NMD is required for timely cell fate transitions by fine-tuning gene expression and regulating translation.

Cell fate transitions depend on balanced rewiring of transcription and translation programs to mediate ordered developmental progression. Components of the nonsense-mediated mRNA decay (NMD) pathway have been implicated in regulating embryonic stem cell (ESC) differentiation, but the exact mechanism is unclear. Here we show that NMD controls expression levels of the translation initiation factor Eif4a2 and its premature termination codon-encoding isoform (Eif4a2PTC ). NMD deficiency leads to translation of the truncated eIF4A2PTC protein. eIF4A2PTC elicits increased mTORC1 activity and translation rates and causes differentiation delays. This establishes a previously unknown feedback loop between NMD and translation initiation. Furthermore, our results show a clear hierarchy in the severity of target deregulation and differentiation phenotypes between NMD effector KOs (Smg5 KO > Smg6 KO > Smg7 KO), which highlights heterodimer-independent functions for SMG5 and SMG7. Together, our findings expose an intricate link between mRNA homeostasis and mTORC1 activity that must be maintained for normal dynamics of cell state transitions.

Nucleolus and chromatin.

The nucleolus as site of ribosome biogenesis holds a pivotal role in cell metabolism. It is composed of ribosomal DNA (rDNA), which is present as tandem arrays located in nucleolus organizer regions (NORs). In interphase cells, rDNA can be found inside and adjacent to nucleoli and the location is indicative for transcriptional activity of ribosomal genes-inactive rDNA (outside) versus active one (inside). Moreover, the nucleolus itself acts as a spatial organizer of non-nucleolar chromatin. Microscopy-based approaches offer the possibility to explore the spatially distinct localization of the different DNA populations in relation to the nucleolar structure. Recent technical developments in microscopy and preparatory methods may further our understanding of the functional architecture of nucleoli. This review will attempt to summarize the current understanding of mammalian nucleolar chromatin organization as seen from a microscopist's perspective.

Morphology of nuclear transcription.

Gene expression control is a fundamental determinant of cellular life with transcription being the most important step. The spatial nuclear arrangement of the transcription process driven by RNA polymerases II and III is nonrandomly organized in foci, which is believed to add another regulatory layer on gene expression control. RNA polymerase I transcription takes place within a specialized organelle, the nucleolus. Transcription of ribosomal RNA directly responds to metabolic requirements, which in turn is reflected in the architecture of nucleoli. It differs from that of the other polymerases with respect to the gene template organization, transcription rate, and epigenetic expression control, whereas other features are shared like the formation of DNA loops bringing genes and components of the transcription machinery in close proximity. In recent years, significant advances have been made in the understanding of the structural prerequisites of nuclear transcription, of the arrangement in the nuclear volume, and of the dynamics of these entities. Here, we compare ribosomal RNA and mRNA transcription side by side and review the current understanding focusing on structural aspects of transcription foci, of their constituents, and of the dynamical behavior of these components with respect to foci formation, disassembly, and cell cycle.

Senescence-associated ß-galactosidase staining over the lifespan differs in a short- and a long-lived fish species.

During the aging process, cells can enter cellular senescence, a state in which cells leave the cell cycle but remain viable. This mechanism is thought to protect tissues from propagation of damaged cells and the number of senescent cells has been shown to increase with age. The speed of aging determines the lifespan of a species and it varies significantly in different species. To assess the progress of cellular senescence during lifetime, we performed a comparative longitudinal study using histochemical detection of the senescence-associated beta-galactosidase as senescence marker to map the staining patterns in organs of the long-lived zebrafish and the short-lived turquoise killifish using light- and electron microscopy. We compared age stages corresponding to human stages of newborn, childhood, adolescence, adult and old age. We found tissue-specific but conserved signal patterns with respect to organ distribution. However, we found dramatic differences in the onset of tissue staining. The stained zebrafish organs show little to no signal at newborn age followed by a gradual increase in signal intensity, whereas the organs of the short-lived killifish show an early onset of staining already at newborn stage, which remains conspicuous at all age stages. The most prominent signal was found in liver, intestine, kidney and heart, with the latter showing the most prominent interspecies divergence in onset of staining and in staining intensity. In addition, we found staining predominantly in epithelial cells, some of which are post-mitotic, such as the intestinal epithelial lining. We hypothesize that the association of the strong and early-onset signal pattern in the short-lived killifish is consistent with a protective mechanism in a fast growing species. Furthermore, we believe that staining in post-mitotic cells may play a role in maintaining tissue integrity, suggesting different roles for cellular senescence during life.

