Association of Mitochondria with Plectin and Desmin Intermediate Filaments in Striated Muscle

Plectin (M(r) > 500,000) is a versatile and widely expressed cytolinker protein. In striated muscle it is predominantly found at the Z-disc level where it colocalizes with the intermediate filament protein desmin. Both proteins show altered labeling patterns in tissues of muscular dystrophy patients. Moreover, mutations in the plectin gene lead to the autosomal recessive human disorder epidermolysis bullosa simplex with muscular dystrophy, and defects in the desmin gene have been shown to cause familiar cardiac and skeletal myopathy. Since intermediate filaments (IFs) in striated muscle tissue have been found to be intimately associated with mitochondria, we investigated whether plectin is involved in this association. Using postembedding immunogold labeling of Lowicryl sections and immunogold labeling of ultrathin cryosections, we show that plectin is associated with desmin IFs linking myofibrils to mitochondria at the level of the Z-disc and along the entire length of the sarcomere. The localization of plectin label at the mitochondrial membrane itself was consistent with a putative linker function of plectin between desmin IFs and the mitochondrial surface. In mitochondrion-rich muscle fibers, both plectin and desmin were part of an ordered arrangement of mitochondrial side branches, which wound around myofibrils adjacent to the Z-discs and were anchored into a filamentous network transversing from one fibril to the other. The association of mitochondria with plectin and IFs was seen also in tissues without regular distribution patterns of mitochondria, such as heart muscle and neonatal skeletal muscle tissues. These data were supplemented with in vitro binding assays showing direct interaction of plectin with desmin via its carboxy-terminal IF-binding domain. As a cytolinker protein associated with mitochondria and desmin IFs, plectin could play an important role in the positioning and shape formation, in particular branching, of mitochondrial organelles in striated muscle tissues.     

Plectin deficiency in liver cancer cells promotes cell migration and sensitivity to sorafenib treatment

Plectin involved in activation of kinases in cell signaling pathway and plays important role in cell morphology and migration. Plectin knockdown promotes cell migration by activating focal adhesion kinase and Rac1-GTPase activity in liver cells. Sorafenib is a multi-targeting tyrosine kinase inhibitor that improves patient survival on hepatocellular carcinoma. The aim of this study is to investigate the correlation between the expression of plectin and cell migration as well as the sensitivity of hepatoma cell lines exposing to sorafenib. Hepatoma cell lines PLC/PRF/5 and HepG2 were used to examine the level of plectin expression and cell migration in comparison with Chang liver cell line. In addition, sensitivity of the 3 cell lines to sorafenib treatment was also measured. Expression of plectin was lower in PLC/PRF/5 and HepG2 hepatoma cells than that of Chang liver cells whereas HepG2 and PLC/PRF/5 cells exhibit higher rate of cell migration in trans-well migration assay. Immunohistofluorecent staining on E-cadherin revealed the highest rate of collective cell migration in HepG2 cells and the lowest was found in Chang liver cells. Likewise, HepG2 cell line was most sensitive to sorafenib treatment and Chang liver cells exhibited the least sensitivity. The drug sensitivity to sorafenib treatment showed inverse correlation with the expression of plectin. We suggest that plectin deficiency and increased E-cadherin in hepatoma cells were associated with higher rates of cell motility, collective cell migration as well as higher drug sensitivity to sorafenib treatment.     

Glucose Transporter Isoform Expression in Huntington's Disease Brain

Abstract: Several reports have suggested a characteristic decrease in glucose use in the striatum of patients with Huntington's disease (HD) may contribute to the cellular atrophy of the caudate and putamen. We examined the expression of the two major glucose transporter isoforms of brain, GLUT1 and GLUT3. GLUT1 is found largely in capillary endothelial cells and to a lesser extent in the brain parenchyma, whereas GLUT3 is localized primarily in neurons. Membranes prepared from postmortem samples of HD caudate and cortex and non-HD caudate and cortex were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and probed with antisera to GLUT1 and GLUT3 by western blotting. Compared with controls, GLUT1 and GLUT3 transporter expression in caudate was decreased by three- and fourfold, respectively, in grade 3 of the disease. At earlier stages (grade 1), there was no significant difference in the expression of the two transporter isoforms compared with nondiseased controls. It is surprising that despite a substantial increase in glial fibrillary acidic protein immunoreactivity (an indicator of the extent of gliosis), glucose transporter expression was diminished significantly in HD caudate. The results suggest in the absence of a significant number of neurons, as in grade 3, glial cell GLUT1 and GLUT3 expression is down-regulated, perhaps reflecting the decreased metabolic demand of this brain region in HD.     

