Cell-specific and lamin-dependent targeting of novel transmembrane proteins in the nuclear envelope

Nuclear envelope complexity is expanding with respect to identification of protein components. Here we test the validity of proteomics results that identified 67 novel predicted nuclear envelope transmembrane proteins (NETs) from liver by directly comparing 30 as tagged fusions using targeting assays. This confirmed 21 as NETs, but 4 only targeted in certain cell types, underscoring the complexity of interactions that tether NETs to the nuclear envelope. Four NETs accumulated at the nuclear rim in normal fibroblasts but not in fibroblasts lacking lamin A, suggesting involvement of lamin A in tethering them in the nucleus. However, intriguingly, for the NETs tested alternative mechanisms for nuclear envelope retention could be found in Jurkat cells that normally lack lamin A. This study expands by a factor of three the number of liver NETs analyzed, bringing the total confirmed to 31, and shows that several have multiple mechanisms for nuclear envelope retention.     

Anandamide extends platelets survival through CB1-dependent Akt signaling

Platelets are stored at 22°C, since incubation at 37°C results in loss of viability. Nonetheless, in our body (37°C), platelets survive for 8–10 days. This discrepancy has been explained in terms of deprivation of viability factors or accumulation of apoptotic factors during storage. We report that the endocannabinoid anandamide (AEA) may be one of the agents allowing platelet survival. In fact, at 37°C, human platelets enhance the expression of pro-apoptotic proteins (caspases, Bax, Bak) and decrease the expression of Bcl-xL, thus changing the Bcl-xL/Bak ratio, a key platelet biological clock. AEA or its non-hydrolyzable analogue, methanandamide, extend platelet life span, without reversing the changes in Bcl-xL/Bak ratio induced by heat stress. Instead, AEA binding to type-1 cannabinoid receptor activates Akt, which regulates, through phosphorylation of Bad, the interactions among different Bcl-2 family members. These findings could have implications for platelet collection and, potentially, for their clinical use.     

The selective BH4-domain biology of Bcl-2-family members: IP3Rs and beyond

Anti-apoptotic Bcl-2-family members not only neutralize pro-apoptotic proteins but also directly regulate intracellular Ca²⁺ signaling from the endoplasmic reticulum (ER), critically controlling cellular health, survival, and death initiation. Furthermore, distinct Bcl-2-family members may selectively regulate inositol 1,4,5-trisphosphate receptor (IP₃R): Bcl-2 likely acts as an endogenous inhibitor of the IP₃R, preventing pro-apoptotic Ca²⁺ transients, while Bcl-X_L likely acts as an endogenous IP₃R-sensitizing protein promoting pro-survival Ca²⁺ oscillations. Furthermore, distinct functional domains in Bcl-2 and Bcl-X_L may underlie the divergence in IP₃R regulation. The Bcl-2 homology (BH) 4 domain, which targets the central modulatory domain of the IP₃R, is likely to be Bcl-2’s determining factor. In contrast, the hydrophobic cleft targets the C-terminal Ca²⁺-channel tail and might be more crucial for Bcl-X_L’s function. Furthermore, one amino acid critically different in the sequence of Bcl-2’s and Bcl-X_L’s BH4 domains underpins their selective effect on Ca²⁺ signaling and distinct biological properties of Bcl-2 versus Bcl-X_L. This difference is evolutionary conserved across five classes of vertebrates and may represent a fundamental divergence in their biological function. Moreover, these insights open novel avenues to selectively suppress malignant Bcl-2 function in cancer cells by targeting its BH4 domain, while maintaining essential Bcl-X_L functions in normal cells. Thus, IP₃R-derived molecules that mimic the BH4 domain’s binding site on the IP₃R may function synergistically with BH3-mimetic molecules selectivity suppressing Bcl-2’s proto-oncogenic activity. Finally, a more general role for the BH4 domain on IP₃Rs, rather than solely anti-apoptotic, may not be excluded as part of a complex network of molecular interactions.     

