Interkinetic nuclear migration: Reciprocal activities of dynein and kinesin

A hallmark of neurogenesis in vertebrates is the apical-basal fluctuation of radial glia nuclei. Such a phenomenon, called INM, has been known for decades and is closely associated with mitosis but still puzzles scientists. An impressive step in the molecular understanding of INM has recently been achieved by Tsai and coworkers. Using RNA interference associated with time-lapse imaging, these authors demonstrated a dual motor system that can push/pull the nuclei accordingly with the cell cycle stages.Key words: interkinetic nuclear migration (INM), radial glial progenitor cells (RGPCs), dynein (Dyn), kinesin (Kin), neurogenesis     

Mechanism and a Peptide Motif for Targeting Peripheral Proteins to the Yeast Inner Nuclear Membrane

Trm1 is a tRNA specific m₂²G methyltransferase shared by nuclei and mitochondria in Saccharomyces cerevisiae . In nuclei, Trm1 is peripherally associated with the inner nuclear membrane (INM). We investigated the mechanism delivering/tethering Trm1 to the INM. Analyses of mutations of the Ran pathway and nuclear pore components showed that Trm1 accesses the nucleoplasm via the classical nuclear import pathway. We identified a Trm1 cis-acting sequence sufficient to target passenger proteins to the INM. Detailed mutagenesis of this region uncovered specific amino acids necessary for authentic Trm1 to locate at the INM. The INM information is contained within a sequence of less than 20 amino acids, defining the first motif for addressing a peripheral protein to this important subnuclear location. The combined studies provide a multi-step process to direct Trm1 to the INM: (i) translation in the cytoplasm; (ii) Ran-dependent import into the nucleoplasm; and (iii) redistribution from the nucleoplasm to the INM via the INM motif. Furthermore, we demonstrate that the Trm1 mitochondrial targeting and nuclear localization signals are in competition with each other, as Trm1 becomes mitochondrial if prevented from entering the nucleus.     

An endogenous calcium-dependent, caspase-independent intranuclear degradation pathway in thymocyte nuclei: antagonism by physiological concentrations of K(+) ions

Calcium ions have been implicated in apoptosis for many years, however the precise role of this ion in the cell death process remains incomplete. We have extensively examined the role of Ca(2+) on nuclear degradation in vitro using highly purified nuclei isolated from non-apoptotic rat thymocytes. We show that these nuclei are devoid of CAD (caspase-activated DNase), and DNA degradation occurs independent of caspase activity. Serine proteases rather than caspase-3 appear necessary for this Ca(2+) -dependent DNA degradation in nuclei. We analyzed nuclei treated with various concentrations of Ca(2+) in the presence of both a physiological (140 mM) and apoptotic (40 mM) concentration of KCl. Our results show that a 5-fold increase in Ca(2+) is required to induce DNA degradation at the physiological KCl concentration compared to the lower, apoptotic concentration of the cation. Ca(2+) -induced internucleosomal DNA degradation was also accompanied by the release of histones, however the apoptotic-specific phosphorylation of histone H2B does not occur in these isolated nuclei. Interestingly, physiological concentrations of K(+) inhibit both Ca(2+) -dependent DNA degradation and histone release suggesting that a reduction of intracellular K(+) is necessary for this apoptosis-associated nuclear degradation in cells. Together, these data define an inherent caspase-independent catabolic pathway in thymocyte nuclei that is sensitive to physiological concentrations of intracellular cations.     

