumann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Model Organism ◽  
Super Resolution ◽  
Nuclear Lamina ◽  
Stimulated Emission ◽  
Structural Level ◽  
Animal Cells ◽  
Nuclear Lamins

Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins. Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells.

Purification and immunological detection of pea nuclear intermediate filaments: evidence for plant nuclear lamins

1992 ◽  
Vol 103 (2) ◽  
pp. 407-414 ◽  
Author(s):  
A.K. McNulty ◽  
M.J. Saunders
Keyword(s):  
Nuclear Envelope ◽  
Intermediate Filaments ◽  
Plant Cells ◽  
Nuclear Lamina ◽  
Animal Cells ◽  
Nuclear Lamins ◽  
Antigenic Characterization ◽  
Major Structural Component ◽  
Nuclear Envelope Assembly ◽  
Type V

A major structural component of the inner face of the nuclear envelope in vertebrates and invertebrates is the nuclear lamina, an array of 1–3 extrinsic membrane proteins, lamins A, B and C. These proteins are highly homologous to intermediate filaments and are classified as type V. We report the first purification, antigenic characterization and immunocytochemical localization of putative plant lamin proteins from pea nuclei. We conclude that plant cells contain this ancestral class of intermediate filaments in their nuclei and that regulation of nuclear envelope assembly/disassembly and mitosis in plants may be similar to that in animal cells.

scholarly journals A role for nuclear lamins in nuclear envelope assembly

2001 ◽  
Vol 154 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Reynold I. Lopez-Soler ◽  
Robert D. Moir ◽  
Timothy P. Spann ◽  
Reimer Stick ◽  
Robert D. Goldman
Keyword(s):  
Nuclear Envelope ◽  
Molecular Interactions ◽  
Nuclear Membrane ◽  
Nuclear Lamina ◽  
Chromatin Decondensation ◽  
Nuclear Lamins ◽  
Nuclear Membranes ◽  
Terminal Domain ◽  
Nuclear Envelope Assembly

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina–pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These “nonfusogenic” vesicles lack lamin B3 (LB3) and do not bind LB3T; however, “fusogenic” vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.

scholarly journals Quantification of Internalized Silica Nanoparticles via STED Microscopy

2015 ◽  
Vol 2015 ◽  
pp. 1-16 ◽  
Author(s):  
Henrike Peuschel ◽  
Thomas Ruckelshausen ◽  
Christian Cavelius ◽  
Annette Kraegeloh
Keyword(s):  
Silica Nanoparticles ◽  
Super Resolution ◽  
Engineered Nanoparticles ◽  
Stimulated Emission ◽  
Alveolar Type ◽  
Serum Free Medium ◽  
Free Medium ◽  
Essential Information ◽  
Serum Free

The development of safe engineered nanoparticles (NPs) requires a detailed understanding of their interaction mechanisms on a cellular level. Therefore, quantification of NP internalization is crucial to predict the potential impact of intracellular NP doses, providing essential information for risk assessment as well as for drug delivery applications. In this study, the internalization of 25 nm and 85 nm silica nanoparticles (SNPs) in alveolar type II cells (A549) was quantified by application of super-resolution STED (stimulated emission depletion) microscopy. Cells were exposed to equal particle number concentrations (9.2×1010particles mL−1) of each particle size and the sedimentation of particles during exposure was taken into account. Microscopy images revealed that particles of both sizes entered the cells after 5 h incubation in serum supplemented and serum-free medium. According to thein vitrosedimentation, diffusion, and dosimetry (ISDD) model 20–27% of the particles sedimented. In comparison, 102-103NPs per cell were detected intracellularly serum-containing medium. Furthermore, in the presence of serum, no cytotoxicity was induced by the SNPs. In serum-free medium, large agglomerates of both particle sizes covered the cells whereas only high concentrations (≥ 3.8 × 1012particles mL−1) of the smaller particles induced cytotoxicity.

