Measuring the regulation of keratin filament network dynamics

The organization of the keratin intermediate filament cytoskeleton is closely linked to epithelial function. To study keratin network plasticity and its regulation at different levels, tools are needed to localize and measure local network dynamics. In this paper, we present image analysis methods designed to determine the speed and direction of keratin filament motion and to identify locations of keratin filament polymerization and depolymerization at subcellular resolution. Using these methods, we have analyzed time-lapse fluorescence recordings of fluorescent keratin 13 in human vulva carcinoma-derived A431 cells. The fluorescent keratins integrated into the endogenous keratin cytoskeleton, and thereby served as reliable markers of keratin dynamics. We found that increased times after seeding correlated with down-regulation of inward-directed keratin filament movement. Bulk flow analyses further revealed that keratin filament polymerization in the cell periphery and keratin depolymerization in the more central cytoplasm were both reduced. Treating these cells and other human keratinocyte-derived cells with EGF reversed all these processes within a few minutes, coinciding with increased keratin phosphorylation. These results highlight the value of the newly developed tools for identifying modulators of keratin filament network dynamics and characterizing their mode of action, which, in turn, contributes to understanding the close link between keratin filament network plasticity and epithelial physiology.     

Apoptosis leads to a degradation of vital components of active nuclear transport and a dissociation of the nuclear lamina

Apoptosis, a physiologically critical process, is characterized by a destruction of the cell after sequential degradation of key cellular components. Here, we set out to explore the fate of the physiologically indispensable nuclear envelope (NE) in this process. The NE mediates the critical nucleocytoplasmic transport through nuclear pore complexes (NPCs). In addition, the NE is involved in gene expression and contributes significantly to the overall structure and mechanical stability of the cell nucleus through the nuclear lamina, which underlies the entire nucleoplasmic face of the NE and thereby interconnects the NPCs, the NE, and the genomic material. Using the nano-imaging and mechanical probing approach atomic force microscopy (AFM) and biochemical methods, we unveiled the fate of the NE during apoptosis. The doomed NE sustains a degradation of both the mediators of the critical selective nucleocytoplasmic transport, namely NPC cytoplasmic filaments and basket, and the nuclear lamina. These observations are paralleled by marked softening and destabilization of the NE and the detection of vesicle-like nuclear fragments. We conclude that destruction of the cell nucleus during apoptosis proceeds in a strategic fashion. Degradation of NPC cytoplasmic filaments and basket shuts down the critical selective nucleocytoplasmic cross-talk. Degradation of the nuclear lamina disrupts the pivotal connection between the NE and the chromatin, breaks up the overall nuclear architecture, and softens the NE, thereby enabling the formation of nuclear fragments at later stages of apoptosis.     

Increased expression of LAP2β eliminates nuclear membrane ruptures in nuclear lamin–deficient neurons and fibroblasts

Defects or deficiencies in nuclear lamins cause pathology in many cell types, and recent studies have implicated nuclear membrane (NM) ruptures as a cause of cell toxicity. We previously observed NM ruptures and progressive cell death in the developing brain of lamin B1–deficient mouse embryos. We also observed frequent NM ruptures and DNA damage in nuclear lamin–deficient fibroblasts. Factors modulating susceptibility to NM ruptures remain unclear, but we noted low levels of LAP2β, a chromatin-binding inner NM protein, in fibroblasts with NM ruptures. Here, we explored the apparent link between LAP2β and NM ruptures in nuclear lamin–deficient neurons and fibroblasts, and we tested whether manipulating LAP2β expression levels would alter NM rupture frequency. In cortical plate neurons of lamin B1–deficient embryos, we observed a strong correlation between low LAP2β levels and NM ruptures. We also found low LAP2β levels and frequent NM ruptures in neurons of cultured Lmnb1^−/− neurospheres. Reducing LAP2β expression in Lmnb1^−/− neurons with an siRNA markedly increased the NM rupture frequency (without affecting NM rupture duration), whereas increased LAP2β expression eliminated NM ruptures and reduced DNA damage. Consistent findings were observed in nuclear lamin–deficient fibroblasts. Reduced LAP2β expression increased NM ruptures, whereas increased LAP2β expression virtually abolished NM ruptures. Increased LAP2β expression nearly abolished NM ruptures in cells subjected to mechanical stress (an intervention that increases NM ruptures). Our studies showed that increasing LAP2β expression bolsters NM integrity in nuclear lamin–deficient cells and markedly reduces NM rupture frequency.