Emery-Dreifuss muscular dystrophy at the nuclear envelope: 10 years on

Emery-Dreifuss muscular dystrophy (EDMD) is a neuromuscular degenerative condition with an associated dilated cardiomyopathy and cardiac conduction defect. It can be inherited in either an X-linked or autosomal manner by mutations in the nuclear proteins emerin and lamin A/C, respectively. Traditionally muscular dystrophies were associated with defects in sarcolemma-associated proteins and, therefore, a nuclear connection suggested the existence of novel signalling pathways associated with this group of diseases. Subsequently, other mutations in the lamin A/C gene were attributed to a range of tissue-specific degenerative conditions, collectively known as the ‘laminopathies’. Therefore, any proposed hypothesis underlying the molecular mechanism of EDMD needs to include this anomaly. As we celebrate the 10th anniversary of the identification of emerin as a component of the nuclear envelope, I discuss here the available evidence that currently implicates EDMD as arising from perturbations in myogenic regulatory pathways, causing temporal delays in both cell cycle progression and muscle regeneration. Received 25 May 2006; received after revision 22 June 2006; accepted 22 August 2006     

Inner nuclear membrane proteins: functions and targeting

We summarize the properties of integral membrane proteins that reside in the inner nuclear membrane, including lamin B receptor (LBR), lamina-associated polypeptide (LAP) 1, LAP2, emerin, MAN1 and nurim. Most of these proteins interact with lamins and chromatin. Some data also suggest more speculative functions such as gene regulation and possibly sterol metabolism. Mutations in emerin and nuclear lamins have been associated with muscular dystrophies and lipodystrophy, raising new questions about the functions of inner nuclear membrane proteins. Integral proteins of the inner nuclear membrane are synthesized on the rough endoplasmic reticulum (ER) and reach the inner nuclear membrane by lateral diffusion in the connected ER and nuclear envelope membranes. Associations with nuclear ligands retain them in the inner nuclear membrane. Further investigation of the functions and targeting of inner nuclear membrane proteins are needed to determine how they are involved in human disease.     

Molecular mechanisms of centrosome and cytoskeleton anchorage at the nuclear envelope

Cell polarization is a fundamental process underpinning organismal development, and tissue homeostasis, which requires an orchestrated interplay of nuclear, cytoskeletal, and centrosomal structures. The underlying molecular mechanisms, however, still remain elusive. Here we report that kinesin-1/nesprin-2/SUN-domain macromolecular assemblies, spanning the entire nuclear envelope (NE), function in cell polarization by anchoring cytoskeletal structures to the nuclear lamina. Nesprin-2 forms complexes with the kinesin-1 motor protein apparatus by associating with and recruiting kinesin light chain1 (KLC1) to the outer nuclear membrane. Similar to nesprin-2, KLC1 requires lamin A/C for proper NE localization. The depletion of nesprin-2 or KLC1, or the uncoupling of nesprin-2/SUN-domain protein associations impairs cell polarization during wounding and dislodges the centrosome from the NE. In addition nesprin-2 loss has profound effects on KLC1 levels, the cytoskeleton, and Golgi apparatus organization. Collectively these data show that NE-associated proteins are pivotal determinants of cell architecture and polarization.     

The structure and function of nuclear lamins: implications for disease

The nuclear lamins polymerize to form the nuclear lamina, a fibrous structure found on the inner face of the nuclear membrane. The lamins also appear to form structures within the nucleoplasm. These various lamin structures help to establish and maintain the shape and strength of the interphase nucleus, but recent work also suggests that the lamins have a role in nuclear processes such as DNA replication. Furthermore, mutations in the human lamin A/C gene have recently been linked to several diseases, including Emery-Dreifuss muscular dystrophy. This review discusses the nature of these mutations and the possible effects of lamin mutations on nuclear function.     

