Living on the edge: the role of Atgolgin-84A at the plant ER-Golgi interface.

The plant Golgi apparatus is responsible for the processing of proteins received from the endoplasmic reticulum (ER) and their distribution to multiple destinations within the cell. Golgi matrix components, such as golgins, have been identified and suggested to function as putative tethering factors to mediate the physical connections between Golgi bodies and the ER network. Golgins are proteins anchored to the Golgi membrane by the C-terminus either through transmembrane domains or interaction with small regulatory GTPases. The golgin N-terminus contains long coiled-coil domains, which consist of a number of α-helices wrapped around each other to form a structure similar to a rope being made from several strands, reaching into the cytoplasm. In animal cells, golgins are also implicated in specific recognition of cargo at the Golgi.Here, we investigate the plant golgin Atgolgin-84A for its subcellular localization and potential role as a tethering factor at the ER-Golgi interface. For this, fluorescent fusions of Atgolgin-84A and an Atgolgin-84A truncation lacking the coiled-coil domains (Atgolgin-84AΔ1-557) were transiently expressed in tobacco leaf epidermal cells and imaged using high-resolution confocal microscopy. We show that Atgolgin-84A localizes to a pre-cis-Golgi compartment that is also labelled by one of the COPII proteins as well as by the tether protein AtCASP. Upon overexpression of Atgolgin-84A or its deletion mutant, transport between the ER and Golgi bodies is impaired and cargo proteins are redirected to the vacuole. LAY DESCRIPTION: The Golgi apparatus is a specialised compartment found in mammalian and plant cells. It is the post office of the cell and packages proteins into small membrane boxes for transport to their destination in the cell. The plant Golgi apparatus consist of many separate Golgi bodies and is responsible for the processing of proteins received from the endoplasmic reticulum (ER) and their distribution to multiple destinations within the cell. Specialised proteins called golgins have been suggested to tether Golgi bodies and the ER. Here we investigate the plant golgin Atgolgin-84A for its exact within the Golgi body and its potential role as a tethering factor at the ER-Golgi interface. For this, we have fused Atgolgin-84A with a fluorescent protein from jellyfish and we are producing this combination in tobacco leaf cells. This allows us to see the protein using laser microscopy. We show that Atgolgin-84A localises to a compartment between the ER and Golgi that is also labelled by the tether protein AtCASP. When Atgolgin-84A is produced in high amounts in the cell, transport between the ER and Golgi bodies is inhibited and proteins are redirected to the vacuole.

Brain Abnormalities in Patients with Germline Variants in H3F3: Novel Imaging Findings and Neurologic Symptoms Beyond Somatic Variants and Brain Tumors.

BACKGROUND AND PURPOSE: Pathogenic somatic variants affecting the genes Histone 3 Family 3A and 3B (H3F3) are extensively linked to the process of oncogenesis, in particular related to central nervous system tumors in children. Recently, H3F3 germline missense variants were described as the cause of a novel pediatric neurodevelopmental disorder. We aimed to investigate patterns of brain MR imaging of individuals carrying H3F3 germline variants. MATERIALS AND METHODS: In this retrospective study, we included individuals with proved H3F3 causative genetic variants and available brain MR imaging scans. Clinical and demographic data were retrieved from available medical records. Molecular genetic testing results were classified using the American College of Medical Genetics criteria for variant curation. Brain MR imaging abnormalities were analyzed according to their location, signal intensity, and associated clinical symptoms. Numeric variables were described according to their distribution, with median and interquartile range. RESULTS: Eighteen individuals (10 males, 56%) with H3F3 germline variants were included. Thirteen of 18 individuals (72%) presented with a small posterior fossa. Six individuals (33%) presented with reduced size and an internal rotational appearance of the heads of the caudate nuclei along with an enlarged and squared appearance of the frontal horns of the lateral ventricles. Five individuals (28%) presented with dysgenesis of the splenium of the corpus callosum. Cortical developmental abnormalities were noted in 8 individuals (44%), with dysgyria and hypoplastic temporal poles being the most frequent presentation. CONCLUSIONS: Imaging phenotypes in germline H3F3-affected individuals are related to brain features, including a small posterior fossa as well as dysgenesis of the corpus callosum, cortical developmental abnormalities, and deformity of lateral ventricles.