We recently examined mouse embryonic fibroblasts (MEFs) that were homozygous for knockout mutations in the three nuclear lamin genes (Lmnb1, Lmnb2, and Lmna) (1). These “triple-knockout fibroblasts” (TKO MEFs) lacked nuclear blebs but nevertheless had frequent nuclear membrane (NM) ruptures (1). The NM ruptures were associated with increased DNA damage (1). The frequency of NM ruptures in TKO MEFs increased when cells were subjected to mechanical stress (uniaxial stretching) and decreased when cells were subjected to interventions that interfere with the transmission of cytoskeletal forces to the nucleus (1). In follow-up studies, we observed frequent NM ruptures, along with DNA damage and cell death, in migrating neurons within the cortical plate of lamin B1–deficient mouse embryos (2). Cortical plate neurons are particularly susceptible to NM ruptures because neuronal migration subjects the cell nucleus to mechanical stress (2) and because embryonic neurons do not express lamin A or lamin C (3–6). In studies of TKO MEFs and Lmnb1^−/− neurons (1, 2), NM ruptures were identified by the escape of a nuclear-localized fluorescent reporter into the cytoplasm.In our studies of NM ruptures in TKO MEFs (1), the outline of the cell nucleus was visualized with an antibody against the inner nuclear membrane protein LAP2β (lamina-associated polypeptide 2, β-isoform). LAP2β contains a transmembrane helix that anchors it to the inner nuclear membrane (7–9), and it interacts, via nucleoplasmic LEM (LAP2, emerin, and Man1) domains (10, 11), with the DNA-bridging protein BAF (barrier-to-autointegration factor). This LEM–BAF complex is thought to tether chromatin to the nuclear periphery (12). In TKO MEFs, we found, by confocal microscopy, reduced amounts of LAP2β in TKO MEFs with NM ruptures. In those cells, there were also gaps in the distribution of LAP2β along the nuclear rim (1). In TKO MEFs without NM ruptures, LAP2β was distributed evenly along the nuclear periphery (1). In our initial study of TKO MEFs (1), we focused on the impact of mechanical stress on NM ruptures and did not pursue the possibility that LAP2β expression might modulate susceptibility of cells to NM ruptures.In the current studies, we investigated the possibility of a link between LAP2β expression and NM ruptures in nuclear lamin–deficient cells. We had two goals. The first was to investigate the potential association between abnormalities in LAP2β expression and distribution and NM ruptures—both in tissues of a nuclear lamin–deficient mouse and in a second nuclear lamin–deficient cell line. We began by studying the developing brain of Lmnb1^−/− embryos. We reasoned that we would be able to assess LAP2β expression, LAP2β distribution, and NM ruptures in migrating neurons of the cortical plate (a process that subjects the nucleus to mechanical stress) and in the germinal cells of the ventricular zone (where stresses on the nucleus are low) (2). We further reasoned that Lmnb1^−/− embryos would be a useful source of primary neurons for cell culture studies of NM ruptures (2).Our second goal was to examine whether LAP2β expression levels in nuclear lamin–deficient cells are directly relevant to the susceptibility to NM ruptures. Specifically, we wanted to test the hypothesis that manipulating LAP2β expression levels would change NM rupture frequency. Initially, we were skeptical that we would find any effect of LAP2β expression levels. Earlier studies identified indirect effects of other LEM domain proteins on the repair of NM ruptures (12–16), but no one had considered the idea that altering LAP2β expression levels would influence the frequency of NM ruptures. Despite our initial skepticism, we tested whether manipulating LAP2β expression would affect the frequency of NM ruptures in TKO fibroblasts and Lmnb1^−/− neurons. To our surprise, we found that reducing LAP2β expression levels markedly increased the frequency—but not the duration—of NM ruptures. Increasing LAP2β expression virtually abolished NM ruptures.     