Human progeroid syndromes,aging and cancer: new genetic and epigenetic insights into old questions

Disorders in which individuals exhibit certain features of aging early in life are referred to as segmental progeroid syndromes. With the progress that has been made in understanding the etiologies of these conditions in the past decade, potential therapeutic options have begun to move from the realm of improbability to initial stages of testing. Among these syndromes, relevant advances have recently been made in Werner syndrome, one of several progeroid syndromes characterized by defective DNA helicases, and Hutchinson-Gilford progeria syndrome, which is characterized by aberrant processing of the nuclear envelope protein lamin A. Although best known for their causative roles in these illnesses, Werner protein and lamin A have also recently emerged as key players vulnerable to epigenetic changes that contribute to tumorigenesis and aging. These advances further demonstrate that understanding progeroid syndromes and introducing adequate treatments will not only prove beneficial to patients suffering from these dramatic diseases, but will also provide new mechanistic insights into cancer and normal aging processes. Received 28 July 2006; received after revision 5 September 2006; accepted 13 October 2006     

Indole-3-carbinol enhances ultraviolet B-induced apoptosis by sensitizing human melanoma cells

Indole-3-carbinol (I3C) has been found to act against several types of cancer, while ultraviolet B (UVB) is known to induce the apoptosis of human melanoma cells. Here, we investigated whether I3C can sensitize G361 human melanoma cells to UVB-induced apoptosis. We examined the effects of combined I3C and UVB (I3C/UVB) at various dosages. I3C (200 μM)/UVB (50 mJ/cm²) synergistically reduced melanoma cell viability, whereas I3C (200 μM) or UVB (50 mJ/cm²), separately, had little effect on cell viability. DNA fragmentation assays indicated that I3C/UVB induced apoptosis. Further results show that I3C/UVB activates caspase-8, −3, and Bid and causes the cleavage of poly(ADP-ribose) polymerase. Moreover, I3C decreased the expression of the anti-apoptotic protein, Bcl-2, whereas UVB increased the translocation of Bax to mitochondria. Thus, an increased Bax/Bcl-2 ratio by I3C/UVB may result in melanoma apoptosis. In conclusion, our study demonstrated that I3C sensitizes human melanoma cells by down-regulating Bcl-2. Received 5 July 2006; received after revision 25 August 2006; accepted 11 September 2006     

刚地弓形虫培养上清抑制THP-1细胞增殖及诱导凋亡的研究

摘要：目的提取弓形虫体外细胞共培养上清，并研究上清对人急性单核细胞白血病细胞THP-1增殖及凋亡的影响。方法收集对数生长期的THP-1细胞以5X10^7／ml细胞浓度接种于不同培养瓶中，对照组加入含10％胎牛血清的RPMll640，实验组加入相同体积不同数量（2×10^7／ml、4X10^7／ml、8×10^7／m1）弓形虫速殖子培养上清，采用四甲基氮噻唑蓝（MTY）法检测吸光度（A490值）并计算THP-1细胞增殖抑制率；倒置显微镜下观察细胞形态变化；Annexin-V-FITC／PI染色细胞后上流式细胞仪检测各个时间点细胞凋亡率变化，以Western印迹方法分析凋亡相关蛋白Bax、Bcl-2的表达或活性。结果MTY法检测结果弓形虫培养上清呈时间剂量依赖性抑制THP-1细胞株增殖，倒置显微镜下观察处理组细胞有发泡现象和凋亡小体出现。流式细胞仪检测弓形虫感染后的THP-1细胞凋亡率较对照组有升高趋势（P〈0．05），呈量效依赖性，Westernblot检测刚地弓形虫培养上清作用于THP-l细胞48h后实验组的Bax、Bcl-2蛋白表达较对照组的比值分别有明显的升高与降低（P〈0．05）。结论刚地弓形虫速殖子培养上清对体外培养THP-l细胞增殖有明显的抑制作用，并可诱导THP-1细胞凋亡。     

Nuclear envelope and nuclear matrix: interactions and dynamics

The peripheral nuclear lamina is located near the nuclear inner membrane and consists of lamin filaments and integral membrane proteins, including the lamin B receptor and various isoforms of lamina-associated polypeptides (LAP) 1 and 2. Several nuclear membrane proteins also interact with chromatin proteins BAF and Hp1. Lamins in the nuclear interior associate with at least one soluble (non-membrane-bound) LAP2 isoform named LAP2alpha. The internal lamins, together with Tpr-based filaments that connect to nuclear pore complexes, are proposed to be major structural elements of the internal nuclear matrix. We describe the structural links between the peripheral lamina and the internal nuclear matrix that are thought to be mediated by LAP2 family members, filament protein Tpr and nucleoporin Nup153. These findings are discussed in relation to human diseases that arise from mutations in nuclear lamina proteins.     

