Cells

937 KiB

Open AccessFeature PaperReview

Telomere Biology—Insights into an Intriguing Phenomenon

by Shriram Venkatesan, Aik Kia Khaw and Manoor Prakash Hande

Cells 2017, 6(2), 15; https://doi.org/10.3390/cells6020015 - 19 Jun 2017

Cited by 20 | Viewed by 7267

Bacteria and viruses possess circular DNA, whereas eukaryotes with typically very large DNA molecules have had to evolve into linear chromosomes to circumvent the problem of supercoiling circular DNA of that size. Consequently, such organisms possess telomeres to cap chromosome ends. Telomeres are [...] Read more.

Bacteria and viruses possess circular DNA, whereas eukaryotes with typically very large DNA molecules have had to evolve into linear chromosomes to circumvent the problem of supercoiling circular DNA of that size. Consequently, such organisms possess telomeres to cap chromosome ends. Telomeres are essentially tandem repeats of any DNA sequence that are present at the ends of chromosomes. Their biology has been an enigmatic one, involving various molecules interacting dynamically in an evolutionarily well-trimmed fashion. Telomeres range from canonical hexameric repeats in most eukaryotes to unimaginably random retrotransposons, which attach to chromosome ends and reverse-transcribe to DNA in some plants and insects. Telomeres invariably associate with specialised protein complexes that envelop it, also regulating access of the ends to legitimate enzymes involved in telomere metabolism. They also transcribe into repetitive RNA which also seems to be playing significant roles in telomere maintenance. Telomeres thus form the intersection of DNA, protein, and RNA molecules acting in concert to maintain chromosome integrity. Telomere biology is emerging to appear ever more complex than previously envisaged, with the continual discovery of more molecules and interplays at the telomeres. This review also includes a section dedicated to the history of telomere biology, and intends to target the scientific audience new to the field by rendering an understanding of the phenomenon of chromosome end protection at large, with more emphasis on the biology of human telomeres. The review provides an update on the field and mentions the questions that need to be addressed. Full article

(This article belongs to the Special Issue DNA Repair Defects and Telomere Dysfunction in Diseases)

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257 KiB

Open AccessReview

Methods for Measuring Autophagy in Mice

by Manon Moulis and Cécile Vindis

Cells 2017, 6(2), 14; https://doi.org/10.3390/cells6020014 - 8 Jun 2017

Cited by 53 | Viewed by 8982

Abstract

Autophagy is a dynamic intracellular process that mediates the degradation of damaged cytoplasmic components by the lysosome. This process plays important roles in maintaining normal cellular homeostasis and energy balance. Measuring autophagy activity is critical and although the determination of autophagic flux in [...] Read more.

Autophagy is a dynamic intracellular process that mediates the degradation of damaged cytoplasmic components by the lysosome. This process plays important roles in maintaining normal cellular homeostasis and energy balance. Measuring autophagy activity is critical and although the determination of autophagic flux in isolated cells is well documented, there is a need to have reliable and quantitative assays to evaluate autophagy in whole organisms. Because mouse models have been precious in establishing the functional significance of autophagy under physiological or pathological conditions, we present in this chapter a compendium of the current available methods to measure autophagy in mice, and discuss their advantages and limitations. Full article

(This article belongs to the Special Issue Assays to Monitor Autophagy in Model Systems)

1510 KiB

Open AccessFeature PaperReview

Activation of the EGF Receptor by Ligand Binding and Oncogenic Mutations: The “Rotation Model”

by Endang R. Purba, Ei-ichiro Saita and Ichiro N. Maruyama

Cells 2017, 6(2), 13; https://doi.org/10.3390/cells6020013 - 2 Jun 2017

Cited by 135 | Viewed by 16585

Abstract

The epidermal growth factor receptor (EGFR) plays vital roles in cellular processes including cell proliferation, survival, motility, and differentiation. The dysregulated activation of the receptor is often implicated in human cancers. EGFR is synthesized as a single-pass transmembrane protein, which consists of an [...] Read more.