A Nonneuronal Isoform of Cell Adhesion Molecule L1: Tissue-Specific Expression and Functional Analysis

Abstract: The cell adhesion molecule L1 is a multifunctional protein in the nervous system characterizing cell adhesion, migration, and neurite outgrowth. In addition to full-length L1, we found an alternatively spliced variant lacking both the KGHHV sequence in the extracellular part and the RSLE sequence in the cytoplasmic part of L1. This L1 variant was expressed exclusively in nonneuronal cells such as Schwann cells, astrocytes, and oligodendrocytes, in contrast to the expression of the full-length L1 in neurons and cells of neuronal origin. To investigate the functions of the L1 variant, we established cell lines transfected with a cytoplasmic short L1 (L1cs) cDNA that lacks only the 12-bp segment encoding for the RSLE sequence. The promoting activities of homophilic cell adhesion, neurite outgrowth, and neuronal cell migration of L1cs-transfected cells (L4-2) were similar to those of full-length L1-transfected cells (L3-1), but the cell migratory activity of L4-2 itself was clearly lower than that of L3-1. In conclusion, the short form of L1 is a nonneuronal type, in contrast to the neuronal type of the full-length L1. Deletion of the four amino acids RSLE in the cytoplasmic region of L1 markedly reduced cell migratory activity, suggesting an importance of the RSLE sequence for the signaling events of neuronal migration mediated by L1.     

Cell-Type-Specific Profiling of Alternative Translation Identifies Regulated Protein Isoform Variation in the Mouse Brain

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Selective Expression of Myosin IC Isoform A in Mouse and Human Cell Lines and Mouse Prostate Cancer Tissues

Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C). Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP) model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate-) cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN) lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.     

Expression Pattern of ALOXE3 in Mouse Brain Suggests Its Relationship with Seizure Susceptibility

Cellular and Molecular Neurobiology - Arachidonic acid (AA), a polyunsaturated fatty acid, is involved in the modulation of neuronal excitability in the brain. Arachidonate lipoxygenase 3 (ALOXE3),...     

Tropomyosin in Preimplantation Mouse Development: Identification, Expression, and Organization during Cell Division and Polarization

Tropomyosin is an actin-binding cytoskeletal protein which has been extensively characterized in a variety of cell types and tissues, with the exception of very early developmental stages during which cellular polarization first occurs. We have identified five polypeptides in mouse preimplantation conceptuses which show many of the characteristics of tropomyosin. They form the major portion of the heat-stable cytoskeletal protein fraction of blastomeres and have the characteristic isoelectric and SDS–PAGE migration characteristics on 1-D and 2-D gels. All five polypeptides were synthesized in late 2- and 4-cell, and all 8-cell stages, with three of the five polypeptides showing lower synthetic levels in fertilized eggs and early 2-cell conceptuses. These heat-stable proteins showed specific differences from proteins isolated from mouse 3T3 fibroblasts by the same method, namely higherM_risoforms were not represented, also some of the isoforms can be labeled by incorporation of [¹⁴C]proline. The cellular distribution of tropomyosin in early stage conceptuses was examined using monoclonal and affinity-purified polyclonal antibodies. Tropomyosin becomes associated both with the blastomere cortex postfertilization and with the cleavage furrow during cytokinesis. The interphase cortical association is uniform until the 8-cell stage, when tropomyosin becomes associated with the developing apical pole and is excluded from the basolateral cortex. This polar localization is inherited along with the pole at the 8- to 16-cell division, but experiments in which cell division is artificially prolonged show that tropomyosin localization does not represent a permanent marking of the pole. We conclude that the early mouse conceptus contains a unique and specific set of tropomyosins which respond to polarizing signals.     

Identification of Atg8 Isoform in Encysting Acanthamoeba

Autophagy-related protein 8 (Atg8) is an essential component of autophagy formation and encystment of cyst-forming parasites, and some protozoa, such as, Acanthamoeba, Entamoeba, and Dictyostelium, have been reported to possess a type of Atg8. In this study, an isoform of Atg8 was identified and characterized in Acanthamoeba castellanii (AcAtg8b). AcAtg8b protein was found to encode 132 amino acids and to be longer than AcAtg8 protein, which encoded 117 amino acids. Real-time PCR analysis showed high expression levels of AcAtg8b and AcAtg8 during encystation. Fluorescence microscopy demonstrated that AcAtg8b is involved in the formation of the autophagosomal membrane. Chemically synthesized siRNA against AcAtg8b reduced the encystation efficiency of Acanthamoeba, confirming that AcAtg8b, like AcAtg8, is an essential component of cyst formation in Acanthamoeba. Our findings suggest that Acanthamoeba has doubled the number of Atg8 gene copies to ensure the successful encystation for survival when 1 copy is lost. These 2 types of Atg8 identified in Acanthamoeba provide important information regarding autophagy formation, encystation mechanism, and survival of primitive, cyst-forming protozoan parasites.     

