scholarly journals Live imaging of chromatin distribution in muscle nuclei reveals novel principles of nuclear architecture and chromatin compartmentalization

2020 ◽  
Author(s):  
Daria Amiad-Pavlov ◽  
Dana Lorber ◽  
Gaurav Bajpai ◽  
Samuel Safran ◽  
Talila Volk
Keyword(s):  
Nuclear Architecture ◽  
Nuclear Lamina ◽  
Nuclear Volume ◽  
Common View ◽  
Over Expression ◽  
Peripheral Layer ◽  
Differentiated Cells ◽  
Fold Reduction ◽  
Transcriptional Output ◽  
Nuclear Compartmentalization

AbstractPackaging of the chromatin within the nucleus serves as an important factor in the regulation of transcriptional output. However, information on chromatin architecture on nuclear scale in fully differentiated cells, under physiological conditions and in live organisms, is largely unavailable. Here, we imaged nuclei and chromatin in muscle fibers of live, intact Drosophila larvae. In contrast to the common view that chromatin is distributed throughout the nuclear volume, we show that the entire chromatin, including active and repressed regions, forms a peripheral layer underneath the nuclear lamina, leaving a chromatin-devoid compartment at the nucleus center. Importantly, visualization of nuclear compartmentalization required imaging of un-fixed nuclei embedded within their intrinsic tissue environment, with preserved nuclear volume. Upon fixation of similar muscle nuclei, we observed an average of three-fold reduction in nuclear volume caused by dehydration and evidenced by nuclear flattening. In these conditions, the peripheral chromatin layer was not detected anymore, demonstrating the importance of preserving native biophysical tissue environment. We further show that nuclear compartmentalization is sensitive to the levels of lamin C, since over-expression of lamin C-GFP in muscle nuclei resulted in detachment of the peripheral chromatin layer from the lamina and its collapse into the nuclear center. Computer simulations of chromatin distribution recapitulated the peripheral chromatin organization observed experimentally, when binding of lamina associated domains (LADs) was incorporated with chromatin self-attractive interactions. Reducing the number of LADs led to collapse of the chromatin, similarly to our observations following lamin C over-expression. Taken together, our findings reveal a novel mode of mesoscale organization of chromatin within the nucleus in a live organism, in which the chromatin forms a peripheral layer separated from the nuclear interior. This architecture may be essential for robust transcriptional regulation in fully differentiated cells.

scholarly journals Live imaging of chromatin distribution reveals novel principles of nuclear architecture and chromatin compartmentalization

2021 ◽  
Vol 7 (23) ◽  
pp. eabf6251
Author(s):  
Daria Amiad-Pavlov ◽  
Dana Lorber ◽  
Gaurav Bajpai ◽  
Adriana Reuveny ◽  
Francesco Roncato ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Physical Modeling ◽  
Transcriptional Control ◽  
Three Dimensional ◽  
Nuclear Architecture ◽  
Nuclear Lamina ◽  
Nuclear Volume ◽  
Current View ◽  
Active Chromatin ◽  
Nuclear Interior

The three-dimensional organization of chromatin contributes to transcriptional control, but information about native chromatin distribution is limited. Imaging chromatin in live Drosophila larvae, with preserved nuclear volume, revealed that active and repressed chromatin separates from the nuclear interior and forms a peripheral layer underneath the nuclear lamina. This is in contrast to the current view that chromatin distributes throughout the nucleus. Furthermore, peripheral chromatin organization was observed in distinct Drosophila tissues, as well as in live human effector T lymphocytes and neutrophils. Lamin A/C up-regulation resulted in chromatin collapse toward the nuclear center and correlated with a significant reduction in the levels of active chromatin. Physical modeling suggests that binding of lamina-associated domains combined with chromatin self-attractive interactions recapitulate the experimental chromatin distribution profiles. Together, our findings reveal a novel mode of mesoscale organization of peripheral chromatin sensitive to lamina composition, which is evolutionary conserved.