A mutation in the human MPDU1 gene causes congenital disorder of glycosylation type If (CDG-If).

We describe a new congenital disorder of glycosylation, CDG-If. The patient has severe psychomotor retardation, seizures, failure to thrive, dry skin and scaling with erythroderma, and impaired vision. CDG-If is caused by a defect in the gene MPDU1, the human homologue of hamster Lec35, and is the first disorder to affect the use, rather than the biosynthesis, of donor substrates for lipid-linked oligosaccharides. This leads to the synthesis of incomplete and poorly transferred precursor oligosaccharides lacking both mannose and glucose residues. The patient has a homozygous point mutation (221T-->C, L74S) in a semiconserved amino acid of MPDU1. Chinese hamster ovary Lec35 cells lack a functional Lec35 gene and synthesize truncated lipid-linked oligosaccharides similar to the patient's. They lack glucose and mannose residues donated by Glc-P-Dol and Man-P-Dol. Transfection with the normal human MPDU1 allele nearly completely restores normal glycosylation, whereas transfection with the patient's MPDU1 allele only weakly restores normal glycosylation. This work provides a new clinical picture for another CDG that may involve synthesis of multiple types of glycoconjugates.

Anticipating endoplasmic reticulum stress. A novel early response before pathogenesis-related gene induction

When it is attacked by a pathogen, a plant produces a range of defense-related proteins. Many of these are synthesized by the rough endoplasmic reticulum (RER) to be secreted from the cell or deposited in vacuoles. Genes encoding endoplasmic reticulum (ER)-resident chaperones, such as the lumenal binding protein (BiP), are also induced under these conditions. Here, we show that BiP induction occurs systemically throughout the plant. Furthermore, this induction occurs rapidly and precedes expression of genes encoding pathogenesis-related (PR) proteins. The underlying signal transduction pathway was shown to be independent of the signaling molecule salicylic acid and the unfolded protein response pathway. In addition, BiP induction was independent of PR gene induction. Overproduction of BiP alone was not sufficient to cause induction of PR gene expression; however, limiting the amount of BiP in the ER lumen via superimposed ER stress inhibited the induction of PR gene expression. We propose that the induction of BiP expression during plant-pathogen interactions is required as an early response to support PR protein synthesis on the RER and that a novel signal transduction pathway exists to trigger this rapid response.

BiP and calreticulin form an abundant complex that is independent of endoplasmic reticulum stress

BiP is found in association with calreticulin, both in the presence and absence of endoplasmic reticulum stress. Although the BiP-calreticulin complex can be disrupted by ATP, several properties suggest that the calreticulin associated with BiP is neither unfolded nor partially or improperly folded. (1) The complex is stable in vivo and does not dissociate during 8 hr of chase. (2) When present in the complex, calreticulin masks epitopes at the C terminus of BiP that are not masked when BiP is bound to an assembly-defective protein. And (3) overproduction of calreticulin does not lead to the recruitment of more BiP into complexes with calreticulin. The BiP-calreticulin complex can be disrupted by low pH but not by divalent cation chelators. When the endoplasmic reticulum retention signal of BiP is removed, complex formation with calreticulin still occurs, and this explains the poor secretion of the truncated molecule. Gel filtration experiments showed that BiP and calreticulin are present in distinct high molecular weight complexes in which both molecules interact with each other. The possible functions of this complex are discussed.

