Characterization of novel members of the LEM-domain containing protein family in mammalian cells

Der Zellkern, das charakteristische Merkmal eukaryotischer Zellen, wird von einer Kernhülle eingeschlossen, die aus einer doppelten Lipidmembran mit eingefügten Kernporenkomplexen und einer filamentösen Kernlamina in mehrzelligen Arten, die der inneren Kernmembran anliegt, besteht. Mehrere integrierte innere Kernmembranenproteine enthalten ein spezifisches Strukturmotiv, die sogenannte LEM-Domäne (LAP2, Emerin, MAN1), die das DNA-bindende Molekül BAF (barrier-to-autointegration factor) bindet. Mutationen in LEM Proteinen wurden mit humanen Pathologien assoziiert, die in der heterogenen Gruppe der sogenannten "Envelopathien" zusammengefaßt werden. Zwei neue LEM Proteine, LEM2 und LEM3, wurden in der vorliegenden Dissertation identifiziert und analysiert. LEM2 ist ubiquitär in Geweben und Zelltypen exprimiert und ist sowohl bezüglich der Primärsequenz als auch der Domänen-Topologie eng mit MAN1 verwandt. Komplementations-assays zeigten, daß Funktionen von LEM2 von der Hefe bis zum Menschen konserviert sind. In Säugetierzellen lokalisiert LEM2 an der inneren Kernhülle, interagiert direkt mit Lamin A/C und BAF und benötigt A-typ Lamine für die Lokalisation an der Kernmembran. Die Lamin A/C-interagierende Region wurde auf einen Teil des N-Terminus eingegrenzt, wohingegen der C Terminus ein DNA-Bindungsmotif enthält. Letzteres ist essentiell für überexprimierte LEM2-Fragmente um Komplexe an der Kernhülle zu formen, die Lamin A und Lamin A-assoziierte Proteine rekrutieren können, jedoch Lamin B und Lamin B-assoziierte Protein exkludieren. Unsere bisherigen Daten weisen auf eine Rolle von LEM2 in der räumlichen Organisation von Komplexen an der Kernhülle und in der Chromatinorganisation an der Kernperipherie hin. LEM3 wurde vor allem in hämatopoietischen Geweben gefunden, so etwa im Knochenmark, Thymus und Milz, sowie in Lymphoma. Eine Analyse der LEM3 Domänenstruktur zeigte eine Gruppe von Ankyrin-Repeats am N-terminus des Proteins sowie ein evolutionär konserviertes GIY-YIG Motif innerhalb des C-Terminus welches zuvor in verschiedenen Proteinen mit Nukleasefunktion beschrieben wurde. Desweiteren habe ich zwei LEM3 Splice-Isoformen identifiziert bei welchen Teile der LEM Domäne fehlen, wobei diese im Gegensatz zum vollständigen LEM3 BAF nicht binden können. LEM3 enthält keine Transmembran-Regionen und wurde als Kern/Zytoplasma-"Shuttling"-Protein identifiziert. In menschlichen Zellen kolokalisiert LEM3 mit cytoplasmatischen Aktin-Filamenten, während es nach pharmakologischer Inhibierung des Kernexports in nukleären "Splicing-Speckles" zu finden ist. Ektopische Expression von LEM3 führte zu einer Mislokalisation von BAF, Zellzyklusarrest und Aktivierung des ATM abhängigen DNA-Schädigungs-Signalweges. Wir