scholarly journals Cytoskeletal proteins in the cell nucleus: a special nuclear actin perspective

2019 ◽  
Vol 30 (15) ◽  
pp. 1781-1785 ◽  
Author(s):  
Piergiorgio Percipalle ◽  
Maria Vartiainen
Keyword(s):  
Gene Expression ◽  
Cell Nucleus ◽  
Cell Biology ◽  
Genome Stability ◽  
Actin Polymerization ◽  
Nuclear Architecture ◽  
Nuclear Lamina ◽  
Cytoskeletal Proteins ◽  
Actin Binding ◽  
Special Focus

The emerging role of cytoskeletal proteins in the cell nucleus has become a new frontier in cell biology. Actin and actin-binding proteins regulate chromatin and gene expression, but importantly they are beginning to be essential players in genome organization. These actin-based functions contribute to genome stability and integrity while affecting DNA replication and global transcription patterns. This is likely to occur through interactions of actin with nuclear components including nuclear lamina and subnuclear organelles. An exciting future challenge is to understand how these actin-based genome-wide mechanisms may regulate development and differentiation by interfering with the mechanical properties of the cell nucleus and how regulated actin polymerization plays a role in maintaining nuclear architecture. With a special focus on actin, here we summarize how cytoskeletal proteins operate in the nucleus and how they may be important to consolidate nuclear architecture for sustained gene expression or silencing.

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 Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase

1997 ◽  
Vol 139 (5) ◽  
pp. 1077-1087 ◽  
Author(s):  
Li Yang ◽  
Tinglu Guan ◽  
Larry Gerace
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Nuclear Lamina ◽  
Normal Function ◽  
S Phase ◽  
Nuclear Volume ◽  
G1 Phase ◽  
Binding Region ◽  
Recombinant Polypeptide ◽  
Volume Increase

Lamina-associated polypeptide 2 (LAP2) is an integral membrane protein of the inner nuclear membrane that binds to both lamin B and chromatin and has a putative role in nuclear envelope (NE) organization. We found that microinjection of a recombinant polypeptide comprising the nucleoplasmic domain of rat LAP2 (residues 1–398) into metaphase HeLa cells does not affect the reassembly of transport-competent nuclei containing NEs and lamina, but strongly inhibits nuclear volume increase. This effect appears to be specifically due to lamin binding, because it also is caused by microinjection of the minimal lamin-binding region of LAP2 (residues 298–373) but not by the chromatin-binding domain (residues 1–88). Injection of the lamin-binding region of rat LAP2 into early G1 phase HeLa cells also strongly affects nuclear growth; it almost completely prevents the threefold nuclear volume increase that normally occurs during the ensuing 10 h. Moreover, injection of the fragment during early G1 phase strongly inhibits entry of cells into S phase, whereas injection during S phase has no apparent effect on ongoing DNA replication. Since the lamin-binding fragment of LAP2 most likely acts by inhibiting dynamics of the nuclear lamina, our results suggest that a normal function of LAP2 involves regulation of nuclear lamina growth. These data also suggest that lamina dynamics are required for growth of the NE and for nuclear volume increase during the cell cycle, and that progression into S phase is dependent on the acquisition of a certain nuclear volume.

Protein 4.1R Exon 16 Splicing Mediated by Regulated Expression of RBM9 and PTB during Erythroid Differentiation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 538-538
Author(s):  
Guang Yang ◽  
Shu-Ching Huang ◽  
Edward J. Benz
Keyword(s):  
Erythroid Differentiation ◽  
Cis Elements ◽  
Cell Type ◽  
Over Expression ◽  
Differentiated Cells ◽  
Regulated Expression ◽  
Cell Type Specific ◽  
Intronic Splicing Enhancer ◽  
Protein 4.1R ◽  
Splicing Enhancer