Resinless section immunogold electron microscopy of karyo-cytoskeletal frameworks of eukaryotic cells cultured in vitro. Absence of a salt-stable nuclear matrix from mouse plasmacytoma MPC-11 cells

1991 ◽  
Vol 98 (1) ◽  
pp. 107-122
Author(s):  
X. Wang ◽  
P. Traub
Keyword(s):  
Electron Microscopy ◽  
Nuclear Matrix ◽  
Protein A ◽  
Mouse Skin ◽  
Nuclear Lamina ◽  
Immunogold Electron Microscopy ◽  
Cell Structures ◽  
Critical Point Drying ◽  
Cells Cultured

The karyo-cytoskeleton of cells cultured in vitro was investigated employing resinless section immunogold electron microscopy. Cells were entrapped in low-melting agarose, sequentially extracted with various buffers and digested with nucleases to obtain karyo-cytoskeletal frameworks and reacted with specific primary and gold-conjugated secondary antibodies or gold-conjugated protein A to decorate structural elements of these frameworks. Following embedment of the gold-labeled residual cell structures in diethylene glycol distearate and their sectioning, the embedding material was removed with organic solvent and the sections were finally subjected to CO2 critical point drying. When this technique was applied to mouse skin fibroblasts (MSF), it revealed a dense and salt-stable intranuclear network of fibrogranular material. Antibodies directed against vimentin and lamin B detected a cytoplasmic meshwork of intermediate filaments (IFs) and a nuclear lamina, respectively; the latter, however, only after removal of chromatin from nuclei by nuclease digestion of DNA. Intranuclear filaments free of adhering globular material were morphologically very similar to cytoplasmic vimentin filaments. By contrast, mouse plasmacytoma MPC-11 cells lacking detectable amounts of cytoplasmic IF proteins and lamins A and C were devoid of a salt-stable internal nuclear matrix. The same holds true for MPC-11 cells that had been treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate to induce vimentin synthesis and establish a cytoplasmically extended IF network. These findings were in accordance with the biochemical behavior of Triton X-100-treated MSF and MPC-11 cells and their appearance in immunofluorescence microscopy upon extraction with high ionic strength buffer. While the chromatin was quantitatively retained in the residual cell structures derived from MSF cells, in those obtained from MPC-11 cells the nuclear lamina was disrupted and the chromatin was released from the nuclei, suggesting that MPC-11 cells lack the salt-stable nuclear scaffold to which chromatin is normally anchored.

scholarly journals A Balance Between Intermediate Filaments and Microtubules Maintains Nuclear Architecture in the Cardiomyocyte

2020 ◽  
Vol 126 (3) ◽  
Author(s):  
Julie Heffler ◽  
Parisha P. Shah ◽  
Patrick Robison ◽  
Sai Phyo ◽  
Kimberly Veliz ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Intermediate Filaments ◽  
Genome Organization ◽  
Contractile Function ◽  
Nuclear Architecture ◽  
Super Resolution ◽  
Nuclear Lamina ◽  
Linc Complex ◽  
Spectrin Repeat ◽  
Balance Of Forces

Rationale: Mechanical forces are transduced to nuclear responses via the linkers of the nucleoskeleton and cytoskeleton (LINC) complex, which couples the cytoskeleton to the nuclear lamina and associated chromatin. While disruption of the LINC complex can cause cardiomyopathy, the relevant interactions that bridge the nucleoskeleton to cytoskeleton are poorly understood in the cardiomyocyte, where cytoskeletal organization is unique. Furthermore, while microtubules and desmin intermediate filaments associate closely with cardiomyocyte nuclei, the importance of these interactions is unknown. Objective: Here, we sought to determine how cytoskeletal interactions with the LINC complex regulate nuclear homeostasis in the cardiomyocyte. Methods and Results: To this end, we acutely disrupted the LINC complex, microtubules, actin, and intermediate filaments and assessed the consequences on nuclear morphology and genome organization in rat ventricular cardiomyocytes via a combination of super-resolution imaging, biophysical, and genomic approaches. We find that a balance of dynamic microtubules and desmin intermediate filaments is required to maintain nuclear shape and the fidelity of the nuclear envelope and lamina. Upon depletion of desmin (or nesprin [nuclear envelope spectrin repeat protein]-3, its binding partner in the LINC complex), polymerizing microtubules collapse the nucleus and drive infolding of the nuclear membrane. This results in DNA damage, a loss of genome organization, and broad transcriptional changes. The collapse in nuclear integrity is concomitant with compromised contractile function and may contribute to the pathophysiological changes observed in desmin-related myopathies. Conclusions: Disrupting the tethering of desmin to the nucleus results in a loss of nuclear homeostasis and rapid alterations to cardiomyocyte function. Our data suggest that a balance of forces imposed by intermediate filaments and microtubules is required to maintain nuclear structure and genome organization in the cardiomyocyte.

scholarly journals High resolution scanning electron microscopy of the nuclear envelope: demonstration of a new, regular, fibrous lattice attached to the baskets of the nucleoplasmic face of the nuclear pores.