The epidermal growth factor receptor (EGFR) plays vital roles in cellular processes including cell proliferation, survival, motility, and differentiation. The dysregulated activation of the receptor is often implicated in human cancers. EGFR is synthesized as a single-pass transmembrane protein, which consists of an extracellular ligand-binding domain and an intracellular kinase domain separated by a single transmembrane domain. The receptor is activated by a variety of polypeptide ligands such as epidermal growth factor and transforming growth factor α. It has long been thought that EGFR is activated by ligand-induced dimerization of the receptor monomer, which brings intracellular kinase domains into close proximity for trans-autophosphorylation. An increasing number of diverse studies, however, demonstrate that EGFR is present as a pre-formed, yet inactive, dimer prior to ligand binding. Furthermore, recent progress in structural studies has provided insight into conformational changes during the activation of a pre-formed EGFR dimer. Upon ligand binding to the extracellular domain of EGFR, its transmembrane domains rotate or twist parallel to the plane of the cell membrane, resulting in the reorientation of the intracellular kinase domain dimer from a symmetric inactive configuration to an asymmetric active form (the “rotation model”). This model is also able to explain how oncogenic mutations activate the receptor in the absence of the ligand, without assuming that the mutations induce receptor dimerization. In this review, we discuss the mechanisms underlying the ligand-induced activation of the preformed EGFR dimer, as well as how oncogenic mutations constitutively activate the receptor dimer, based on the rotation model. Full article

(This article belongs to the Special Issue Receptor Tyrosine Kinases in Health and Disease)

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Figure 1
Schematic diagrams of the domains of EGFR and oncogenic mutation sites. (a) Exons encoding the human EGFR protein (EMBL/GenBank Accession No. AF288738; NCBI Accession No. NM_005228.4). Nucleotide sequence numbers of exon boundaries are shown above the exon diagram; (b) Domain structure of EGFR. Oncogenic mutation sites are also shown below the structure. Amino acid residue numbers (a.a. residue #), including the signal peptide sequences, are also shown below the domain diagram. Mutation sites are shown using a.a. residue numbers, including the signal peptide sequences. Full article ">Figure 2
Model for the activation of EGFR by ligand binding. EGFR exists in dimeric form, stabilized through the interaction of the intracellular domains, the transmembrane domains, and the extracellular C-terminal regions of Subdomain IV. The intracelluar kinase domains with a symmetric configuration are further stabilized through interactions with the AP-2 helix. The extracellular domain of the receptor dimer adopts flexible configurations between “tethered” and “extended” forms through the interaction of Subdomains II and IV. The ligand has high affinity for the “extended” structure, and stabilizes the extracellular ligand binding domain for exposure of the “β-hairpin.” Interaction between the two “β-hairpins” in the dimer induces rotation of the transmembrane domains, which dissociates the intracellular symmetric inactive kinase dimer, resulting in an asymmetric active kinase dimer. In this asymmetric active configuration, the C-lobe of the “activator” kinase domain interacts with the N-lobe of the “receiver” kinase for the activation of the latter. Note that structures (a) and (b) indicate a tethered inactive dimer and an extended active dimer, respectively. Full article ">

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Open AccessReview

Major Tumor Suppressor and Oncogenic Non-Coding RNAs: Clinical Relevance in Lung Cancer

by Kentaro Inamura

Cells 2017, 6(2), 12; https://doi.org/10.3390/cells6020012 - 9 May 2017

Cited by 82 | Viewed by 12347

Abstract

Lung cancer is the leading cause of cancer deaths worldwide, yet there remains a lack of specific and sensitive tools for early diagnosis and targeted therapies. High-throughput sequencing techniques revealed that non-coding RNAs (ncRNAs), e.g., microRNAs and long ncRNAs (lncRNAs), represent more than [...] Read more.

Lung cancer is the leading cause of cancer deaths worldwide, yet there remains a lack of specific and sensitive tools for early diagnosis and targeted therapies. High-throughput sequencing techniques revealed that non-coding RNAs (ncRNAs), e.g., microRNAs and long ncRNAs (lncRNAs), represent more than 80% of the transcribed human genome. Emerging evidence suggests that microRNAs and lncRNAs regulate target genes and play an important role in biological processes and signaling pathways in malignancies, including lung cancer. In lung cancer, several tumor suppressor/oncogenic microRNAs and lncRNAs function as biomarkers for metastasis and prognosis, and thus may serve as therapeutic tools. In this review, recent work on microRNAs and lncRNAs is introduced and briefly summarized with a focus on potential biological and therapeutic applications. Full article

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2238 KiB

Open AccessFeature PaperReview

Taking a Bad Turn: Compromised DNA Damage Response in Leukemia

by Nadine Nilles and Birthe Fahrenkrog

Cells 2017, 6(2), 11; https://doi.org/10.3390/cells6020011 - 4 May 2017

Cited by 15 | Viewed by 7260

Abstract

Genomic integrity is of outmost importance for the survival at the cellular and the organismal level and key to human health. To ensure the integrity of their DNA, cells have evolved maintenance programs collectively known as the DNA damage response. Particularly challenging for [...] Read more.