Barrier-to-autointegration factor is required to segregate and enclose chromosomes within the nuclear envelope and assemble the nuclear lamina

Barrier-to-autointegration factor (BAF) binds dsDNA, LEM-domain proteins, and lamins. Caenorhabditis elegans BAF requires Ce-lamin and two LEM-domain proteins (Ce-emerin and Ce-MAN1) to localize during nuclear assembly. It was unknown whether Ce-lamin and LEM proteins, in turn, depend on Ce-BAF (mutually dependent structural roles). RNA interference-mediated down-regulation of Ce-BAF caused gross defects in chromosome segregation, chromatin decondensation, and mitotic progression as early as the two-cell stage, and embryos died at the approximately 100-cell stage. Nuclear pores reassembled, whereas Ce-lamin, Ce-emerin, and Ce-MAN1 bound chromatin but remained patchy and disorganized. The nuclear membranes formed but failed to enclose anaphase-bridged chromatin. Time-lapse imaging showed two phenotypes: anaphase-bridged chromatin that eventually resolved, and segregated chromatin that returned to the midzone. Thus, the assembly of BAF, lamins, and LEM-domain proteins is mutually dependent, and is required to capture segregated chromosomes within the nascent nuclear envelope. Embryos that escaped lethality by down-regulation of Ce-BAF grew into sterile adults with misplaced distal tip cells and gonads, further suggesting that mild postembryonic reductions in BAF disrupt tissue-specific functions.     

Autoimmune cytopenias associated with autoantibodies to nuclear envelope polypeptides

A subset of anti-nuclear autoantibodies (ANA) are directed against nuclear envelope (NE) polypeptides and display by indirect immunofluorescence (IIF) a ring-like fluorescent pattern. We report herein 19 patients with autoimmune cytopenias associated with antibodies (Abs) to NE polypeptides. Anti-NE specificity was determined by immunoblot, using NE preparations and purified lamina fractions. Eleven sera reacted with lamin B(1), and two reacted with both lamin B(1) and an unidentified 150-kDa protein (p150). One serum reacted with only p150. Four sera reacted with lamins A and C, and one reacted with and an unidentified 52-kDa NE polypeptide (p52). Autoimmune cytopenias included hemolytic anemia (7 cases), thrombocytopenia (13 cases), and neutropenia (6 cases). Five patients had 2 (3 cases) or 3 (2 cases) different cytopenias. Antiphospholipid antibodies (APLA) were detected in 14 patients, 2 of whom experienced thromboembolic events. A liver disorder was present in 7 patients. Systemic lupus erythematosus and lupus-like syndrome were diagnosed in 11 and 2 patients, respectively. Cytopenias responded to steroids alone (13 patients), or together with intravenous immunoglobulins (2 patients), or cyclophosphamide (2 patients). Two patients did not require treatment. Our results suggest that anti-NE Abs need to be sought for in patients with peripheral cytopenias, particularly when they are associated with APLA and/or liver disorders. Their detection strongly suggests an autoimmune process. Such cytopenias are often manifestations of a lupus or lupus-like disease and are responsive to steroids.     

The coronavirus E protein: assembly and beyond

The coronavirus E protein is a small membrane protein that has an important role in the assembly of virions. Recent studies have indicated that the E protein has functions during infection beyond assembly, including in virus egress and in the host stress response. Additionally, the E protein has ion channel activity, interacts with host proteins, and may have multiple membrane topologies. The goal of this review is to highlight the properties and functions of the E protein, and speculate on how they may be related.     