青蒿水提液对肺癌A549细胞的影响

目的研究青蒿水提液对肺癌A549细胞株增殖的影响和诱导凋亡的情况。方法不同浓度青蒿水提液作用于细胞不同时间，四甲基氮噻唑蓝（MTT）法检测吸光度值（A490nm）并计算增殖抑制率；AnnexinV-FITC／PI荧光染色后流式细胞仪检测细胞凋亡率；并以荧光显微镜观察细胞形态改变情况；蛋白质印迹法分析细胞凋亡相关蛋白Bax、Bcl-2的表这。结果青蒿水提液呈时间和剂量依赖性抑制A549细胞增殖；荧光显微镜下A549细胞出现不同时期凋亡特征性改变；流式细胞仪检测细胞凋亡率随着药物浓度增加而升高；A549细胞株的Bax蛋白表达量增多、Bcl-2蛋白表达量下降。结论青蒿水提液促进体外培养的A549细胞株增殖抑制并诱导凋亡，其机制可能与A549细胞Bax表达上调和Bcl-2表达下调有关。     

MPTP慢性帕金森病小鼠海马内氧化应激与细胞凋亡蛋白的表达

目的 观察 MPTP慢性帕金森病小鼠海马氧化应激指标及细胞凋亡相关蛋白的表达.方法 应用MPTP制备慢性帕金森病模型,观察行为学改变,Morris水迷宫实验,检测模型组和对照组小鼠海马内超氧化物歧化酶（SOD）、还原型谷胱甘肽（GSH）及丙二醛（MDA）舍量变化,以及免疫组织化学方法、RT-PCR方法、Western blot方法检测海马Bax和Bel-2蛋白表达.结果 慢性帕金森病模型小鼠行为学特点为震颤、活动减少;水迷宫实验结果提示认知功能降低;与对照组海马比较：模型组海马SOD、GSH含量均下降,而MDA含量升高（P〈0.05）;模型组海马Bax蛋白表达增高,而Bel-2蛋白表达下降（P〈0.05）.结论 氧化应激在慢性帕全森病认知功能障碍发病机制中起重要作用,而Bax和Bcl-2蛋白参与了氧化应激诱导海马神经元凋亡的调控过程.     

Microtubule dynamics alter the interphase nucleus

Microtubules are known to drive chromosome movements and to induce nuclear envelope breakdown during mitosis and meiosis. Here we show that microtubules can enforce nuclear envelope folding and alter the levels of nuclear envelope-associated heterochromatin during interphase, when the nuclear envelope is intact. Microtubule reassembly, after chemically induced depolymerization led to folding of the nuclear envelope and to a transient accumulation of condensed chromatin at the site nearest the microtubule organizing center (MTOC). This microtubule-dependent chromatin accumulation next to the MTOC is dependent on the composition of the nuclear lamina and the activity of the dynein motor protein. We suggest that forces originating from simultaneous polymerization of microtubule fibers deform the nuclear membrane and the underlying lamina. Whereas dynein motor complexes localized to the nuclear envelope that slide along the microtubules transfer forces and/or signals into the nucleus to induce chromatin reorganization and accumulation at the nuclear membrane folds. Thus, our study identified a molecular mechanism by which mechanical forces generated in the cytoplasm reshape the nuclear envelope, alter the intranuclear organization of chromatin, and affect the architecture of the interphase nucleus.     

The plant nuclear envelope

This review summarizes our present knowledge about the composition and function of the plant nuclear envelope. Compared with animals or yeast, our molecular knowledge of the nuclear envelope in higher plants is in its infancy. However, there are fundamental differences between plants and animals in the structure and function of the nuclear envelope. This review will compare and contrast these differences for nuclear pore complexes, nuclear transport, inner nuclear envelope proteins and the role of the nuclear envelope during mitosis. In some cases, seemingly 'novel' aspects of plant nuclear envelope function may provide new insight into the animal cell nucleus.     