The Drosophila melanogaster LEM-domain protein MAN1

Here we describe the Drosophila melanogaster LEM-domain protein encoded by the annotated gene CG3167 which is the putative ortholog to vertebrate MAN1. MAN1 of Drosophila (dMAN1) and vertebrates have the following properties in common. Firstly, both molecules are integral membrane proteins of the inner nuclear membrane (INM) and share the same structural organization comprising an N-terminally located LEM motif, two transmembrane domains in the middle of the molecule, and a conserved RNA recognition motif in the C-terminal region. Secondly, dMAN1 has similar targeting domains as it has been reported for the human protein. Thirdly, immunoprecipitations with dMAN1-specific antibodies revealed that this Drosophila LEM-domain protein is contained in protein complexes together with lamins Dm0 and C. It has been previously shown that human MAN1 binds to A- and B-type lamins in vitro. During embryogenesis and early larval development LEM-domain proteins dMAN1 and otefin show the same expression pattern and are much more abundant in eggs and the first larval instar than in later larval stages and young pupae whereas the LEM-domain protein Bocksbeutel is uniformly expressed in all developmental stages. dMAN1 is detectable in the nuclear envelope of embryonic cells including the pole cells. In mitotic cells of embryos at metaphase and anaphase, LEM-domain proteins dMAN1, otefin and Bocksbeutel were predominantly localized in the region of the two spindle poles whereas the lamin B receptor and lamin Dm0 were more homogeneously distributed. Downregulation of dMAN1 by RNA interference (RNAi) in Drosophila cultured Kc167 cells has no obvious effect on nuclear architecture, viability of RNAi-treated cells and the intracellular distribution of the LEM-domain proteins Bocksbeutel and otefin. In contrast, the localization of dMAN1, Bocksbeutel and otefin at the INM is supported by lamin Dm0. We conclude that the dMAN1 protein is not a limiting component of the nuclear architecture in Drosophila cultured cells.     

An update on non-peptide angiotensin receptor antagonists and related RAAS modulators

The renin-angiotensin-aldosterone-system (RAAS) is an important regulator of blood pressure and fluid-electrolyte homeostasis. RAAS has been implicated in pathogenesis of hypertension, congestive heart failure, and chronic renal failure. Aliskiren is the first non-peptide orally active renin inhibitor approved by FDA. Angiotensin Converting Enzyme (ACE) Inhibitors are associated with frequent side effects such as cough and angio-oedema. Recently, the role of ACE2 and neutral endopeptidase (NEP) in the formation of an important active metabolite/mediator of RAAS, ang 1-7, has initiated attempts towards development of ACE2 inhibitors and combined ACE/NEP inhibitors. Furukawa and colleagues developed a series of low molecular weight nonpeptide imidazole analogues that possess weak but selective, competitive AT1 receptor blocking property. Till date, many compounds have exhibited promising AT1 blocking activity which cause a more complete RAAS blockade than ACE inhibitors. Many have reached the market for alternative treatment of hypertension, heart failure and diabetic nephropathy in ACE inhibitor intolerant patients and still more are waiting in the queue. But, the hallmark of this area of drug research is marked by a progress in understanding molecular interaction of these blockers at the AT1 receptor and unraveling the enigmatic influence of AT2 receptors on growth/anti-growth, differentiation and the regeneration of neuronal tissue. Different modeling strategies are underway to develop tailor made molecules with the best of properties like Dual Action (Angiotensin And Endothelin) Receptor Antagonists (DARA), ACE/NEP inhibitors, triple inhibitors, AT2 agonists, AT1/TxA2 antagonists, balanced AT1/AT2 antagonists, and nonpeptide renin inhibitors. This abstract gives an overview of these various angiotensin receptor antagonists.     

Interkinetic nuclear migration: a mysterious process in search of a function

During interkinetic nuclear migration (INM), the nuclei in many epithelial cells migrate between the apical and basal surfaces, coordinating with the cell cycle, and undergoing cytokinesis at the apical surface. INM is observed in a wide variety of tissues and species. Recent advances in time-lapse microscopy have provided clues about the mechanisms and functions of INM. Whether actin or microtubules are responsible for nuclear migration is controversial. How mitosis is initiated during INM is poorly understood, as is the relationship between the cell cycle and nuclear movement. It is possible that the disagreements stem from differences in the tissues being studied, since epithelia undergoing INM vary greatly in terms of cell height and cell fates. In this review we examine the reports addressing the mode and mechanisms that regulate INM and suggest possible functions for this dramatic event.     