1992 ◽  
Vol 119 (6) ◽  
pp. 1429-1440 ◽  
Author(s):  
M W Goldberg ◽  
T D Allen
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
High Resolution ◽  
Nuclear Envelope ◽  
Nuclear Lamina ◽  
Objective Lens ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Triturus Cristatus ◽  
Scanning Electron

The nuclear envelope (NE) of amphibian oocytes can be readily isolated in relatively structurally intact and pure form and has been used extensively for structural studies. Using high resolution scanning electron microscopy (HRSEM), both surfaces of the NE can be visualized in detail. Here, we demonstrate the use of HRSEM to obtain high resolution information of NE structure, confirming previous data and providing some new information. NEs, manually isolated from Triturus cristatus oocytes, have been mounted on conductive silicon chips, fixed, critical point dried and coated with a thin, continuous film of chromium or tantalum and viewed at relatively high accelerating voltage in a field emission scanning electron microscope with the sample within the objective lens. Both nucleoplasmic and cytoplasmic surfaces of the nuclear pore complexes (NPC) have been visualized, revealing the cytoplasmic coaxial ring, associated particles, central plug/transporter and spokes. The nucleoplasmic face is dominated by the previously described basketlike structure attached to the nucleoplasmic coaxial ring. In Triturus, a novel, highly regular flat sheet of fibers, termed the NE lattice (NEL) has been observed attached to the distal ring of the NPC basket. The NEL appears to be distinct from the nuclear lamina. Evidence for the NEL is also presented in thin TEM sections from Triturus oocytes and GVs and in spread NEs from Xenopus. A model is presented for NEL structure and its interaction with the NPCs is discussed.

DNA from Drosophila melanogaster β-heterochromatin binds specifically to nuclear lamins in vitro and the nuclear envelope in situ

Gene ◽  
1996 ◽  
Vol 171 (2) ◽  
pp. 171-176 ◽  
Author(s):  
Ella A. Baricheva ◽  
Miguel Berrios ◽  
Sergei S. Bogachev ◽  
Igor V. Borisevich ◽  
Eugenia R. Lapik ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Nuclear Envelope ◽  
Nuclear Lamins

scholarly journals HSV-1 UL34 mutants that affect membrane budding regulation and nuclear lamina disruption

2021 ◽  
Author(s):  
Amber Vu ◽  
Shaowen White ◽  
Tiffany Cassmann ◽  
Richard J. Roller
Keyword(s):  
Virus Replication ◽  
Nuclear Lamina ◽  
Structural Basis ◽  
Structural Requirement ◽  
Nuclear Lamins ◽  
Nuclear Egress ◽  
Host Cell Proteins ◽  
Membrane Budding ◽  
The Individual

Nuclear envelope budding in herpesvirus nuclear egress may be negatively regulated, since the pUL31/pUL34 nuclear egress complex heterodimer can induce membrane budding without capsids when expressed ectopically or on artificial membranes in vitro, but not in the infected cell. We have previously described a pUL34 mutant that contained alanine substitutions at R158 and R161, and that showed impaired growth, impaired pUL31/pUL34 interaction, and unregulated budding. Here we determine the phenotypic contributions of the individual substitutions to these phenotypes. Neither substitution alone was able to reproduce the impaired growth or NEC interaction phenotypes. Either substitution, however, could fully reproduce the unregulated budding phenotype, suggesting that mis-regulated budding may not substantially impair virus replication. Additionally, the R158A substitution caused re-localization of the NEC to intranuclear punctate structures and recruited lamin A/C to those structures, suggesting that this residue might be important for recruitment of kinases for dispersal of nuclear lamins. Importance Herpesvirus nuclear egress is a complex, regulated process coordinated by two virus proteins that are conserved among the herpesviruses that form a heterodimeric nuclear egress complex (NEC). The NEC drives budding of capsids at the inner nuclear membrane, and recruits other viral and host cell proteins for disruption of the nuclear lamina, membrane scission and fusion. The structural basis of individual activities of the NEC, apart from membrane budding, are not clear, nor is the basis of the regulation of membrane budding. Here we explore the properties of NEC mutants that have an unregulated budding phenotype, determine the significance of that regulation for virus replication, and also characterize a structural requirement for nuclear lamina disruption.