Saturation of the endoplasmic reticulum retention machinery reveals anterograde bulk flow

We have studied the possible mechanisms of endoplasmic reticulum (ER) export and retention by using natural residents of the plant ER. Under normal physiological conditions, calreticulin and the lumenal binding protein (BiP) are efficiently retained in the ER. When the ER retention signal is removed, truncated calreticulin is much more rapidly secreted than truncated BiP. Calreticulin carries two glycans of the typical ER high-mannose form. Both glycans are competent for Golgi-based modifications, as determined from treatment with brefeldin A or based on the deletion of the ER retention motif. In contrast to BiP, calreticulin accumulation is strongly dependent on its retention signal, thereby allowing us to test whether saturation of the retention mechanism is possible. Overexpression of calreticulin led to 100-fold higher levels in dilated globular ER cisternae as well as dilated nuclear envelopes and partial secretion of both BiP and calreticulin. This result shows that both molecules are competent for ER export and supports the concept that proteins are secreted by default. This result also supports previous data suggesting that truncated BiP devoid of its retention motif can be retained in the ER by association with calreticulin. Moreover, even under these saturating conditions, cellular calreticulin did not carry significant amounts of complex glycans, in contrast to secreted calreticulin. This result shows that calreticulin is rapidly secreted once complex glycans have been synthesized in the medial/trans Golgi apparatus and that the modified protein does not appear to recycle back to the ER.

Sorting of soluble proteins in the secretory pathway of plants.

The secretory pathway of plants is a network of organelles that communicate via vesicle transport. This process involves budding on donor membranes followed by their targeting to, recognition by and fusion with the acceptor membrane. Protein sorting through the plant secretory pathway is a process that requires the specific recognition of signals by receptor molecules. For soluble proteins, recognition takes place in the lumen of the secretory pathway. The sorting receptors must mediate signal transduction across the membrane to convey the information about the presence of cargo molecules to cytosolic factors, which regulate the formation of transport vesicles. Recently, a number of key elements in this process have been identified, providing tools to study protein sorting at the molecular level.

Dexamethasone induces partial resistance to cisplatinum in C6 glioma cells.

Malignant glioma patients are sometimes treated with cisplatinum (CDDP) and dexamethasone (DEX). The question, was addressed as to whether DEX induces cellular resistance to CDDP using the C6 glioma cell line in MTT-tests. 50% of the cells were killed by 2 x 10(-5) M, 5 x 10(-6) M, and 7 x 10(-7) M CDDP after 2, 24, and 72 hours of incubation, respectively. 10-6M DEX treatment protected C6 cells from CDDP 5 x 10(-5) M 72 hours, resulting in twice as many surviving cells, [p<0.01(t-test)]. This protection was also observed in human TE671 rhabdomyosarcoma and T98G human glioma cells but not in A172 human glioma cells. It was mediated by glucocorticoid receptors and increased glutathione. DEX reduced the sensitivity of C6 cells also to carboplatinum, doxorubicin, actinomycin D, cytosine-arabinoside and methotrexate but not to 4-hydroxyifosfamide, vincristine, radiation, 6-mercaptopurine or thioguanine. These data suggest a more restricted use of DEX during chemotherapy of brain tumour patients.

Falsification of tetrazolium dye (MTT) based cytotoxicity assay results due to mycoplasma contamination of cell cultures.

Mycoplasma contamination of cell cultures is a frequently observed problem. Due to the inconspicuous growth in cell cultures, periodical screening procedures represent the only protection. Many influences of mycoplasma on cell culture parameters have been described. We addressed the question of whether mycoplasma contamination affects the most frequently used cytotoxicity assay, the tetrazolium based MTT assay. We contaminated C6 glioma cells with mycoplasma and performed MTT assays with doxorubicin, vincristine, etoposide and cisplatinum under various conditions. Contaminated cells demonstrated significant different results when tested with the MTT assay than mycoplasma free controls. Differences were not detectable when cells were counted as toxicity assay. Due to an additional reduction of tetrazolium by mycoplasmas, contaminated cells appeared up to 15 fold resistant to doxorubicin, vincristine and etoposide, but not to cisplatinum. Differences decreased with decreasing drug doses and decreasing plated cell count. Our findings confirm the compelling need for periodical mycoplasma screening, especially when tetrazolium based cytotoxicity assay (MTT) are used.