CX-5461 causes nucleolar compaction, alteration of peri- and intranucleolar chromatin arrangement, an increase in both heterochromatin and DNA damage response.

In this study, we characterize the changes in nucleolar morphology and its dynamics induced by the recently introduced compound CX-5461, an inhibitor of ribosome synthesis. Time-lapse imaging, immunofluorescence and ultrastructural analysis revealed that exposure of cells to CX-5461 has a profound impact on their nucleolar morphology and function: nucleoli acquired a compact, spherical shape and display enlarged, ring-like masses of perinucleolar condensed chromatin. Tunnels consisting of chromatin developed as transient structures running through nucleoli. Nucleolar components involved in rRNA transcription, fibrillar centres and dense fibrillar component with their major constituents ribosomal DNA, RNA polymerase I and fibrillarin maintain their topological arrangement but become reduced in number and move towards the nucleolar periphery. Nucleolar changes are paralleled by an increased amount of the DNA damage response indicator γH2AX and DNA unwinding enzyme topoisomerase I in nucleoli and the perinucleolar area suggesting that CX-5461 induces torsional stress and DNA damage in rDNA. This is corroborated by the irreversibility of the observed altered nucleolar phenotypes. We demonstrate that incubation with CX-5461, apart from leading to specific morphological alterations, increases senescence and decreases cell replication. We discuss that these alterations differ from those observed with other drugs interfering with nucleolar functions.

Chromosomal dynamics of cell cycle regulator gene p21 during transcriptional activation.

The radial position of a gene within its chromosome territory (CT) in the interphase nucleus is thought to depend on the transcriptional activity of the gene and on transcriptional activity, gene density, and conformation of the chromosomal surrounding. In this study we analyzed the position of the cell cycle regulator gene p21 within the CT of human chromosome 6 (HSA6) upon transcriptional activation. Whereas the majority of active p21 genes is located in the interior of the CT of HSA6, induction of p21 transcription correlates with increased variation of gene localization within the CT and with a higher percentage of p21 genes located at the periphery of the CT. Additionally it demonstrates once more that transcription can take place throughout CTs. Comparison of the p21 locus with two non-coding regions on HSA6 showed that both non-coding sequences are located more frequently in the interior of the CT than p21 genes although they are situated in chromosomal neighborhoods with widely differing gene density and regional transcriptional activity. Thus our data support models describing an influence of the transcriptional activity of a gene on the localization within its CT. However, our data also indicate that additional factors such as chromatin remodeling are implicated in the positioning of genes within the respective chromosome territory.

Immunohistochemical expression analysis of Cx43, Cx26, c-KIT and PlAP in contralateral testis biopsies of patients with non-seminomatous testicular germ cell tumor.

Non seminomatous testicular germ cell tumors (NSTGCTs) express fetal stem cell markers and display dysregulation of connexin 43 expression. Persistence of fetal spermatogonial characteristics was implicated in the emergence of testicular germ cell tumors. The objective of this study was to analyze the tubular architecture in contralateral testes of patients with NSTGCT. We studied morphologic alterations, expression patterns of markers for the integrity of the germinal epithelium (gap junction proteins connexin 43 and 26), as well as of the embryonic markers c-KIT and placental alkaline phosphatase (PlAP), both established markers to detect carcinoma in situ (CIS). In all samples, tubules showing maturation of germ cells up to spermatozoa were observed. In addition, tubules with alterations in tubular architecture and with impaired spermatogenesis occurred. In tubules showing aberrant spermatogenesis, connexin 43 (Cx43) signal was down-regulated and a shift of signal from gap junctions to the cytoplasm occurred. Concomitantly, Cx26 was found highly up-regulated in tubules with incomplete and aberrant germ cell maturation. All testes exhibited single spermatogonia with positive reaction for c-KIT and a significant positive correlation was found between the mean number of c-KIT positive spermatogonia per tubule and the percentage of tubules presenting severely impaired spermatogenesis. Our data show alterations of the normal architecture of the germinal epithelium and disturbances of spermatogenesis in the contralateral testes of patients with NSTGCT in all cases evaluated. The concomitant occurrence of c-KIT positive spermatogonia and defects in tubular architecture is in line with the hypothesis that patients with NSTGCT suffer from disturbed germ cell development.