Differential roles of HIC-5 isoforms in the regulation of cell death and myotube formation during myogenesis

Hic-5 is a LIM-Only member of the paxillin superfamily of focal adhesion proteins. It has been shown to regulate a range of biological processes including: senescence, tumorigenesis, steroid hormone action, integrin signaling, differentiation, and apoptosis. To better understand the roles of Hic-5 during development, we initiated a detailed analysis of Hic-5 expression and function in C(2)C(12) myoblasts, a well-established model for myogenesis. We have found that: (1) myoblasts express at least 6 distinct Hic-5 isoforms; (2) the two predominant isoforms, Hic-5alpha and Hic-5beta, are differentially expressed during myogenesis; (3) any experimentally induced change in Hic-5 expression results in a substantial increase in apoptosis during differentiation; (4) ectopic expression of Hic-5alpha is permissive to differentiation while expression of either Hic-5beta or antisense Hic-5 blocks myoblast fusion but not chemodifferentiation; (5) Hic-5 localizes to focal adhesions in C(2)C(12) myoblasts and perturbation of Hic-5 leads to defects in cell spreading; (6) alterations in Hic-5 expression interfere with the normal dynamics of laminin expression; and (7) ectopic laminin, but not fibronectin, can rescue the Hic-5-induced blockade of myoblast survival and differentiation. Our data demonstrate differential roles for individual Hic-5 isoforms during myogenesis and support the hypothesis that Hic-5 mediates these effects via integrin signaling.     

Two wrongs make a right: deficits in reversal learning after orbitofrontal damage are improved by amygdala ablation

The nuclei of dividing neural progenitors undergo a cell-cycle-dependent change in position along the apico-basal axis known as interkinetic nuclear migration (INM). The functional relationship between INM and the mode of division of neural progenitors remains elusive, in part because its regulation at the molecular level is poorly understood. In this issue of Neuron, Xie et al. identify two centrosomal proteins (Cep120 and TACCs) regulating the INM of cortical neural progenitors.     

Regulation of interkinetic nuclear migration by cell cycle-coupled active and passive mechanisms in the developing brain

A hallmark of neurogenesis in the vertebrate brain is the apical-basal nuclear oscillation in polarized neural progenitor cells. Known as interkinetic nuclear migration (INM), these movements are synchronized with the cell cycle such that nuclei move basally during G1-phase and apically during G2-phase. However, it is unknown how the direction of movement and the cell cycle are tightly coupled. Here, we show that INM proceeds through the cell cycle-dependent linkage of cell-autonomous and non-autonomous mechanisms. During S to G2 progression, the microtubule-associated protein Tpx2 redistributes from the nucleus to the apical process, and promotes nuclear migration during G2-phase by altering microtubule organization. Thus, Tpx2 links cell-cycle progression and autonomous apical nuclear migration. In contrast, in vivo observations of implanted microbeads, acute S-phase arrest of surrounding cells and computational modelling suggest that the basal migration of G1-phase nuclei depends on a displacement effect by G2-phase nuclei migrating apically. Our model for INM explains how the dynamics of neural progenitors harmonize their extensive proliferation with the epithelial architecture in the developing brain.     

The inner nuclear membrane protein Lem2 is critical for normal nuclear envelope morphology

The inner nuclear membrane (INM) of eukaryotic cells is characterized by a unique set of transmembrane proteins which interact with chromatin and/or the nuclear lamina. The number of identified INM proteins is steadily increasing, mainly as a result of proteomic and computational approaches. However, despite a link between mutation of several of these proteins and disease, the function of most transmembrane proteins of the INM remains unknown and depletion of many of these proteins from a variety of systems did not produce an obvious phenotype in the affected cells. Here, we report that depletion of the conserved INM protein Lem2 from human cell lines leads to abnormally shaped nuclei and severely reduces cell survival. We suggest that interactions of Lem2 with lamins or chromatin are critical for maintaining the integrity of the nuclear envelope.     