Isoform switching facilitates period control in the Neurospora crassa circadian clock

A striking and defining feature of circadian clocks is the small variation in period over a physiological range of temperatures. This is referred to as temperature compensation, although recent work has suggested that the variation observed is a specific, adaptive control of period. Moreover, given that many biological rate constants have a Q₁₀ of around 2, it is remarkable that such clocks remain rhythmic under significant temperature changes. We introduce a new mathematical model for the Neurospora crassa circadian network incorporating experimental work showing that temperature alters the balance of translation between a short and long form of the FREQUENCY (FRQ) protein. This is used to discuss period control and functionality for the Neurospora system. The model reproduces a broad range of key experimental data on temperature dependence and rhythmicity, both in wild‐type and mutant strains. We present a simple mechanism utilising the presence of the FRQ isoforms (isoform switching) by which period control could have evolved, and argue that this regulatory structure may also increase the temperature range where the clock is robustly rhythmic.     

Expression of Alternative Ago2 Isoform Associated with Loss of microRNA-Driven Translational Repression in Mouse Oocytes

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Effects of Naltrexone and Acamprosate on Alcohol-Induced NGFI-A Expression in Mouse Brain

In search for the substrate of naltrexone and acamprosate action on alcohol craving, we investigated the effects of ethanol alone and combined with naltrexone or acamprosate on expression of nerve growth factor-inducible clone A (NGFI-A; zif268). In Experiments 1 and 3, alcohol (2 g/kg) alone or in combination with naltrexone (15 mg/kg) or acamprosate (300 mg/kg) was injected intraperitoneally into mice. In Experiment 2, treatment was nor-BNI (0.5 mg/kg) to investigate whether the effect of naltrexone involved blockade of κ-opioid receptors. Both ethanol and naltrexone alone induced NGFI-A in the central amygdala, but not in several other areas; these effects were additive. However, acamprosate alone or in combination with ethanol had no effect on NGFI-A mRNA, while nor-BNI induced NGFI-A mRNA in the basolateral amygdala. The central amygdala appears to be an important target of both alcohol and naltrexone. Acamprosate may not share the site of action with naltrexone despite being used for the same therapeutic purpose. Special issue article in honor of Dr.Ji-Sheng Han.     

干扰素调节因子新剪接异构体IRF7-e的结构及功能

干扰素调节因子7是诱导玉型干扰素表达的最主要的转录因子。寻找IRF7新的剪接异构体,研究其结构及功能,为探索IRF7参与调控玉型干扰素机制的多样性提供基础。通过PCR和Sanger测序获得了IRF7一种新的剪接形式IRF7-e,并通过RACE获取了IRF7-e 基因全长。IRF7-e全长为1994 bp,含5'-UTR 410 bp,3'-UTR 120 bp,开放阅读框1464 bp,编码487个氨基酸的蛋白,预测其等电点为6.659,蛋白分子量为52.8 kD。双荧光素酶报告分析表明过表达IRF7-e能够提高玉型干扰素IFNα和IFNβ启动子的活性,其中对IFNα启动子活性提高了12.18倍,对IFNβ的启动子活性提高了2.99倍。表明IRF7-e可能参与玉型干扰素的调控。     

Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1^−/− and ROCK2^−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1^−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2^−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.     

Cell-Type-Specific Gene Expression Profiling in Adult Mouse Brain Reveals Normal and Disease-State Signatures

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Efficient Down-Regulation of Glia Maturation Factor Expression in Mouse Brain and Spinal Cord

Long-lasting siRNA-based down-regulation of gene of interest can be achieved by lentiviral-based expression vectors driving the production of short hairpin RNA (shRNA). We investigated an attractive therapeutic approach to target the expression of proinflammatory GMF by using lentiviral vector encoding GMF-specific shRNA to reduce GMF levels in the spinal cord and brain of mice. To determine the effect of GMF-shRNA on GMF protein levels, we performed quantitative ELISA analysis in brain and in thoracic, cervical and lumbar regions of spinal cord from mice followed by GMF-shRNA (G-shRNA) or control shRNA (C-shRNA) treatments. Our results show a marked reduction of GMF protein levels in brain and spinal cord of mice treated with GMF-shRNA compared to control shRNA treatment. Consistent with the GMF protein analysis, the immunohistochemical examination of the spinal cord sections of EAE mice treated with GMF-shRNA showed significantly reduced GMF-immunoreactivity. Thus, the down-regulation of GMF by GMF-shRNA was efficient and wide spread in CNS as evident by the significantly reduced levels of GMF protein in the brain and spinal cord of mice.