Sodium-calcium exchanger complexed with GM1 ganglioside in nuclear membrane transfers calcium from nucleoplasm to endoplasmic reticulum

The inner membrane of the nuclear envelope (NE) was previously shown to contain a Na/Ca exchanger (NCX) tightly linked to GM1 ganglioside that mediates transfer of nucleoplasmic Ca²⁺ to the NE lumen and constitutes a cytoprotective mechanism. This transfer was initially observed with isolated nuclei and is now demonstrated in living cells in relation to subcellular Ca²⁺ dynamics. Four cell lines with varying expression of NCX and GM1 in the NE were transfected with cameleon-fluorescent Ca²⁺ indicators genetically targeted to NE/endoplasmic reticulum (ER) and nucleoplasm to monitor [Ca²⁺]_ne/er and [Ca²⁺]_n respectively. Cytosolic Ca²⁺ ([Ca²⁺]_cyt) was indicated with fura-2. Thapsigargin caused progressive loss of [Ca²⁺]_ne/er, which was rapidly replaced on addition of extrinsic Ca²⁺ to those cells containing fully functional NCX/GM1: differentiated NG108–15 and C6 cells. Reduced elevation of [Ca²⁺]_ne/er following thapsigargin depletion occurred in cells containing little or no GM1 in the NE: undifferentiated NG108–15 and NG-CR72 cells. No change in [Ca²⁺]_ne/er due to applied Ca²⁺ was seen in Jurkat cells, which entirely lack NCX. Ca²⁺ entry to NE/ER was also blocked by KB-R7943, inhibitor of NCX. [Ca²⁺]_n and [Ca²⁺]_cyt were elevated independent of [Ca²⁺]_ne/er and remained in approximate equilibrium with each other. Ca²⁺ rise in the ER originated in the NE region and extended to the entire ER network. These results indicate the nuclear NCX/GM1 complex acts to gate Ca²⁺ transfer from cytosol to ER, an alternate route to the sarcoplasmic/endoplasmic reticulum calcium ATPase pump. They also suggest a possible contributory mechanism for independent regulation of nuclear Ca²⁺.     

HIV Rev response element (RRE) directs assembly of the Rev homooligomer into discrete asymmetric complexes

RNA is a crucial structural component of many ribonucleoprotein (RNP) complexes, including the ribosome, spliceosome, and signal recognition particle, but the role of RNA in guiding complex formation is only beginning to be explored. In the case of HIV, viral replication requires assembly of an RNP composed of the Rev protein homooligomer and the Rev response element (RRE) RNA to mediate nuclear export of unspliced viral mRNAs. Assembly of the functional Rev-RRE complex proceeds by cooperative oligomerization of Rev on the RRE scaffold and utilizes both protein-protein and protein-RNA interactions to organize complexes with high specificity. The structures of the Rev protein and a peptide-RNA complex are known, but the complete RNP is not, making it unclear to what extent RNA defines the composition and architecture of Rev-RNA complexes. Here we show that the RRE controls the oligomeric state and solubility of Rev and guides its assembly into discrete Rev-RNA complexes. SAXS and EM data were used to derive a structural model of a Rev dimer bound to an essential RRE hairpin and to visualize the complete Rev-RRE RNP, demonstrating that RRE binding drives assembly of Rev homooligomers into asymmetric particles, reminiscent of the role of RNA in organizing more complex RNP machines, such as the ribosome, composed of many different protein subunits. Thus, the RRE is not simply a passive scaffold onto which proteins bind but instead actively defines the protein composition and organization of the RNP.     