Pathophysiology of mitochondrial cell death control

Mitochondria have been recently recognized to play a major role in the control of apoptosis or programmed cell death. Permeabilization of mitochondrial membranes, a decisive feature of early cell death, is regulated by members of the Bcl-2 family which interact with the permeability transition pore complex (PTPC). Thus, the cytoprotective oncoprotein Bcl-2 stabilizes the mitochondrial membrane barrier function, whereas the tumor suppressor protein Bax permeabilizes mitochondrial membranes. The regulation of membrane permeabilization is intertwined with that of the bioenergetic and redox functions of mitochondria. The implications of alterations in the composition of the PTPC and in mitochondrial function for the pathophysiology of cancer (reduced apoptosis) and neurodegeneration (enhanced apoptosis) are discussed.     

The nuclear envelope: emerging roles in development and disease

The chromosomes of eukaryotic cells are separated from the cytoplasm by the nuclear envelope. The nuclear envelope includes two riveted membranes, plus embedded pore complexes that mediate nuclear import and export. In this sense, the nuclear envelope is truly a border zone. However, the envelope also links directly to chromosomes, and anchors two major infrastructures--the nuclear lamina and Tpr filaments--to the nuclear perimeter. Proteins of the nuclear envelope mediate a variety of fundamental activities, including DNA replication, gene expression and silencing, chromatin organization, cell division, apoptosis, sperm nuclear remodeling, the behavior of pronuclei, cell fate determination, nuclear migration and cell polarity. Furthermore, mutations in nuclear lamins and lamin-binding proteins cause tissue-specific inherited diseases. This special issue of Cell and Molecular Life Sciences is devoted to recent major advances in the characterization of nuclear envelope proteins and their roles. We offer here an overview of the topics covered in this issue of CMLS, and also discuss the emerging recognition that the nuclear envelope is an organelle critical for a wide range of genetic and developmental activity in multicellular organisms.     

Dynamics of the nuclear envelope at mitosis and during apoptosis

The nuclear envelope is a highly dynamic structure that reversibly disassembles and reforms at mitosis. The nuclear envelope also breaks down--irreversibly--during apoptosis, a process essential for development and tissue homeostasis. Analyses of fixed cells, time-lapse, imaging studies of live cells and the development of powerful cell-free extracts derived from gametes or mammalian somatic cells have provided insights on the fate of nuclear envelope proteins during mitosis and apoptosis, and on the mechanisms behind nuclear envelope modifications in these processes. In this review, we discuss evidence leading to our understanding of the dynamics of the nuclear envelope alterations at mitosis and during apoptosis. We also present novel imaging and genetic approaches to the study of nuclear envelope dynamics and function.     

Menadione-induced apoptosis and the degradation of lamin-like proteins in tobacco protoplasts

Detection of stereotypic hallmarks of apoptosis during cell death induced by menadione, including DNA laddering and the formation of apoptotic bodies, is reported. Comet assay and the TdT-mediated dUTP nick end labelling (TUNEL) procedure were also performed to detect DNA fragmentation. Inhibition of DNA fragmentation by Ac-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and phenylmethylsulfosyl (PMSF) implicated the involvement of caspase-like proteases in menadione-induced apoptosis in plants. We further studied the cleavage of lamin-like proteins during apoptosis in menadione-treated tobacco protoplasts. In animals, it has been reported that the solubilization of nuclear lamina and lamin degradation occurs during apoptotic cell death. However, little is known about the fate of lamins in apoptotic plant cells. Our study provided evidence that lamin-like proteins degraded into 35-kDa fragments in tobacco protoplasts induced by menadione, and this preceded DNA fragmentation. The results thus indicated that proteolytic cleavage of nuclear lamins was also conserved in programmed cell death in plants. Received 16 November 1998; received after revision 21 December 1998; accepted 23 December 1998