Oligochitosan induces programmed cell death in tobacco suspension cells

Oligochitosan has been proved to trigger plant cell death. To gain some insights into the mechanisms of oligochitosan-induced cell death, the nature of oligochitosan-induced cell death and the role of calcium (Ca²⁺), nitric oxide (NO) and hydrogen peroxide (H₂O₂) were studied in tobacco suspension cells. Oligochitosan-induced cell death occurred in cytoplasmic shrinkage, phosphatidylserine externalization, chromatin condensation, TUNEL-positive nuclei, cytochrome c release and induction of programmed cell death (PCD)-related gene hsr203J, suggesting the activation of PCD pathway. Pretreatment cells with cyclosporin A, resulted in reducing oligochitosan-induced cytochrome c release and cell death, indicating oligochitosan-induced PCD was mediated by cytochrome c. In the early stage, cells undergoing PCD showed an immediate burst in free cytosolic Ca²⁺ ([Ca²⁺]_cyt) elevation, NO and H₂O₂ production. Further study showed that these three signals were involved in oligochitosan-induced PCD, while Ca²⁺ and NO played a negative role in this process by modulating cytochrome c release.     

Insights on the structure of native CNF,an endogenous phospholipase A2 inhibitor from Crotalus durissus terrificus, the South American rattlesnake

Several snake species possess endogenous phospholipase A₂ inhibitors (sbPLIs) in their blood plasma, the primary role of which is protection against an eventual presence of toxic phospholipase A₂ (PLA₂) from their venom glands in the circulation. These inhibitors have an oligomeric structure of, at least, three subunits and have been categorized into three classes (α, β and γ) based on their structural features. SbγPLIs have been further subdivided into two subclasses according to their hetero or homomeric nature, respectively. Despite the considerable number of sbγPLIs described, their structures and mechanisms of action are still not fully understood. In the present study, we focused on the native structure of CNF, a homomeric sbγPLI from Crotalus durissus terrificus, the South American rattlesnake. Based on the results of different biochemical and biophysical experiments, we concluded that, while the native inhibitor occurs as a mixture of oligomers, tetrameric arrangement appears to be the predominant quaternary structure. The inhibitory activity of CNF is most likely associated with this oligomeric conformation. In addition, we suggest that the CNF tetramer has a spherical shape and that tyrosinyl residues could play an important role in the oligomerization. The carbohydrate moiety, which is present in most sbγPLIs, is not essential for the inhibitory activity, oligomerization or complex formation of the CNF with the target PLA₂. A minor component, comprising no more than 16% of the sample, was identified in the CNF preparations. The amino-terminal sequence of that component is similar to the B subunits of the heteromeric sbγPLIs; however, the role played by such molecule in the functionality of the CNF, if any, remains to be determined.     

Regulation of adaptor protein GIT1 in platelets, leading to the interaction between GIT1 and integrin alpha(IIb)beta3

GIT1 is an adaptor protein, which links signaling proteins to focal adhesion, thereby regulating cytoskeletal reorganization. Platelets undergo dynamic cytoskeletal reorganization during platelet activation, for which a large number of adaptor proteins are required. However, there has been no report of GIT1 in platelets. We found that GIT1 was abundantly expressed in platelets and underwent tyrosine phosphorylation downstream of integrin α_IIbβ₃, which was inhibited by the Src kinase inhibitor PP2. Furthermore, GIT1 constitutively associated with βPIX, a guanine nucleotide exchange factor (GEF) for Rac. The GIT1/βPIX complex associated with α_IIbβ₃, concomitantly with GIT1 tyrosine phosphorylation. Moreover, both GIT1 and α_IIbβ₃ rapidly translocated to the cytoskeletal fraction during platelet aggregation, which was not observed in the absence of aggregation. These results suggest that tyrosine phosphorylation of GIT1 by Src kinases may regulate cytoskeletal reorganization downstream of α_IIbβ₃ by bringing the Rac GEF βPIX to the vicinity of the integrin.     

The binding properties of the human receptor for the cellular uptake of vitamin B12

Cellular uptake of vitamin B(12) (cobalamin, Cbl) is mediated by a receptor expressed on the plasma membrane that binds transcobalamin (TC) saturated with Cbl and internalizes the TC-Cbl by endocytosis. A few reports have described the characterization of the receptor protein. However, many discrepancies have emerged in the functional and structural properties of the receptor and therefore, the identity and primary structure of this protein remains unconfirmed. In this report, we provide evidence of a 58 kDa monomeric protein as the likely receptor for the uptake of TC-Cbl and that the functional activity is not associated with a 72/144 kDa monomer/dimer with immunoglobulin Fc structural domain that has been purported to be the receptor in a number of publications.     