Genomic integrity is of outmost importance for the survival at the cellular and the organismal level and key to human health. To ensure the integrity of their DNA, cells have evolved maintenance programs collectively known as the DNA damage response. Particularly challenging for genome integrity are DNA double-strand breaks (DSB) and defects in their repair are often associated with human disease, including leukemia. Defective DSB repair may not only be disease-causing, but further contribute to poor treatment outcome and poor prognosis in leukemia. Here, we review current insight into altered DSB repair mechanisms identified in leukemia. While DSB repair is somewhat compromised in all leukemic subtypes, certain key players of DSB repair are particularly targeted: DNA-dependent protein kinase (DNA-PK) and Ku70/80 in the non-homologous end-joining pathway, as well as Rad51 and breast cancer 1/2 (BRCA1/2), key players in homologous recombination. Defects in leukemia-related DSB repair may not only arise from dysfunctional repair components, but also indirectly from mutations in key regulators of gene expression and/or chromatin structure, such as p53, the Kirsten ras oncogene (K-RAS), and isocitrate dehydrogenase 1 and 2 (IDH1/2). A detailed understanding of the basis for defective DNA damage response (DDR) mechanisms for each leukemia subtype may allow to further develop new treatment methods to improve treatment outcome and prognosis for patients. Full article

(This article belongs to the Special Issue DNA Repair Defects and Telomere Dysfunction in Diseases)

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Schematic presentation of the major steps of the different double-strand break (DSB) repair pathways. (A) Non-homologous end joining starts with break recognition by the Mre11-Rad50-Nbs1 (MRN) complex and subsequent phosphorylation of and by ataxia telangiectasia mutated (ATM) to signal the break and recruit further repair components. End processing is mediated by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)/Ku70/80 complex and Artemis is recruited to prepare the DNA end for ligation, which is performed by the XRCC4-like factor (XLF)/ X-ray repair cross-complementing protein 4 (XRCC4)/DNA ligase 4 (LIG4) complex. (B) Homologous recombination (HR) equally starts with break recognition by the MRN complex and subsequent phosphorylation of and by ATM. Mre11 and other nucleases form single-strand DNA (ssDNA) overhangs that become coated by replication protein A (RPA). Strand invasion, DNA synthesis and resolution is mediated by Rad51, breast cancer 1/2 (BRCA1/2) and ligation by the DNA polymerase and Rad54. (C) Single-strand annealing starts alike HR, but after end processing Rad52 simply anneals the ssDNA ends and the non-homologous tails are cut off by excision repair 1 (ERCC1) and Xeroderma pigmentosum complementation group F protein (XPF). (D) The Fanconi anemia pathway starts with break recognition by the E3 ubiquitin core complex that ubiquitylates Fanconi anemia complementation group protein D2 (FANCD2). The FANCD2/BRCA2 complex can relocalize to the break and act together with components of the HR machinery to repair the break. SSA: single-strand annealing; NHEJ: non-homologous end joining. Full article ">Figure 2
Affected DSB repair components in leukemia. (A) Upregulated PARP1 leads to upregulation of DNA ligase III (LIG3) and thus a more active alternative NHEJ (alt-NHEJ) (black arrows). The presence of an oncogenic Kirsten ras oncogene (K-RAS) mutant leads to upregulation of PARP1 and consequently upregulated XRCC1 and LIG3 and more active alt-NHEJ (blue arrows). The presence of the fusion protein between the Rho guanine nucleotide exchange factor (RhoGEF) and GTPase activating protein BCR and the non-receptor tyrosine kinase ABL1 (BCR-ABL1) leads to a decrease of Artemis and LIG4 and consequently LIG3 is upregulated and the repair directed towards alt-NHEJ instead of NHEJ (green arrows). BCR-ABL1 or the colony stimulating factor 1 receptor (CSF1R or FMS)-like tyrosine kinase 3 internal tandem duplication (FLT3/ITD) leads to a decrease in Ku70/80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and thus decreased NHEJ (brown arrows). Increased DNA-PKcs activity due to changes in Ku70/80 leads to enhanced NHEJ activity (white arrows). A chromosome deletion affecting MRE11 expression leads to a decrease in both NHEJ and HR (gray arrows; brown arrows in C). (B) SIRT1 overexpression leads to higher Ku70/80 activity and an increase in NHEJ (blue arrows). (C) The presence of BCR-ABL1 provokes increased Rad51 levels, which result in higher HR activity (yellow arrows), whereas downregulation of BRCA1/2 leads to a decrease in HR activity (gray arrows). (D) The presence of mutant IDH1/2 or mutated ten-eleven translocation 2 (TET2) leads to reduced 5-hydroxymethylcytosine (5hmC), which indirectly affects DSB repair. Identical colors indicate that the different components are affected together or have an effect on one another. (2A–C: adapted from [<a href="#B12-cells-06-00011" class="html-bibr">12</a>]; 2D: adapted from [<a href="#B93-cells-06-00011" class="html-bibr">93</a>]). Full article ">