Congenital hypofibrinogenemia: characterization of two missense mutations affecting fibrinogen assembly and secretion

Congenital hypofibrinogenemia is a rare bleeding disorder characterized by abnormally low levels of fibrinogen in plasma, generally due to heterozygous mutations in one of the three fibrinogen genes (FGA, FGB, and FGG, coding for Aα, Bβ, and γ chain, respectively). Hypofibrinogenemic patients are usually asymptomatic, whereas individuals bearing similar mutations in the homozygous or compound heterozygous state develop a severe bleeding disorder: afibrinogenemia. The mutational spectrum of these quantitative fibrinogen disorders includes large deletions, point mutations causing premature termination codons, and missense mutations affecting fibrinogen assembly or secretion, distributed throughout the 50-kb fibrinogen gene cluster. In this study, we report the mutational screening of two unrelated hypofibrinogenemic patients leading to the identification of two missense mutations, one hitherto unknown (αCys45Phe), and one previously described (γAsn345Ser). The involvement of αCys45Phe and γAsn345Ser in the pathogenesis of hypofibrinogenemia was investigated by in-vitro expression experiments. Both mutations were demonstrated to cause a severe impairment of intracellular fibrinogen processing, either by affecting half-molecule dimerization (αCys45Phe) or by hampering hexamer secretion (γAsn345Ser).     

肝细胞核因子4α在肝脏疾病发生发展中的作用

肝细胞核因子(HNF)4α属于细胞核受体超家族成员,在分化成熟的肝细胞中高表达。HNF4α在转录水平上调控肝脏特异基因的表达,促进肝细胞的发育和分化,参与肝细胞极性的建立和维持,增强肝脏的合成、代谢以及解毒功能。HNF4α可通过抑制肝星状细胞的活化、逆转上皮间质细胞转化、抑制肝癌细胞增殖、侵袭和转移等机制,参与肝纤维化、肝硬化、肝细胞癌等疾病的发生和变化过程。阐述了HNF4α的生物学功能,对其参与多种主要肝脏疾病信号通路机制的研究进展进行了分析总结,旨在为进一步发掘这一潜在的肝脏疾病靶向治疗途径提供参考。     

Efficient plant male fertility depends on vegetative nuclear movement mediated by two families of plant outer nuclear membrane proteins

Increasing evidence suggests that nuclear migration is important for eukaryotic development. Although nuclear migration is conserved in plants, its importance for plant development has not yet been established. The most extraordinary plant nuclear migration events involve plant fertilization, which is starkly different from that of animals. Instead of evolving self-propelled sperm cells (SCs), plants use pollen tubes to deliver SCs, in which the pollen vegetative nucleus (VN) and the SCs migrate as a unit toward the ovules, a fundamental but barely understood process. Here, we report that WPP domain-interacting proteins (WIPs) and their binding partners the WPP domain-interacting tail-anchored proteins (WITs) are essential for pollen nuclear migration. Loss-of-function mutations in WIT and/or WIP gene families resulted in impaired VN movement, inefficient SC delivery, and defects in pollen tube reception. WIPs are Klarsicht/ANC-1/Syne-1 Homology (KASH) analogs in plants. KASH proteins are key players in animal nuclear migration. Thus, this study not only reveals an important nuclear migration mechanism in plant fertilization but also, suggests that similar nuclear migration machinery is conserved between plants and animals.Nuclear migration is essential for cell differentiation, polarization, and migration, which influence organism development (1–3). Examples range from Caenorhabditis elegans P-cell development to mammalian neural development (1–3). The key players in opisthokont nuclear migration are the inner nuclear membrane Sad1/UNC-84 (SUN) proteins and outer nuclear membrane Klarsicht/ANC-1/Syne-1 Homology (KASH) proteins. SUN and KASH proteins form the linkers of the nucleoskeleton and the cytoskeleton complexes at the nuclear envelope (NE) and transfer cytoplasmic forces to the nucleus (1–3). In plants, nuclear migration is associated with a number of developmental events and environmental responses, including fertilization, root and leaf hair formation, and plant–microbe interactions (4, 5). So far, little is known about the mechanism of plant nuclear migration. Although SUN proteins are conserved in plants (6, 7), absence of animal KASH homologs in plants suggests that plants may have evolved different molecular solutions to achieve nuclear migration. Recently, WPP domain-interacting proteins (WIPs) were identified as KASH proteins in plants (8), and their outer nuclear membrane binding partners WPP domain-interacting tail anchored proteins (WITs) were shown to interact with myosin XI-I (9). The WIT–myosin XI-I complexes regulate nuclear movement in root and mesophyll cells, but no developmental events have been linked to these nuclear movements (9).Essential for plant fertility, pollen tube growth harbors the most dramatic nuclear movement in plants. Unlike animals, which have sperm cells (SCs) that travel through self-propelled flagellum, flowering plants use pollen tubes to deliver SCs to ovules (10–13). In Arabidopsis, pollen tube growth is guided by chemical cues in carpel tissues and attracted by small peptides secreted by synergid cells in the vicinity of ovules (14–18). Pollen tube reception is completed by pollen tube burst, SC release, and degeneration of synergid cells (12). If this process fails, a second pollen tube can be attracted to the same ovule for a second attempt, resulting in polytubey (19). The SCs [or their progenitor the generative cell (GC)] are enclosed by an endocytic membrane tethered to the pollen vegetative nucleus (VN) (20). During pollen tube elongation, the VN and the SCs/GC are usually closely associated and move as a male germ unit (MGU) (13, 21). For decades, the movement of the MGU has been analyzed using cytoskeleton-depolymerizing reagents or heterogeneous antimyosin antibodies (22–27). However, no genes have been implicated in MGU movement, and the function of the joint migration of VN and GC/SC remains hypothetical.Here, we have identified the Arabidopsis WIT and WIP protein families as key players in VN movement. WIP1 and WIT1 are localized at the vegetative nuclear envelope (VNE). Loss of either WIT or WIP family proteins impaired VN movement, resulting in defective pollen tube reception and inefficient SC-to-ovule migration. This study has not only identified a molecular mechanism regulating the VN movement but also, revealed an important function of the VN in plant fertilization.     