c-erbB-3: a nuclear protein in mammary epithelial cells.

c-erbB receptors are usually located in cell membranes and are activated by extracellular binding of EGF-like growth factors. Unexpectedly, using immunofluorescence we found high levels of c-erbB-3 within the nuclei of MTSV1-7 immortalized nonmalignant human mammary epithelial cells. Nuclear localization was mediated by the COOH terminus of c-erbB-3, and a nuclear localization signal was identified by site-directed mutagenesis and by transfer of the signal to chicken pyruvate kinase. A nuclear export inhibitor caused accumulation of c-erbB-3 in the nuclei of other mammary epithelial cell lines as demonstrated by immunofluorescence and biochemical cell fractionation, suggesting that c-erbB-3 shuttles between nuclear and nonnuclear compartments in these cells. Growth of MTSV1-7 on permeable filters induced epithelial polarity and concentration of c-erbB-3 within the nucleoli. However, the c-erbB-3 ligand heregulin beta1 shifted c-erbB-3 from the nucleolus into the nucleoplasm and then into the cytoplasm. The subcellular localization of c-erbB-3 obviously depends on exogenous stimuli and on the stage of epithelial polarity and challenges the specific function of c-erbB-3 as a transmembrane receptor protein arguing for additional, as yet unidentified, roles of c-erbB-3 within the nucle(ol)us of mammary epithelial cells.

Gene dynamics and nuclear architecture during differentiation.

Recent advances have demonstrated that placing genes in a specific nuclear context plays an important role in the regulation of coordinated gene expression, thus adding an additional level of complexity to the mechanisms of gene regulation. Differentiation processes are characterized by dynamic changes in gene activation and silencing. These alterations are often accompanied by gene relocations in relation to other genomic regions or to nuclear compartments. Unraveling of mechanisms and dynamics of chromatin positioning will thus expand our knowledge about cellular differentiation.

LEM4/ANKLE-2 deficiency impairs post-mitotic re-localization of BAF, LAP2α and LaminA to the nucleus, causes nuclear envelope instability in telophase and leads to hyperploidy in HeLa cells.

The human LEM-domain protein family is involved in fundamental aspects of nuclear biology. The LEM-domain interacts with the barrier-to-autointegration factor (BAF), which itself binds DNA. LEM-domain proteins LAP2, emerin and MAN1 are proteins of the inner nuclear membrane; they have important functions: maintaining the integrity of the nuclear lamina and regulating gene expression at the nuclear periphery. LEM4/ANKLE-2 has been proposed to participate in nuclear envelope reassembly after mitosis and to mediate dephosphorylation of BAF through binding to phosphatase PP2A. Here, we used CRISPR/Cas9 to create several cell lines deficient in LEM4/ANKLE-2. By using time-lapse video microscopy, we show that absence of this protein severely compromises the post mitotic re-association of the nuclear proteins BAF, LAP2α and LaminA to chromosomes. These defects give rise to a strong mechanical instability of the nuclear envelope in telophase and to a chromosomal instability leading to increased number of hyperploid cells. Reintroducing LEM4/ANKLE-2 in the cells by transfection could efficiently restore the telophase association of BAF and LAP2α to the chromosomes. This rescue phenotype was abolished for N- or C-terminally truncated mutants that had lost the capacity to bind PP2A. We demonstrate also that, in addition to binding to PP2A, LEM4/ANKLE-2 binds BAF through its LEM-domain, providing further evidence for a generic function of this domain as a principal interactor of BAF.

GJB2 mutations in hearing impairment: identification of a broad clinical spectrum for improved genetic counseling.