4932 KiB

Open AccessArticle

Distinct Fiber Type Signature in Mouse Muscles Expressing a Mutant Lamin A Responsible for Congenital Muscular Dystrophy in a Patient

by Alice Barateau, Nathalie Vadrot, Onnik Agbulut, Patrick Vicart, Sabrina Batonnet-Pichon and Brigitte Buendia

Cells 2017, 6(2), 10; https://doi.org/10.3390/cells6020010 - 24 Apr 2017

Cited by 4 | Viewed by 6787

Abstract

Specific mutations in LMNA, which encodes nuclear intermediate filament proteins lamins A/C, affect skeletal muscle tissues. Early-onset LMNA myopathies reveal different alterations of muscle fibers, including fiber type disproportion or prominent dystrophic and/or inflammatory changes. Recently, we identified the p.R388P LMNA mutation [...] Read more.

Specific mutations in LMNA, which encodes nuclear intermediate filament proteins lamins A/C, affect skeletal muscle tissues. Early-onset LMNA myopathies reveal different alterations of muscle fibers, including fiber type disproportion or prominent dystrophic and/or inflammatory changes. Recently, we identified the p.R388P LMNA mutation as responsible for congenital muscular dystrophy (L-CMD) and lipodystrophy. Here, we asked whether viral-mediated expression of mutant lamin A in murine skeletal muscles would be a pertinent model to reveal specific muscle alterations. We found that the total amount and size of muscle fibers as well as the extent of either inflammation or muscle regeneration were similar to wildtype or mutant lamin A. In contrast, the amount of fast oxidative muscle fibers containing myosin heavy chain IIA was lower upon expression of mutant lamin A, in correlation with lower expression of genes encoding transcription factors MEF2C and MyoD. These data validate this in vivo model for highlighting distinct muscle phenotypes associated with different lamin contexts. Additionally, the data suggest that alteration of muscle fiber type identity may contribute to the mechanisms underlying physiopathology of L-CMD related to R388P mutant lamin A. Full article

(This article belongs to the Collection Lamins and Laminopathies)