Abstract Protein 4.1R (4.1R), a vital component of the red cell membrane cytoskeleton, stabilizes the spectrin-actin lattice and attaches it to the embedded membrane proteins. The inclusion of exon 16, which encodes peptides critical for spectrin/actin binding, occurs via an intricate interplay between the auxiliary cis-elements and transacting factors. An intronic splicing enhancer, UGCAUG, is present in triplicate and is situated between two polypyrimidinetract-binding (PTB) sites, TCTT, in the intron downstream of exon 16. In addition, PTB binding sites are also present in triplicate in the upstream intron of exon 16. In this study, we characterized the splicing factors that orchestrate the erythroid differentiation stage-specific switch in exon 16 splicing through these cis-elements using two cell systems: mouse erythroleukemia cells (MELC) that can be induced to erythroid differentiation and G1E-ER cells that undergo synchronous erythroid maturation after induced GATA-1 expression. We identified two RBM9 isoforms (RBM9-1A and RBM9-1F) with distinct amino-termini that interact with the intronic splicing enhancer UGCAUG. The expression of RBM9-1A is erythroid-specific while RBM9-1F can be detected in a wide variety of cell types. Real-time PCR and Western blot analyses showed that RBM9-1A expression is significantly increased while RBM9-1F is reduced during induced erythroid differentiation in both MELC and G1E-ER4 cells. The up-regulation of RBM9-1A correlated with exon 16 inclusion in differentiated cells. Furthermore, the inhibition of RBM9 expression by isoform specific-shRNA reversed 1A enhancing activity, but not that of 1F on exon 16 inclusion in differentiated cells. Thus, exon 16 splicing is mediated by a cell type-specific RBM9 isoform and its up-regulation in late erythroid differentiation is vital for exon 16 splicing. However, over-expression of PTB completely diminished the enhancing effect of RBM9-1A on exon 16 splicing in both differentiated MELC and G1E-ER4 cells, suggesting that PTB plays a role in exon 16 splicing. We analyzed PTB expression and its effect on the exon 16 splicing switch during erythroid differentiation. PTB, a repressive regulator of alternative splicing, binds to the exon 16 upstream and downstream intronic silencers. Its over-expression reduced exon 16 inclusion in both endogenous 4.1R and transfected exon 16 minigenes. Moreover, PTB expression was down-regulated and coincided with increased exon 16 splicing during erythroid differentiation suggesting that regulated expression of repressor PTB mediates exon 16 splicing. Our results further suggest that the differentiation-specific exon 16 splicing switch is achieved by varying the amount of either ubiquitously expressed or cell-type specific activators and inhibitors, and hence the relative efficiency of spliceosome recruitment in the exon inclusion pathway.

scholarly journals Investigating the Central Dogma of Molecular Biology in the Context of Nuclear Architecture and Cell Cycle

2019 ◽  
Author(s):  
Shivnarayan Dhuppar ◽  
Aprotim Mazumder
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Nuclear Architecture ◽  
Nuclear Lamina ◽  
Gene Copy ◽  
A Cell ◽  
Cycle Dependent

AbstractNuclear architecture is the organization of the genome within a cell nucleus with respect to different nuclear landmarks such as nuclear lamina, matrix or nucleoli. Lately it has emerged as a major regulator of gene expression in mammalian cells. The studies connecting nuclear architecture with gene expression are largely population-averaged and do not report on the heterogeneity in genome organization or in gene expression within a population. In this report we present a method for combining 3D DNA Fluorescence in situ Hybridization (FISH) with single molecule RNA FISH (smFISH) and immunofluorescence to study nuclear architecture-dependent gene regulation on a cell-by-cell basis. We further combine it with an imaging-based cell cycle staging to correlate nuclear architecture with gene expression across the cell cycle. We present this in the context of Cyclin A2 (CCNA2) gene for its known cell cycle-dependent expression. We show that, across the cell cycle, the expression of a CCNA2 gene copy is stochastic and depends neither on its sub-nuclear position—which usually lies close to nuclear lamina—nor on the expression from the other copies.