OBJECTIVES/HYPOTHESIS: Hearing impairment has a high prevalence affecting approximately 1 in 1000 newborn children. Alterations in the gap junction protein beta 2 (GJB2) and gap junction protein beta 6 (GJB6) are associated with nonsyndromic hearing impairment and should have a significant impact on genetic counseling. STUDY DESIGN: Various cases of nonsyndromic hearing impairment were screened for alterations in GJB2 and GJB6 in this clinical study. METHODS: The prevalence of mutations in GJB2 encoding for connexin 26 in a patient group with nonsyndromic hearing impairment comprising 45 families and 57 sporadic cases was initially determined by sequencing. The role of GJB2 was then assessed in individuals with hearing impairment (3 families and 20 sporadic cases) who are usually excluded from analysis because of the presence of additional symptoms or in cases in which a role for nongenetic factors cannot be eliminated. In hearing-impaired individuals with heterozygous GJB2 mutations the recently identified 342-kb deletion truncating GJB6 called del(GJB6-D13S1830) as a digenetic component in hearing impairment was excluded by polymerase chain reaction. RESULTS: Autosomal recessively inherited GJB2 mutations induced hearing impairment in 25.5% of individuals in the nonsyndromic hearing impairment group. GJB2 alterations were also seen in 17.4% of individuals in whom additional symptoms or a role for nongenetic involvement could not be excluded. In all, 15 different alterations in GJB2 were detected, including the previously unknown 154G>C, 557C>T, and 682C>T mutations, and these were correlated to clinical parameters. CONCLUSION: Improved genetic counseling can be performed by screening for GJB2 alterations in patients with nonsyndromic hearing impairment including patients within groups for which a role for exogenetic factors cannot be excluded. Specific genetic counseling for GJB2-linked hearing impairment in heterozygotes will depend on future research.

Connexin 26 mutations in cases of sensorineural deafness in eastern Austria.

Mutations in the connexin 26 (Cx26) gene (GJB2) are associated with autosomal nonsyndromic sensorineural hearing loss. This study describes mutations in the Cx26 gene in cases of familial and sporadic hearing loss (HL) by gene sequencing and identifies the allelic frequency of the most common mutation leading to HL (35delG) in the population of eastern Austria. For this purpose we have developed and applied a molecular beacon based real-time mutation detection assay. Mutation frequencies in the Cx26 gene of individuals from affected families (14 out of 46) and sporadic cases (11 out of 40) were 30.4% and 27.5%, respectively. In addition to known disease related alterations, a novel mutation 262 G-->T (A88S) was also identified. 35delG accounted for almost 77% of all Cx26 mutations detected and displayed an allelic frequency in the normal hearing population of 1.7% (2 out of 120). The high prevalence of the 35delG mutation in eastern Austria would therefore allow screening of individuals and family members with Cx26 dependent deafness by a highly specific and semi-automated method.

Distinct chromatin signature of histone H3 variant H3.3 in human cells.

Actively transcribed regions of the genome have been found enriched for the histone H3 variant H3.3. This variant is incorporated into nucleosomes throughout the cell cycle whereas the canonical isoforms are predominately deposited in association with replication. In order to obtain a global picture of the deposition pattern at the single cell level we expressed H3.3 in both normal and malignant human cells and analyzed nuclei using conventional and structured illumination imaging (SIM). We found that the distribution pattern of H3.3 in interphase differs from that of the canonical histone H3 variants and this difference is conveyed to mitotic chromosomes which display a distinct H3.3 banding pattern. Histone H3.3 localization positively correlated with markers for transcriptionally active chromatin and, notably, H3.3 was almost completely absent from the inactive X chromosome. Collectively, our data show that histone variant H3.3 occupies distinct intranuclear chromatin domains and that these genomic loci are associated with gene expression.

Expression patterns of protein C inhibitor in mouse development.

Proteolysis of extracellular matrix is an important requirement for embryonic development and is instrumental in processes such as morphogenesis, angiogenesis, and cell migration. Efficient remodeling requires controlled spatio-temporal expression of both the proteases and their inhibitors. Protein C inhibitor (PCI) effectively blocks a range of serine proteases, and recently has been suggested to play a role in cell differentiation and angiogenesis. In this study, we mapped the expression pattern of PCI throughout mouse development using in situ hybridization and immunohistochemistry. We detected a wide-spread, yet distinct expression pattern with prominent PCI levels in skin including vibrissae, and in fore- and hindgut. Further sites of PCI expression were choroid plexus of brain ventricles, heart, skeletal muscles, urogenital tract, and cartilages. A strong and stage-dependent PCI expression was observed in the developing lung. In the pseudoglandular stage, PCI expression was present in distal branching tubules whereas proximal tubules did not express PCI. Later in development, in the saccular stage, PCI expression was restricted to distal bronchioli whereas sacculi did not express PCI. PCI expression declined in postnatal stages and was not detected in adult lungs. In general, embryonic PCI expression indicates multifunctional roles of PCI during mouse development. The expression pattern of PCI during lung development suggests its possible involvement in lung morphogenesis and angiogenesis.