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Figure 1
Impact of Adeno-associated virus (AAV)-mediated lamin A (LA) expression on phenotypes of mouse Tibialis anterior (TA) muscles. (a) Western blot analysis of whole protein extracts of TA transduced with AAV-LA wild-type (WT) or mutant R388P (RP) tagged with FLAG. Ectopic lamin A (arrows) was detected using either anti-FLAG or anti-lamin A/C antibodies. Endogenous lamin A/C (brackets) was detected using anti-lamin A/C antibodies. Desmin and GAPDH were used as loading controls. (b) In situ detection of ectopic lamin A using anti-FLAG antibodies on transverse sections of TA expressing WT or mutant lamin A. Scale bar represents 20 μm. (c) Representative images show in situ detection of ectopic lamins, perlecan, and DNA, in a TA muscle section upon expression of WT lamin A. Scale bar represents 500 μm. Areas indicated by white squares are magnified in right panels, where scale bar represents 100 μm. Graph shows the mean percentage of FLAG-positive areas detected on transverse sections (n = 8–9). (d) Quantification of mouse body weight, TA muscle weight, mean number of fibers, mean minimal Feret’s diameter, and mean variance coefficient of myofiber diameter in transverse TA muscle sections upon expression of WT or mutant lamin A (n = 10–11). Full article ">Figure 2
Inflammation, regeneration, and fibrosis in mouse TA muscles expressing AAV-mediated lamin A. (a) examples of hematoxylin and eosin staining of transverse muscle sections expressing WT (upper) or R388P mutant (lower) lamin A. (b) in situ detection of A-type lamins (ectopic and endogenous; green) and perlecan (grey) on transverse muscle sections expressing WT or R388P lamin A. (c) mean percentage ± s.e.m. of myofibers with central nuclei (n = 6–7). (d) mean percentage ± s.e.m of muscle section areas showing patterns of inflammation (n = 7–8). (e) examples of Sirius Red staining of transverse muscle sections expressing WT (upper) or mutant (lower) lamin A. Scale bars represent 100 μm. Full article ">Figure 3
Fiber type distribution analyses in mouse TA muscles expressing AAV-mediated lamin A. (a) Representative image show the staining for succinate dehydrogenase (SDH) activity in a transverse sections of TA muscle upon expression of mutant lamin A. Labels of s, i, and w indicating strong, intermediate, and weak SDH staining, respectively. Scale bar represents 100 μm. (b) Mean percentage ± s.e.m of fibers with SDH activity relative to total number of fibers in a muscle section expressing WT or R388P mutant lamin A (n = 8–11 muscles). (c,d) Representative images show the staining in successive transverse sections of TA muscle upon expression of mutant lamin A for SDH and Myosin heavy chain (MHC) isoforms MHC I, MHC IIA, MHC IIX, or MHC IIB. Perlecan staining delimited individual fibers. Scale bars represent 50 μm. (e) Mean percentage ± s.e.m of fibers expressing MHC I, MHC IIA, MHC IIX, or MHC IIB relative to total number of fibers in a muscle section expressing WT or R388P mutant lamin A (n = 8–11 muscles). ** p < 0.05 (Mann–Whitney test). Full article ">Figure 4
Gene expression related to fiber type determination in TA expressing WT or R388P mutant lamin A. Graphs depict mRNA levels normalized to 18S rRNA for (a) Myh2, Myh1, and Myh4 mRNAs encoding myosin heavy chains isoforms MHC IIA, MHC IIX, and MHC IIB, respectively; (b) Mef2c, Mef2d, and Myog mRNAs encoding key muscle transcription factors MEF2C, MEF2d and Myogenin, respectively; (c) Hdac1, Hdac9, and Sirt1 mRNAs encoding histone deacetylases HDAC 1, HDAC 9 and SIRT1, respectively; (d) MyoD, Six1, and Eya1 mRNAs encoding Myod and related co-factors SIX1 and Eya1, respectively; and (e) Ppargc1a and Dtna mRNAs encoding two proteins related to muscular atrophy and dystrophy, PGC1α and Dystrobrevin α, respectively. Data represent mean ± s.e.m (n = 12 for WT lamin A; n = 10 for mutant lamin A). * p = 0.05; ** p < 0.05. Full article ">Figure 4 Cont.
Gene expression related to fiber type determination in TA expressing WT or R388P mutant lamin A. Graphs depict mRNA levels normalized to 18S rRNA for (a) Myh2, Myh1, and Myh4 mRNAs encoding myosin heavy chains isoforms MHC IIA, MHC IIX, and MHC IIB, respectively; (b) Mef2c, Mef2d, and Myog mRNAs encoding key muscle transcription factors MEF2C, MEF2d and Myogenin, respectively; (c) Hdac1, Hdac9, and Sirt1 mRNAs encoding histone deacetylases HDAC 1, HDAC 9 and SIRT1, respectively; (d) MyoD, Six1, and Eya1 mRNAs encoding Myod and related co-factors SIX1 and Eya1, respectively; and (e) Ppargc1a and Dtna mRNAs encoding two proteins related to muscular atrophy and dystrophy, PGC1α and Dystrobrevin α, respectively. Data represent mean ± s.e.m (n = 12 for WT lamin A; n = 10 for mutant lamin A). * p = 0.05; ** p < 0.05. Full article ">Figure 5
Expression pathways for genes encoding key factors related to Myh2 and MHC IIA expression that are induced by expression of mutant lamin A in mouse TA. Bold, italic, green text indicates no change in mRNA expression; bold, italic, red text indicates reduced mRNA expression. Dashed lines indicate hypothetical altered pathways that decrease levels of Myh2 mRNA and MHC IIA. Full article ">

16850 KiB

Open AccessArticle

Microinjection of Antibodies Targeting the Lamin A/C Histone-Binding Site Blocks Mitotic Entry and Reveals Separate Chromatin Interactions with HP1, CenpB and PML

by Charles R. Dixon, Melpomeni Platani, Alexandr A. Makarov and Eric C. Schirmer

Cells 2017, 6(2), 9; https://doi.org/10.3390/cells6020009 - 25 Mar 2017

Cited by 9 | Viewed by 7700

Abstract

Lamins form a scaffold lining the nucleus that binds chromatin and contributes to spatial genome organization; however, due to the many other functions of lamins, studies knocking out or altering the lamin polymer cannot clearly distinguish between direct and indirect effects. To overcome [...] Read more.