scholarly journals A molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei

2017 ◽  
Author(s):  
Anneliese Hoffmann ◽  
Sandro Käser ◽  
Martin Jakob ◽  
Simona Amodeo ◽  
Camille Peitsch ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mitochondrial Genome ◽  
Trypanosoma Brucei ◽  
De Novo ◽  
Molecular Model ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Kinetoplast Dna ◽  
Exclusion Zone ◽  
Super Resolution Microscopy

AbstractIn almost all eukaryotes mitochondria maintain their own genome. Despite the discovery more than 50 years ago still very little is known about how the genome is properly segregated during cell division. The protozoan parasite Trypanosoma brucei contains a single mitochondrion with a singular genome the kinetoplast DNA (kDNA). Electron microscopy studies revealed the tripartite attachment complex (TAC) to physically connect the kDNA to the basal body of the flagellum and to ensure proper segregation of the mitochondrial genome via the basal bodies movement, during cell cycle. Using super-resolution microscopy we precisely localize each of the currently known unique TAC components. We demonstrate that the TAC is assembled in a hierarchical order from the base of the flagellum towards the mitochondrial genome and that the assembly is not dependent on the kDNA itself. Based on biochemical analysis the TAC consists of several non-overlapping subcomplexes suggesting an overall size of the TAC exceeding 2.8 mDa. We furthermore demonstrate that the TAC has an impact on mitochondrial organelle positioning however is not required for proper organelle biogenesis or segregation.Significance StatementMitochondrial genome replication and segregation are essential processes in most eukaryotic cells. While replication has been studied in some detail much less is known about the molecular machinery required distribute the replicated genomes. Using super-resolution microscopy in combination with molecular biology and biochemistry we show for the first time in which order the segregation machinery is assembled and that it is assembled de novo rather than in a semi conservative fashion in the single celled parasite Trypanosoma brucei. Furthermore, we demonstrate that the mitochondrial genome itself is not required for assembly to occur. It seems that the physical connection of the mitochondrial genome to cytoskeletal elements is a conserved feature in most eukaryotes, however the molecular components are highly diverse.Abbreviation(EZF)Exclusion zone filaments(ULF)Unilateral filament(TAC)tripartite attachment complex(OM)outer mitochondrial(IM)inner mitochondrial(BSF)bloodstream form(PCF)procyclic form(kDNA)kinetoplast DNA(gRNA)guide RNA(SBFSEM)Serial block face-scanning electron microscopy(Tet)tetracyclin(STED)Stimulated Emission Depletion

scholarly journals Nucleoplasmic Lamin C Rapidly Accumulates at Sites of Nuclear Envelope Rupture with BAF and cGAS

2022 ◽  
Author(s):  
Yohei Kono ◽  
Stephen A. Adam ◽  
Karen Reddy ◽  
Yixian Zheng ◽  
Ohad Medalia ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Nuclear Lamina ◽  
Structural Components ◽  
Cell Nuclei ◽  
Embryonic Fibroblasts ◽  
Nuclear Lamins ◽  
Laser Microirradiation

In mammalian cell nuclei, the nuclear lamina (NL) underlies the nuclear envelope (NE) to maintain nuclear structure. The nuclear lamins, the major structural components of the NL, are involved in the protection against NE rupture induced by mechanical stress. However, the specific role of the lamins in repair of NE ruptures has not been fully determined. Our analyses using immunofluorescence and live-cell imaging revealed that lamin C but not the other lamin isoforms rapidly accumulated at sites of NE rupture induced by laser microirradiation in mouse embryonic fibroblasts. The immunoglobulin-like fold domain and the NLS were required for the recruitment from the nucleoplasm to the rupture sites with the Barrier-to-autointegration factor (BAF). The accumulation of nuclear BAF and cytoplasmic cyclic GMP-AMP (cGAMP) synthase (cGAS) at the rupture sites was in part dependent on lamin A/C. These results suggest that nucleoplasmic lamin C, BAF and cGAS concertedly accumulate at sites of NE rupture for repair.

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