Structure,variation, and assembly of the root-associated microbiomes of rice

Plants depend upon beneficial interactions between roots and microbes for nutrient availability, growth promotion, and disease suppression. High-throughput sequencing approaches have provided recent insights into root microbiomes, but our current understanding is still limited relative to animal microbiomes. Here we present a detailed characterization of the root-associated microbiomes of the crop plant rice by deep sequencing, using plants grown under controlled conditions as well as field cultivation at multiple sites. The spatial resolution of the study distinguished three root-associated compartments, the endosphere (root interior), rhizoplane (root surface), and rhizosphere (soil close to the root surface), each of which was found to harbor a distinct microbiome. Under controlled greenhouse conditions, microbiome composition varied with soil source and genotype. In field conditions, geographical location and cultivation practice, namely organic vs. conventional, were factors contributing to microbiome variation. Rice cultivation is a major source of global methane emissions, and methanogenic archaea could be detected in all spatial compartments of field-grown rice. The depth and scale of this study were used to build coabundance networks that revealed potential microbial consortia, some of which were involved in methane cycling. Dynamic changes observed during microbiome acquisition, as well as steady-state compositions of spatial compartments, support a multistep model for root microbiome assembly from soil wherein the rhizoplane plays a selective gating role. Similarities in the distribution of phyla in the root microbiomes of rice and other plants suggest that conclusions derived from this study might be generally applicable to land plants.Land plants grow in soil, placing them in direct proximity to a high abundance of microbial diversity (1). Plants and microbes have both adapted to use their close association for their mutual benefit. Critical nutrients are converted to more usable forms by microbes before assimilation by plants (2–4). In turn, bacteria in the rhizosphere receive carbon metabolites from the plant through root exudates (5). Beneficial soil microbes also contribute to pathogen resistance, water retention, and synthesis of growth-promoting hormones (6–8).Recent studies have used high-throughput sequencing to provide new insights into the bacterial composition and organization of different plant microbiomes, including Arabidopsis, Populus, and maize (9–14). Detailed characterization of the core root microbiome of Arabidopsis (9–11) showed that the dominant phyla inside the root (the endosphere) are much less diverse than the phyla in the soil around the root (the rhizosphere), and a potential core root microbiome could be identified. In Arabidopsis, the endophytic microbiome exhibits some genotype-dependent variation within the species and an increased variation when other related species are examined (9–11). A recent study in maize examined microbiome variation across many different inbred lines at different sites and found a large variation arising from geographical location between three different states in the United States and a relatively smaller dependence on the genotype (12). Although the microbiomes examined in the maize study consisted of combined rhizospheric and endospheric microbes (12), a study in poplar found that the variation between locations in two different states affected both rhizospheric and endospheric microbes (14).These studies have opened the way toward a new understanding of the composition and structure of plant microbiomes and the factors that affect them. However, this understanding is still at the initial stages, and several key questions are as yet unanswered. One such question regards the mechanism of microbiome acquisition and assembly in plants. Unlike animals, where the gut microbiome is assembled internally and is transmissible through birth (15, 16), the root microbiome is predominantly assembled from the external microbes in the soil. Based on the composition of the endospheric and