Lamins form a scaffold lining the nucleus that binds chromatin and contributes to spatial genome organization; however, due to the many other functions of lamins, studies knocking out or altering the lamin polymer cannot clearly distinguish between direct and indirect effects. To overcome this obstacle, we specifically targeted the mapped histone-binding site of A/C lamins by microinjecting antibodies specific to this region predicting that this would make the genome more mobile. No increase in chromatin mobility was observed; however, interestingly, injected cells failed to go through mitosis, while control antibody-injected cells did. This effect was not due to crosslinking of the lamin polymer, as Fab fragments also blocked mitosis. The lack of genome mobility suggested other lamin-chromatin interactions. To determine what these might be, mini-lamin A constructs were expressed with or without the histone-binding site that assembled into independent intranuclear structures. HP1, CenpB and PML proteins accumulated at these structures for both constructs, indicating that other sites supporting chromatin interactions exist on lamin A. Together, these results indicate that lamin A-chromatin interactions are highly redundant and more diverse than generally acknowledged and highlight the importance of trying to experimentally separate their individual functions. Full article

(This article belongs to the Collection Lamins and Laminopathies)

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Lamin A/C histone binding site antibodies are specific, target in vivo and block H2A binding in vitro. (a) Schematic of lamin A domain structure highlighting the head, rod and tail domains, as well as the mapped chromatin binding site (HBS; Taniura et al., 1995). (b) Western blot on total HeLa cell lysates using a pan-lamin antibody and each histone-binding site antibody (A/C, B1 and B2) revealed that all antibodies prepared against the chromatin binding sites of lamins were each highly specific for each subtype. (c) The affinity-purified antibodies were conjugated to an SV40 NLS peptide and microinjected into either the nuclei or cytoplasm of U2OS cells. In both cases, subsequent fixation and visualization with fluorophore-conjugated secondary antibodies revealed nuclear rim staining, consistent with their binding the expected target on lamin A in the polymer underlying the inner nuclear membrane. (d) To test for antibody blocking of histone binding, lamin A∆rod protein was coupled to beads, incubated with either no antibodies (Lane 2), antibodies generated against BSA (Lane 3) or the lamin A histone-binding site antibodies (Lane 4). Uncoupled beads were also tested as a control for background binding to the beads because histones tend to be sticky (Lane 1). After washing, each was incubated with histones, eluted with SDS and analysed for the amount of bound histones by Western blot. Three technical replicates were quantified for fluorescence intensity using a LiCor Odyssey and plotted (n = 3). Standard deviations are shown, paired t-test shows a significant reduction in H2A bound by lamin A∆rod in the presence of the histone-binding site antibody compared to the BSA control. Scale bars, 10 µm. Full article ">Figure 2
Microinjection of lamin A/C chromatin binding site antibodies does not increase the mobility of centromeres. (a) The affinity-purified antibodies were conjugated to an SV40 NLS peptide and microinjected into either the nuclei or cytoplasm of U2OS cell stably expressing CenpB-GFP. With images taken every 10 min the positions of centromeres were essentially unchanged after 2 h. (b) Movies covering a period of 10 min with images taken every 1 min were analysed using Imaris 8 tracking software. The data for mobility were plotted using a Tukey box-plot with the median represented by a line and the mean by an x. The p-value from a Kolmogorov–Smirnov test is given above. (c) The same movies were also analysed after tracking to determine the two most distal points in the area covered by the centre of the CenpB-GFP signal. The data for distance were plotted using a Tukey box-plot with the median represented by a line and the mean by an x. The p-value from a Kolmogorov–Smirnov test is given above. No significant differences were observed between histone-binding site antibody injected and non-injected cells. (d) Tracking of the movement of the CenpB-GFP centromere spots was followed for each frame in the 10-min movies. The position of centromeres at the start of the movie is shown in the first panel, while the middle panel shows the final position with the tracking for the centre of the GFP signal generated using Imaris 8 software. Centromeres were false coloured in red to make it easier to visualize the tracking, which is colour-coded for time from blue to green to yellow and then to red and terminating at the white dot at the end of the movies (see the colour bar at the bottom of the figure). In the far right panel, an enlargement from the middle image is shown. (e) The same is shown for a non-injected cell. Scale bars, 10 µm. Full article ">Figure 3