postulieren eine Funktion von LEM3 in der DNA Rekombination oder im DNA Reparatur Signalweg.The nucleus in eukaryotic cells is enclosed by a nuclear envelope consisting of an inner and outer membrane, nuclear pore complexes, and in metazoans a filamentous lamina meshwork underlying the inner nuclear membrane. The lamina consists of intermediate filament-type proteins, the lamins, and numerous integral inner nuclear membrane proteins. Among these, a family of membrane proteins contain a conserved structural motif, called LEM domain (LAP2, Emerin, MAN1), which interacts with the DNA binding molecule BAF (barrier-to-autointegration factor). LEM proteins have been implicated in chromatin organization and gene expression control and have been linked to a heterogeneous group of inherited human diseases, collectively termed "envelopathies". This PhD thesis describes the identification and initial characterization of two novel LEM proteins, LEM2 and LEM3. LEM2 is ubiquitously expressed in many tissues and cell types and is closely related to MAN1 in primary sequence and domain topology. Complementation assays revealed that LEM2 is functionally conserved from yeast to man. In mammalian cells, it localizes at the inner nuclear membrane, interacts directly with A-type lamins and with BAF, and requires lamin A/C for nuclear envelope localization. The lamin A/C interaction domain was mapped to the N-terminus, while the C-terminus contains a conserved DNA binding motif. The latter was required for the ability of overexpressed LEM2 fragments to form patches at the nuclear envelope that recruit lamin A and lamin A-binding proteins, but exclude lamin B and associated proteins. Our data suggest a role of LEM2 in the spatial organization of protein complexes at the nuclear envelope and in chromatin organization at the nuclear periphery. LEM3 is primarily expressed in hematopoietic tissues such as bone marrow, thymus and spleen, and in lymphoma-derived cell lines, suggesting a B-cell related function. Analysis of LEM3 domain topology revealed a cluster of Ankyrin repeats at the N-terminus and a conserved C terminal GIY-YIG motif previously described in proteins with nuclease activity. I identified two LEM3 splice-isoforms lacking parts of the LEM domain. Unlike these isoforms full length LEM3 bound BAF. LEM3 misses a transmembrane domain and was found to shuttle between nucleoplasm and cytoplasm. In human cells ectopic LEM3 co-localizes with cytoplasmic actin filaments, while it is in nuclear splicing speckles upon pharmacological inhibition of nuclear export. Ectopic expression of LEM3 in the nucleus causes a mislocalization of BAF, cell cycle arrest, and activation of the ATM-dependent DNA damage pathway. We propose that LEM3 may be involved in DNA recombination or repair pathways