rhizopheric microbiomes, it has been proposed that plants might assemble their microbiomes in two steps, with the first step involving a general recruitment to the vicinity of the root and a second step for entry inside the root that involves species-specific genetic factors (7). Although this is a plausible hypothesis, direct support for this model through detailed dynamic studies has not yet been provided. Additionally, the role of the root surface or rhizoplane, which forms the critical interface between plants and soil, remains poorly understood, and the microbial composition of the rhizoplane in relation to those of the rhizosphere and endosphere is unknown.To address some of these questions, we have undertaken an exhaustive characterization of the root-associated microbiome of rice. Rice is a major crop plant and a staple food for half of the world’s population. Metagenomic and proteomic approaches have been used to identify different microbial genes present in the rice microbiome (17, 18), but an extensive characterization of microbiome composition and variation has not been performed. Rice cultivation also contributes to global methane, accounting for an estimated 10–20% of anthropogenic emissions, due to the growth of methanogenic archaea in the vicinity of rice roots (19). Here we have used deep sequencing of microbial 16S rRNA genes to detect over 250,000 operational taxonomic units (OTUs), with a structural resolution of three distinct compartments (rhizosphere, rhizoplane, and endosphere) and extending over multiple factors contributing to variation, both under controlled greenhouse conditions as well as different field environments. The large datasets from the different conditions sampled in this study were used for identification of putative microbial consortia involved in processes such as methane cycling. Through dynamic studies of the microbiome composition, we provide insights into the process of root microbiome assembly.     

Abnormal development of the cerebral cortex and cerebellum in the setting of lamin B2 deficiency

Nuclear lamins are components of the nuclear lamina, a structural scaffolding for the cell nucleus. Defects in lamins A and C cause an array of human diseases, including muscular dystrophy, lipodystrophy, and progeria, but no diseases have been linked to the loss of lamins B1 or B2. To explore the functional relevance of lamin B2, we generated lamin B2-deficient mice and found that they have severe brain abnormalities resembling lissencephaly, with abnormal layering of neurons in the cerebral cortex and cerebellum. This neuronal layering abnormality is due to defective neuronal migration, a process that is dependent on the organized movement of the nucleus within the cell. These studies establish an essential function for lamin B2 in neuronal migration and brain development.     

Serum amino acids in patients with mutations in the hepatocyte nuclear factor-1 alpha gene.

AIMS: Knockout mice lacking both copies of the hepatocyte nuclear factor 1 (HNF1) gene have altered serum levels of amino acids and generalized aminoaciduria. The aim of our study was to test whether alterations in serum amino acid levels were found in patients with mutations in the hepatocyte nuclear factor-1 alpha (HNF-1alpha) gene compared with controls. METHODS: Fasting serum from 20 patients with HNF-1alpha mutations and 20 age, sex and body mass index-matched controls was analysed for 16 amino acids. Means were compared between the two groups and Z scores calculated. RESULTS: There was no significant difference between patients with HNF-1alpha mutations and controls in serum levels of phenylalanine, arginine, citrulline or lysine as suggested by knockout mice models. Although serum levels of eight amino acids were different in the two groups, these were not significant after Bonferroni correction. CONCLUSIONS: The alterations in serum amino acid levels seen in mice models are not seen in patients with mutations in the HNF-1alpha gene. This suggests differences in mouse and man in the regulation of amino acid transport and has not provided us with a phenotypic marker to use before confirmatory genetic testing.     