The lack of a mobility effect upon microinjection of lamin A/C histone-binding site antibodies is observed for both euchromatic and heterochromatic regions. (a) An HT1080 cell line with a 254 copy LacO array integrated into a euchromatic region of chromosome 5 that tended to be in the nuclear interior was microinjected with lamin A/C histone-binding site antibodies. At 3 h post-injection, the cell was imaged live for 70 min with Z-stacks taken every 10 min. For comparison, a non-injected cell in the same field is shown in the bottom panels. No visual increase in mobility was observed in this or any of several other injected cells observed. (b) A similar array integrated into a heterochromatic region of HT1080 chromosome 13 that tended to be at the nuclear periphery was similarly analysed, except that imaging was started at 1 h post-injection and continued for 3 h. Arrowheads indicate the LacO array in the bottom movie, as the spot is more difficult to distinguish in this movie. Again, no difference was observed between the injected and non-injected cells, and very little mobility was observed at all. Scale bars, 10 µm. Full article ">Figure 4
Microinjected lamin A/C HBS antibodies blocked mitotic entry and delayed DNA replication. (a–c) Synchronized cells were injected within 1 h from release at G1/S with HBS or control antibodies conjugated to an SV40 NLS peptide using fluorescent dextran to identify injected cells. (a) Control antibody injected cells (αBSA) visualized from 6 h post-G1/S release (5 h post-injection). Images at times shown follow cells through mitosis with arrows. The control-injected cells went through mitosis at similar rates as non-injected cells in the same fields (c). (b) Lamin A/C HBS antibody-injected cells were followed longer as no cells entered mitosis in the timeframe of controls. (c) Numbers of cells followed for each condition, including also cells injected with Fab fragments of the lamin A/C HBS antibodies. Scale bars, 10 µm. (d) HBS antibody effects on DNA replication. HeLa nuclei were microinjected within 30 min after G1/S release and pulsed with BrdU starting at 3 h post-release for 1 h. The percentage of BrdU-positive cells at 4 and 6 h post-G1/S release for each condition is given in the table. To ensure that microinjection itself did not affect DNA replication, control antibodies (BSA-NLS) were injected into a parallel culture. A representative image of an injected, BrdU-labelled cell is shown. Full article ">Figure 5
A lamin A mutant generated to increase the density of histone-binding sites assembles intranuclear structures independent of the peripheral lamina. (a) The lamin A/C rod domain is similar in mass to the globular tail domain of lamin A that contains the mapped lamin A/C histone-binding site. Thus, in A∆rod, most of the rod was deleted to roughly double the mass density of histone-binding sites. As a negative control, A∆rod∆hbs also deleted the major mapped histone-binding site. (b) When A∆rod was expressed in U2OS cells, it formed circular structures both in the nucleus and in the cytoplasm. In most cells, these structures neither integrated with nor perturbed the peripheral lamin structural network, as indicated by co-staining for the lamin A∆rod mutants and for endogenous lamins with a pan-lamin antibody or an antibody to the similar region in lamin B2. (c) Expressing the lamin A constructs in COS-7 cells to match the earlier B1∆rod experiments, some A∆rod and A∆rod∆hbs structures were formed by 20 h post-transfection and most by 40 h. (d,e) 3D reconstructions of cells, produced from serial z-sections (0.2-μm step size) after deconvolution using Imaris 8 software revealed that the A∆ structures tend to be completely internal without connecting to the membrane. (f) Co-staining with the membrane dye DiOC6 further confirmed that the internal structures were free from membrane, and thus, results could not be attributed to nuclear membrane proteins. All scale bars, 10 µm. Full article ">Figure 6
General chromatin effects of A∆rod and A∆rod∆hbs. (a) DNA staining (DAPI) in U2OS cells expressing A∆rod. Chromatin staining tended to be stronger on or around lamin A∆rod structures. (b) pRb is a known binding partner of lamin A, and so, it was co-stained as a positive control, revealing strong co-localization with the A∆ structures. (c) No co-localization between A∆rod structures and 53BP1 in HeLa cells indicates that chromatin is not damaged by the structures. (d) Similarly, γH2AX did not accumulate on A∆ structures. Full article ">Figure 6 Cont.
General chromatin effects of A∆rod and A∆rod∆hbs. (a) DNA staining (DAPI) in U2OS cells expressing A∆rod. Chromatin staining tended to be stronger on or around lamin A∆rod structures. (b) pRb is a known binding partner of lamin A, and so, it was co-stained as a positive control, revealing strong co-localization with the A∆ structures. (c) No co-localization between A∆rod structures and 53BP1 in HeLa cells indicates that chromatin is not damaged by the structures. (d) Similarly, γH2AX did not accumulate on A∆ structures. Full article ">Figure 7