A hydrogel model of the human blood-brain barrier using differentiated stem cells

An in vitro model of the human blood-brain barrier was developed, based on a collagen hydrogel containing astrocytes, overlaid with a monolayer of endothelium, differentiated from human induced pluripotent stem cells (hiPSCs). The model was set up in transwell filters allowing sampling from apical and basal compartments. The endothelial monolayer had transendothelial electrical resistance (TEER) values >700Ω.cm2 and expressed tight-junction markers, including claudin-5. After differentiation of hiPSCs the endothelial-like cells expressed VE-cadherin (CDH5) and von-Willebrand factor (VWF) as determined by immunofluorescence. However, electron microscopy indicated that at set-up (day 8 of differentiation), the endothelial-like cells still retained some features of the stem cells, and appeared immature, in comparison with primary brain endothelium or brain endothelium in vivo. Monitoring showed that the TEER declined gradually over 10 days, and transport studies were best carried out in a time window 24-72hrs after establishment of the model. Transport studies indicated low permeability to paracellular tracers and functional activity of P-glycoprotein (ABCB1) and active transcytosis of polypeptides via the transferrin receptor (TFR1)

The blood-brain barrier is dysregulated in COVID-19 and serves as a CNS entry route for SARS-CoV-2.

Neurological complications are common in COVID-19. Although SARS-CoV-2 has been detected in patients' brain tissues, its entry routes and resulting consequences are not well understood. Here, we show a pronounced upregulation of interferon signaling pathways of the neurovascular unit in fatal COVID-19. By investigating the susceptibility of human induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (BCECs) to SARS-CoV-2 infection, we found that BCECs were infected and recapitulated transcriptional changes detected in vivo. While BCECs were not compromised in their paracellular tightness, we found SARS-CoV-2 in the basolateral compartment in transwell assays after apical infection, suggesting active replication and transcellular transport of virus across the blood-brain barrier (BBB) in vitro. Moreover, entry of SARS-CoV-2 into BCECs could be reduced by anti-spike-, anti-angiotensin-converting enzyme 2 (ACE2)-, and anti-neuropilin-1 (NRP1)-specific antibodies or the transmembrane protease serine subtype 2 (TMPRSS2) inhibitor nafamostat. Together, our data provide strong support for SARS-CoV-2 brain entry across the BBB resulting in increased interferon signaling

Study of the Museum in the 20th Century Japan

研究科: 千葉大学大学院人文社会科学研究科修了年:2011年(著者注)原本に添付した正誤表の正誤箇所は修正済みです

The GIY-YIG Type Endonuclease Ankyrin Repeat and LEM Domain-Containing Protein 1 (ANKLE1) Is Dispensable for Mouse Hematopoiesis

<div><p>Ankyrin repeat and LEM-domain containing protein 1 (ANKLE1) is a GIY-YIG endonuclease with unknown functions, mainly expressed in mouse hematopoietic tissues. To test its potential role in hematopoiesis we generated <i>Ankle1</i>-deficient mice. <i>Ankle1</i>^Δ/Δ mice are viable without any detectable phenotype in hematopoiesis. Neither hematopoietic progenitor cells, myeloid and lymphoid progenitors, nor B and T cell development in bone marrow, spleen and thymus, are affected in <i>Ankle1</i>^Δ/Δ<i>-</i>mice. Similarly embryonic stress erythropoiesis in liver and adult erythropoiesis in bone marrow and spleen appear normal. To test whether ANKLE1, like the only other known GIY-YIG endonuclease in mammals, SLX1, may contribute to Holliday junction resolution during DNA repair, <i>Ankle1</i>-deficient cells were exposed to various DNA-damage inducing agents. However, lack of <i>Ankle1</i> did not affect cell viability and, unlike depletion of <i>Slx1</i>, <i>Ankle1</i>-deficiency did not increase sister chromatid exchange in Bloom helicase-depleted cells. Altogether, we show that lack of <i>Ankle1</i> does neither affect mouse hematopoiesis nor DNA damage repair in mouse embryonic fibroblasts, indicating a redundant or non-essential function of ANKLE1 in mouse.</p></div

Flow cytometric analysis of lymphoid differentiation in 8 weeks old <i>Ankle1</i>^Δ/Δ mice.

<p>(A) Representative dot plots of B cell progenitors (B220⁺ IgM^-) and B cells (B220⁺ IgM⁺) in white blood cells (WBC) from bone marrow of an <i>Ankle1</i>^<i>+/+</i> and an <i>Ankle1</i>^Δ/Δ mouse and relative quantification of the B220⁺ IgM^- and B220⁺ IgM⁺ cells in a bar graph; (B) Representative dot plots of consecutive developmental stages of T cell progenitors in thymus of an <i>Ankle1</i>^<i>+/+</i> and an <i>Ankle1</i>^Δ/Δ mouse and its relative quantification; Relative quantification of (C) B220⁺ and (D) CD4⁺ and CD8⁺ WBCs in spleen; error bars: standard deviation, paired students t test was used for determination of statistical significant differences (p≤0.05), n = 5.</p

Flow cytometric analysis of early hematopoietic development in 9–10 months old <i>Ankle1</i>^Δ/Δ mice.