Nuclear lamina and regulation of nuclear envelofe structure

1987 ◽  
Vol 45 ◽  
pp. 806-807
Author(s):  
Larry Gerace ◽  
Ueli Aebi ◽  
Brian Burke ◽  
Frank Suprynowicz
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Xenopus Oocytes ◽  
Nuclear Periphery ◽  
Nuclear Architecture ◽  
Supramolecular Assembly ◽  
Nuclear Lamina ◽  
Eukaryotic Cells ◽  
Functional Studies ◽  
Tetragonal Lattice

The nuclear lamina is a protein meshwork that lines the nucleoplasmic surface of the nuclear envelope. In numerous higher eukaryotic cells, the lamina is known to contain a polymer of 1-3 major polypeptides (“lamins“) that form an insoluble supramolecular assembly during interphase. The lamina is thought to provide both a skeletal framework for the nuclear envelope (that regulates its disassembly and reformation during mitosis) and an anchoring site at the nuclear periphery for interphase chromosomes. Recent structural and functional studies on the nuclear lamina have yielded important new insight on its roles in nuclear architecture.Using electron microscopy, we found that the nuclear lamina of Xenopus oocytes is a meshwork of intermediate-sized (8-12 nm) filaments arranged in a near-tetragonal lattice having a spacing of 52 nm.

Abstract 673: KLF10 is a Critical Mediator of Wnt Signaling in Aortic Valve Interstitial Cells

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Nalini M Rajamannan ◽  
Muzaffer Cicek ◽  
John Hawse ◽  
Thomas Spelsberg ◽  
Malayannan Subramaniam
Keyword(s):  
Wnt Signaling ◽  
Wnt Signaling Pathway ◽  
Fold Increase ◽  
Interstitial Cells ◽  
Aortic Valves ◽  
Valve Interstitial Cells ◽  
Over Expression ◽  
Differentiated Cells ◽  
Undifferentiated Cells

We have previously demonstrated that β-catenin plays important roles in valve calcification with a specific osteogenic phenotype defined by increased bone mineral content and overall valve thickening. Recent studies indicate that KLF10 may be involved in mediating the Wnt signaling pathway in bone, which is known to play critical roles in osteoblast differentiation and mineralization. Therefore, we sought to test the role of KLF10 in mediating Wnt signaling, as well as differentiation and mineralization, in valve interstitial cells (VICs) isolated from porcine valves. Exposure of VICs to differentiation media led to increased expression of Runx2, Sox9 and osteocalcin. Differentiated cells also stained positive with Von Kossa while undifferentiated cells stained negative confirming the induction of an osteogenic phenotype. As expected, over-expression of both Lef1 and β-catenin led to activation of the top-flash reporter when transfected into VICs. Interestingly, over-expression of KLF10 also significantly up-regulated the top-flash reporter alone and further enhanced the activity of both Lef1 and β-catenin when co-transfected. We further confirmed the role of TIEG in an atherosclerotic mouse model ApoE/LRP5 double KO and found a two-fold increase in KLF10, Lrp6, and Runx2 expression in the cholesterol treated aortic valves as compared to controls. These data suggested that KLF10, Lef1 and β-catenin interact with each other to form a transcriptionally active protein complex leading to enhanced Wnt signaling in VICs. This possibility was further confirmed by the observation that KLF10 and β-catenin co-localize with one another in the nucleus of VICs following stimulation with LiCl and/or TGF-β. Taken together, these data implicate an important role for KLF10 in mediating Wnt signaling and Lef1 transcriptional activity in VICs, and implicate a potential role for canonical Wnt signaling in the observed osteogenic bone phenotype of cardiac aortic valves.

scholarly journals Condensins Exert Force on Chromatin-Nuclear Envelope Tethers to Mediate Nucleoplasmic Reticulum Formation in Drosophila melanogaster