Heterochromatin effects of A∆rod and A∆rod∆hbs. (a) H3K9me2 staining in lamin A∆rod expressing U2OS cells. No notable accumulation of these marks was observed on the A∆rod structures. In fact, the greater concentrations of the epigenetic mark tended to occur in regions devoid of the A∆rod structures. (b) H3K9me3 staining in lamin A∆rod and A∆rod∆hbs expressing U2OS cells. Clear accumulations of histones carrying this mark were observed around the structures formed by the A∆ proteins. (c) HP1 staining revealed concentrations that co-localized with the A∆rod structures in U2OS cells. To test if this was specific to the mapped histone-binding site, cells expressing A∆rod∆hbs were also stained for HP1. The interaction appears to be from a distinct region in the mini-lamins because a similar co-localization was observed. This co-localization with the A∆rod∆hbs structures was observed in both U2OS (upper panels) and HeLa (lower panels) cells. Deconvolved images are shown in this panel to better clarify the co-localization. All scale bars, 10 µm. Full article ">Figure 7 Cont.
Heterochromatin effects of A∆rod and A∆rod∆hbs. (a) H3K9me2 staining in lamin A∆rod expressing U2OS cells. No notable accumulation of these marks was observed on the A∆rod structures. In fact, the greater concentrations of the epigenetic mark tended to occur in regions devoid of the A∆rod structures. (b) H3K9me3 staining in lamin A∆rod and A∆rod∆hbs expressing U2OS cells. Clear accumulations of histones carrying this mark were observed around the structures formed by the A∆ proteins. (c) HP1 staining revealed concentrations that co-localized with the A∆rod structures in U2OS cells. To test if this was specific to the mapped histone-binding site, cells expressing A∆rod∆hbs were also stained for HP1. The interaction appears to be from a distinct region in the mini-lamins because a similar co-localization was observed. This co-localization with the A∆rod∆hbs structures was observed in both U2OS (upper panels) and HeLa (lower panels) cells. Deconvolved images are shown in this panel to better clarify the co-localization. All scale bars, 10 µm. Full article ">Figure 8
Potential centromere interaction with lamin A. (a) U2OS cells stably expressing CenpB-GFP were transfected with A∆rod and imaged. The CenpB-GFP protein accumulated around A∆rod structures both in association with centromere spots and separately, notably at the nuclear periphery. (b) To determine if aberrant distributions of the CenpB-GFP also affected endogenous centromere proteins, cells were co-stained with an antibody to CenpC. The CenpC did not display a similar aberrant distribution at the nuclear periphery, but in some cases where CenpB-GFP spots were distended, a similar distension in the CenpC staining pattern was observed. Arrows point to such a distended centromere spot, and both individual and double and triple merged images are shown. (c) A similar redistribution of CenpB-GFP is observed in cells expressing A∆rod∆hbs. Notably, the CenpB-GFP signal could typically be observed within the circular structures formed by A∆rod∆hbs. (d) To test whether the indicated interaction is an artefact of the exogenously-expressed GFP fusion protein, HeLa cells expressing A∆rod∆hbs and not expressing the GFP protein were stained for endogenous CenpB. Considerable co-localization was observed in roughly half of cells examined, while very little co-localization was observed in the other half. (e) HeLa cells expressing A∆rod∆hbs were also stained for CenpA with similar results. All scale bars, 10 µm. Full article ">Figure 9
Potential PML interaction with lamin A. (a) HeLa cells expressing A∆rod and A∆rod∆hbs were stained with an antibody for PML. Nearly all PML foci were associated with the mini-lamin structures. Scale bar, 10 µm. (b) Numbers of observed PML foci increase in cells expressing A∆rod∆hbs. Left panels, untransfected cells exhibited relatively few PML foci. Middle panel, co-localization between A∆rod∆hbs and PML was observed even at early time points before the larger circular structures had formed. Right panel, later cells with fully-formed intranuclear A∆rod∆hbs circular structures exhibited both co-localization with PML and a considerable increase in the number of PML foci. Scale bars, 10 µm. (c) PML foci were often distended on the mini-lamin A structures. Left panels show an A∆rod∆hbs expressing cell with two more distended PML foci indicated by arrows that curve around the A∆rod∆hbs circular structures instead of appearing as a normal spot. Right panels show that, in HeLa cells co-expressing A∆rod and PML fused to CFP, extremely long distended PML structures (arrows) were observed, even in cells without significant development of the A∆rod circular structures. Scale bars, 10 µm. Full article ">

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Cells, Volume 6, Issue 2 (June 2017) – 7 articles

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