H2O2-NOX系统: 一种植物体内重要的发育调控与胁迫响应机制

以H₂O₂为中心的活性氧(reactive oxygen species, ROS)的产生是动植物发育与响应外界生物与非生物胁迫的普遍 特征, 其在生理和分子2个水平上调控植物的发育和对外界胁迫的响应, 并与一系列信号转导过程相关联。作为关键的ROS产生酶, 质膜NADPH氧化酶(plasma membrane NADPH oxidase, PM-NOX)在植物应对各种生物和非生物胁迫中具有重要作用, 被广泛认为是胁迫条件下植物细胞ROS产生并积累的主要来源。该文简要综述了近年来人们在植物细胞ROS产生、清除、生理功能以及PM-NOX酶的结构特征与功能等方面的研究进展, 并认为H₂O₂-NOX系统是一种植物体内普遍存在的重要发育调控与胁迫响应机制。     

Purification of a post-synaptic neurotoxic phospholipase A2 from Naja naja venom and its inhibition by a glycoprotein from Withania somnifera

A post-synaptic neurotoxic phospholipase A(2) (PLA(2)) has been purified from Indian cobra Naja naja venom. It was associated with a peptide in the venom. The association was disrupted using 8 M urea. It is denoted to be a basic protein by its behavior on both ion exchange chromatography and electrophoresis. It is toxic to mice, LD(50) 1.9 mg/kg body weight (ip). It is proved to be post-synaptic PLA(2) by chymographic experiment using frog nerve-muscle preparation. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera. The WSG inhibited the phospholipase A(2) activity of NN-XIa-PLA(2,) isolated from the cobra venom, completely at a mole-to-mole ratio of 1:2 (NN-XIa-PLA(2): WSG) but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA(2) isoforms from the venom to varying extent. The interaction of the WSG with the PLA(2) is confirmed by fluorescence quenching and gel-permeation chromatography. Chemical modification of the active histidine residue of PLA(2) using p-brophenacyl bromide resulted in the loss of both catalytic activity as well as neurotoxicity of the molecule. These findings suggest that the venom PLA(2) has multiple sites on it; perhaps some of them are overlapping. Application of the plant extract on snakebite wound confirms the medicinal value associated with the plant.     

μ- and μ4-η coordination of A2H2 (A = C, Si, Ge, Sn and Pb) ligands with transition metals

The heavier analogs of C₂H₂ have been studied at the B3LYP level for their μ and μ₄-η² coordination properties with the transition metals. Based on known alkyne compounds, transition metal fragments [W₂(μ-NH)(Cp)₂(Cl)₂] and [Fe₄(CO)₁₂] have been chosen. The SBKJC relativistic effective core potentials and their associated basis sets were used on W, Fe, Sn and Pb, and the 6-31G(d) basis set was used on all other elements. All the complexes of Si₂H₂, Ge₂H₂, Sn₂H₂ and Pb₂H₂ are found to be local minima. The trans-twist nature of the ligand A₂H₂ (A = Si-Pb) is large in μ-coordinated complexes of W, and it is very small in μ₄-η² coordinated complexes of Fe. The electronic structure of these complexes was investigated using fragment molecular orbital method (FMO).     

The Melting Phase Transition in Small Carbon Dioxide Clusters

Abstract

Small isolated carbon dioxide clusters have been studied by canonical and microcanonical Molecular Dynamics simulation. The phenomenon of “dynamical coexistence” between solidlike and liquidlike forms of the (CO₂)₁₃ cluster was found, in a finite energy range. An animation of this phenomenon is shown. Coexistence is associated with bimodality in the probability distribution of the short-time averaged internal kinetic energy of the system in the microcanonical ensemble. This phenomenon has also been observed in (CO₂)_12,14. It is interpreted in terms of the energy distribution of the Potential Energy Surface minima. This statistical mechanics tool enables to understand why dynamical coexistence has also been observed in previous works on (Ar)₁₃ and (N₂)₁₃ but not in the case of (Ar)_8,14 and (SF₆)₁₃.