High incidence of GJB2 mutations during screening of newborns for hearing loss in Austria.

OBJECTIVES: The aim of the present study was to evaluate gap junction protein beta2 (GJB2) genetic testing within a national neonate screening program for hearing loss (HL) in a European population. DESIGN: Neonatal cases of nonsyndromic HL (N = 21) were identified by postpartal otoacoustic emissions (OAE) and brain stem electric response audiometry (BERA) analysis. GJB2 testing was performed by direct sequencing. RESULTS: Mutations in GJB2 were found in 15 of 21 children (71.4%) identified by neonatal audiological screening. The 35delG mutation in GJB2 was found homozygous in 10 cases (47.6%) and also as a clear cause of HL as the heterozygous alterations 35delG/del311-324 and 35delG/L90P. In a single case, L90P/R143Q was also identified as a cause of HL. In 3 HL cases that were not identifiable during initial OAE testing, homozygous 35delG and 35delG/R184P defined the genetic basis for HL in 2 cases, whereas one case had wild-type GJB2. CONCLUSIONS: Our findings of the high mutation rate in the Austrian population, especially in neonates identified during the newborn screening program, confirm the importance of screening for mutations in GJB2.

Male fertility and protein C inhibitor/plasminogen activator inhibitor-3 (PCI): localization of PCI in mouse testis and failure of single plasminogen activator knockout to restore spermatogenesis in PCI-deficient mice.

OBJECTIVE: To investigate the mechanisms responsible for the testicular abnormalities and infertility of previously generated male protein C inhibitor (PCI)-deficient mice. DESIGN: Determination of the localization of PCI in the reproductive organs of wild-type males. Generation of double knockout mice lacking the protease inhibitor PCI and one plasminogen activator, either urokinase (uPA) or tissue plasminogen activator (tPA), both of which are PCI-target proteases. SETTING: Animal research and histologic analysis. ANIMAL(S): Male mice of desired genotype. INTERVENTION(S): Fertility testing of double knockout mice. MAIN OUTCOME MEASURE(S): Infertility of PCI(-/-)uPA(-/-) and PCI(-/-)tPA(-/-) double knockout mice. RESULT(S): In the testes of wild-type males PCI was detected in spermatocytes of prophase I, as well as in late spermatids and mature spermatozoa, but absent from somatic cells. All PCI(-/-) uPA(-/-) and PCI(-/-) tPA(-/-) male mice were infertile and histologic analysis of testis showed similar alterations as previously described for PCI(-/-) mice. CONCLUSION(S): The abnormal spermatogenesis of PCI (plasminogen activator inhibitor-3)-deficient mice cannot be rescued by single plasminogen activator knockout.

Mapping of cellular compartments based on ultrastructural immunogold labeling.

Ultrastructural identification of subcellular morphologically inconspicuous compartments is based on detection of specific molecules or by a presence of specific functions. Such compartments are detected using antibodies with attached label, usually gold particles. However, the gold particles have a point pattern, while a compartment is a coherent area. In addition, some background labeling is always present that complicates identification of the labeled compartments. The aim of this study was therefore to develop a stereological method that would enable us to define cellular compartments based on delineating the borders of gold particle clusters, and to test the practical use of the method using biological experimental data. New computer program plug-ins were developed to facilitate the practical use of the stereological method. The kernel estimation method was successfully tested by detection of ribosomal rRNA over morphologically recognizable nucleoli. In a next step, we successfully detected individual chromosomal domains-nuclear compartments that cannot be distinguished in cell nuclei morphologically. The results show that the new stereological/image analysis method is well able to discriminate cellular compartments based on density of immunogold particles. The plug-ins were made available to scientific community at http://nucleus.biomed.cas.cz/gold.

A novel connexin 26 mutation associated with autosomal recessive sensorineural deafness.

Mutations in the connexin 26 (Cx26) gene (GJB2) are a common cause of hereditary hearing impairment which affects approximately 1 in 2000 newborn children. We report the identification of a novel Cx26 point mutation (439 G-->A) linked to familial, autosomal recessive, sensorineural hearing loss. This missense mutation (E147K) is located in the highly conserved, putative K(+) channel lining sequence of the third transmembrane domain (TM3) of Cx26. Hearing impairment associated with this mutation was congenital, moderate to profound and showed no signs of progressive deterioration.