2015 ◽  
Vol 5 (3) ◽  
pp. 341-352 ◽  
Author(s):  
Julianna Bozler ◽  
Huy Q Nguyen ◽  
Gregory C Rogers ◽  
Giovanni Bosco
Keyword(s):  
Nuclear Envelope ◽  
Chromatin Condensation ◽  
Three Dimensional ◽  
Nuclear Architecture ◽  
Nuclear Lamina ◽  
Dimensional Structure ◽  
Interphase Chromosome ◽  
Chromatin Compaction ◽  
Three Dimensional Structure ◽  
Nucleoplasmic Reticulum

Abstract Although the nuclear envelope is known primarily for its role as a boundary between the nucleus and cytoplasm in eukaryotes, it plays a vital and dynamic role in many cellular processes. Studies of nuclear structure have revealed tissue-specific changes in nuclear envelope architecture, suggesting that its three-dimensional structure contributes to its functionality. Despite the importance of the nuclear envelope, the factors that regulate and maintain nuclear envelope shape remain largely unexplored. The nuclear envelope makes extensive and dynamic interactions with the underlying chromatin. Given this inexorable link between chromatin and the nuclear envelope, it is possible that local and global chromatin organization reciprocally impact nuclear envelope form and function. In this study, we use Drosophila salivary glands to show that the three-dimensional structure of the nuclear envelope can be altered with condensin II-mediated chromatin condensation. Both naturally occurring and engineered chromatin-envelope interactions are sufficient to allow chromatin compaction forces to drive distortions of the nuclear envelope. Weakening of the nuclear lamina further enhanced envelope remodeling, suggesting that envelope structure is capable of counterbalancing chromatin compaction forces. Our experiments reveal that the nucleoplasmic reticulum is born of the nuclear envelope and remains dynamic in that they can be reabsorbed into the nuclear envelope. We propose a model where inner nuclear envelope-chromatin tethers allow interphase chromosome movements to change nuclear envelope morphology. Therefore, interphase chromatin compaction may be a normal mechanism that reorganizes nuclear architecture, while under pathological conditions, such as laminopathies, compaction forces may contribute to defects in nuclear morphology.

scholarly journals Loss of a-Type Lamin Expression Compromises Nuclear Envelope Integrity Leading to Muscular Dystrophy

1999 ◽  
Vol 147 (5) ◽  
pp. 913-920 ◽  
Author(s):  
Teresa Sullivan ◽  
Diana Escalante-Alcalde ◽  
Harshida Bhatt ◽  
Miriam Anver ◽  
Narayan Bhat ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Mammalian Cells ◽  
Nuclear Architecture ◽  
Nuclear Lamina ◽  
Postnatal Growth ◽  
Developmentally Regulated ◽  
Inner Nuclear Membrane ◽  
Cardiac Muscles

The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the inner nuclear membrane and represents an important determinant of interphase nuclear architecture. Its major components are the A- and B-type lamins. Whereas B-type lamins are found in all mammalian cells, A-type lamin expression is developmentally regulated. In the mouse, A-type lamins do not appear until midway through embryonic development, suggesting that these proteins may be involved in the regulation of terminal differentiation. Here we show that mice lacking A-type lamins develop to term with no overt abnormalities. However, their postnatal growth is severely retarded and is characterized by the appearance of muscular dystrophy. This phenotype is associated with ultrastructural perturbations to the nuclear envelope. These include the mislocalization of emerin, an inner nuclear membrane protein, defects in which are implicated in Emery-Dreifuss muscular dystrophy (EDMD), one of the three major X-linked dystrophies. Mice lacking the A-type lamins exhibit tissue-specific alterations to their nuclear envelope integrity and emerin distribution. In skeletal and cardiac muscles, this is manifest as a dystrophic condition related to EDMD.

Sign in / Sign up

Export Citation Format

Share Document