Mechanical stress-induced sarcomere assembly for cardiac muscle growth in length and width

A ventricular myocyte experiences changes in length and load during every beat of the heart and has the ability to remodel cell shape to maintain cardiac performance. Specifically, myocytes elongate in response to increased diastolic strain by adding sarcomeres in series, and they thicken in response to continued systolic stress by adding filaments in parallel. Myocytes do this while still keeping the resting sarcomere length close to its optimal value at the peak of the length-tension curve. This review focuses on the little understood mechanisms by which direction of growth is matched in a physiologically appropriate direction. We propose that the direction of strain is detected by differential phosphorylation of proteins in the costamere, which then transmit signaling to the Z-disc for parallel or series addition of thin filaments regulated via the actin capping processes. In this review, we link mechanotransduction to the molecular mechanisms for regulation of myocyte length and width.     

Identification of new mutations in the hepatocyte nuclear factor 4alpha gene among families with early onset Type 2 diabetes mellitus.

AIMS: Mutations in hepatocyte nuclear factor (HNF)-4alpha gene located on chromosome 20q have been found to be responsible for the development of early onset Type 2 diabetes mellitus (DM). Through a national campaign, 53 families with autosomal dominant, early onset Type 2 DM (n=654) were assembled to determine the frequency of mutations in the HNF-4alpha gene and their contribution to the development of diabetes. METHODS: Twelve exons and the promoter region of the HNF-4alpha gene were screened in probands of the families by a double gradient, denaturing gradient gel electrophoresis (DG-DGGE) protocol combined with automated bi-directional sequencing of the PCR products of all heterozygous individuals. RESULTS: We detected two new mutations in the HNF-4alpha gene that changed the amino-acid sequence. The first mutation was a Gly-->Ser substitution in codon 115 within a highly conserved DNA binding domain, and all six carriers of this mutation had diabetes and low insulin secretion. The second mutation was an Ile-->Val substitution in codon 454 within the transactivation domain. It was carried by four family members, two of whom also carried a mutation in the HNF-1alpha gene. Of those having only the mutation in HNF-4alpha one had diabetes and the other had normal glucose tolerance and both were obese and hyperinsulinaemic. Thus, it is uncertain that this mutation is responsible for any of the diabetes in this family. CONCLUSION: We have found that mutations in the HNF-4alpha gene account for a small proportion, about 2-4%, of families with early onset, autosomal dominant, Type 2 DM in US Caucasians.     

Distribution of melatonin in mammalian tissues: The relative importance of nuclear versus cytosolic localization

Abstract: Besides its presence in the pineal gland, melatonin has been found in a variety of other tissues as well. The indoleamine also has been identified in invertebrates including an unicellular organism where it exhibits a diurnal rhythm. Although melatonin is mainly known for its effects on seasonal reproduction and endocrine physiology, there is evidence showing that this ubiquitously acting hormone is also a potent free radical scavenger, thereby providing protection from oxidative attack to DNA and other biomolecules. Through the years, melatonin was thought to be exclusively cytosolic. However, careful examination of some of these pioneering reports revealed a nuclear localization of melatonin in different tissues including the retina and Harderian glands. Using a very sensitive immunocytochemical method, we have also found that melatonin is located in the nucleus of many cells where it may bind to nuclear components. The use of cell fractionation studies followed by radioimmunoassay confirmed these results. The administration of exogenous melatonin resulted in a marked increase in the nuclear melatonin content without a concomitant change in the cytosolic fraction. In addition to its ability to scavenge free radicals, its location in the nucleus suggests possible genomic actions.