<p>(A) Representative dot plot of lineage negative bone marrow cells (BMCs) of an <i>Ankle1</i>^<i>+/+</i> and an <i>Ankle1</i>^Δ/Δ mouse and relative quantification of the KLS cells (c-Kit+ and Sca-1+, boxed) depicted in a scatter plot; (B) Representative dot plot of c-Kit+ Lin- IL7Rα- Sca-1- BMCs of an <i>Ankle1</i>^<i>+/+</i> and an <i>Ankle1</i>^Δ/Δ mouse and relative quantification of the boxed, CD16/32^low CD34⁺ common myeloid progenitors (CMPs) in a scatter plot; (C) Representative dot plot of IL7Rα⁺ Lin^- BMC of an <i>Ankle1</i>^<i>+/+</i> and an <i>Ankle1</i>^Δ/Δ mouse and relative quantification of the c-Kit⁺ Sca-1⁺, common lymphoid progenitors (CLPs) depicted in a scatter plot; scatter plots indicate the mean (n = 5), error bars: standard deviation, paired students t test was used for determination of statistical significant differences (p≤0.05).</p

<i>Ankle1</i> knock-out mouse model.

<p>(A) Schematic drawing of the <i>Ankle1</i> wild-type locus with exons 1 to 9, the knock-out allele carrying the complete deletion cassette (<i>Ankle1</i>^<i>-</i>) consisting of FRT recombination sites (semi circle), splice acceptors site (En2 SA), Internal Ribosome Entry Site (IRES), LacZ gene (<i>LacZ</i>), polyadenylation site (pA), loxP recombination site (triangle), β-actin promoter (β -act.-p.) and aminoglycoside phosphotransferase gene (neo) and the knock-out allele after CRE-recombination (<i>Ankle1</i>^Δneo) carrying the reporter gene cassette only and the knock-out allele after FLP-mediated recombination (<i>Ankle1</i>^Δ); (B) Birth statistics of different <i>Ankle1</i>-deficient mouse strains (for organization of the genomic locus see Fig 2A) from heterozygous pairings, for determination of statistical significant differences between the observed and the expected birth ratios (25% wildtype, 50% heterozygous, 25% knock-out) a Chi square test was performed, p≤0.05 statistical significant, p≤0.01 very statistical significant; (C) Weight of <i>Ankle1</i>^<i>+/+</i> and <i>Ankle1</i>^Δ/Δ animals 1–4 months and 4–8 months old, paired students t-test, statistical significance p≤0.05, 17 ≤ n ≤ 19; (D) Survival curve for <i>Ankle1</i>^<i>+/+</i> and <i>Ankle1</i>^Δ/Δ mice, log rank (Mantel-Cox) test p = 0.11, n = 18.</p

Growth and DNA damage sensitivity of <i>Ankle1</i>-deficient mouse embryonic fibroblasts (MEFs).

<p>(A) Growth curve of primary MEFs (pMEFs), n = 3, error bars: standard deviation; (B-D) Survival of pMEFs after chronic exposure to indicated DNA damaging agents for 72 h, n = 3, error bars: standard deviation; (E) Survival of pMEFs 72 h after single exposure to increasing doses of UV-light, n = 3, error bars: standard deviation; (F-I) Clonogenic survival assay of immortalized MEFs (iMEFs) after chronic exposure to indicated DNA damaging agents for 10–14 days, n = 2–3, error bars: standard error of the mean.</p

Flow cytometric analysis of erythroid differentiation in <i>Ankle1</i>^Δ/Δ mice.

<p>Cells were labeled with antibodies against Ter119 and CD71 (A) Representative dot plots of fetal liver cells from <i>Ankle1</i>^<i>+/+</i> and <i>Ankle1</i>^Δ/Δ embryos (E 13.5), regions 1–5 (R1–R5) represent five erythroid maturation stages, bar graphs show the percentage of labeled cells in each differentiation stage, n _litter = 3; Representative dot plots of (B) Bone marrow and (C) Splenic cells from an 9–10 months old <i>Ankle1</i>^<i>+/+</i> and an <i>Ankle1</i>^Δ/Δ mouse, regions 1–4 (R1–R4) represent four erythroid maturation stages, bar graphs show the percentage of labeled cells in each differentiation stage, n = 5; error bars: standard deviation, students t test was used for determination of statistical significant differences (p≤0.05).</p