Multiple drug-resistant C6 glioma cells cross-resistant to irradiation.

Although many advances in antineoplastic therapy have taken place, a clinical breakthrough in the therapy of malignant gliomas is still required. One of the reasons for this is the poor response to cytotoxic drugs and irradiation. We established a subline of the rat glioma cell line C6, named C6,5 x 10(-7) Dox, by exposure to increasing doses of doxorubicin for 5 months. C6,5 x 10(-7) Dox cells expressed high levels of P-glycoprotein (Pgp), known to function as an energy-dependent efflux pump for lipophilic drugs causing the multidrug resistance phenotype. Pgp, which normally has a molecular weight of 170 to 180 kd, appears in C6,5 x 10(-7) Dox cells as two bands with a molecular weight of 140 and 120 kd in western blots. In addition to the typical cross-resistance to doxorubicin, daunorubicin, vincristine and etoposide, we observed a significant resistance of the C6,5 x 10(-7) Dox cell line to irradiation, which cannot be explained by Pgp-expression.

Frontal lobe-dependent functions in treated phenylketonuria: blood phenylalanine concentrations and long-term deficits in adolescents and young adults.

Even early-treated phenylketonurics may suffer from phenylalanine-related deficits. Elevated phenylalanine concentrations can interfere with the development and function of the CNS. Outcome beyond childhood has not been extensively investigated. This long-term study was performed to determine whether adolescents and young adults with PKU show frontal lobe-dependent deficits when compared to diabetic patients. The comparative study covered 35 PKU patients, 13-21 years of age (mean 17.8 years), and 35 diabetic patients matched for sex, age and socioeconomic status. Patients were assessed for IQ (Culture Fair Intelligence Test), information processing (Trail Making Test), and selective and sustained attention (Stroop Task, Test d-2). Assessments were repeated within a 3-year follow-up. PKU patients showed no increase in blood phenylalanine concentrations at follow-up. They had significantly poorer test results than the diabetic patients at both assessment times. Within the tests, however, this was due to reduced performance speed but not to deficits in specific frontal lobe-dependent functions. Elevated phenylalanine concentrations seem to exert a global effect slowing performance speed. This effect is enduring in adolescence and early adulthood.

Congenital disorders of glycosylation: review of their molecular bases, clinical presentations and specific therapies.

Congenital disorders of glycosylation (CDG, formerly named carbohydrate-deficient glycoprotein syndromes) are a rapidly growing family of inherited disorders affecting the assembly or processing of glycans on glycoconjugates. The clinical spectrum of the different types of CDG discovered so far is variable, ranging from severe multisystemic disorders to disorders restricted to specific organs. This review deals with clinical, diagnostic, and biochemical aspects of all characterized CDGs, including a disorder affecting the N-glycosylation of erythrocytes, congenital dyserythropoietic anemia type II (CDA II/HEMPAS), and the first disorders affecting O-glycosylation. Since the clinical spectrum of symptoms in CDG is variable and may be unspecific, a generous selective screening for the presence of CDG is recommended.

Protein secretion in plant cells can occur via a default pathway.

To study protein secretion in plant cells, we established and evaluated a model system based on transient synthesis of heterologous proteins in tobacco protoplasts. We show that the nonsecretory enzymes phosphinothricin acetyl transferase, neomycin phosphotransferase II, and beta-glucuronidase are secreted when targeted to the lumen of the endoplasmic reticulum by signal peptide-mediated translocation. These data are consistent with the view that secretion can occur independent of active sorting mechanisms by nonspecific migration through the exocytic pathway. However, the rate of secretion differs significantly among these enzymes. Furthermore, the presence of signal sequences was found to be correlated with a reduction of the levels of the encoded gene products. This is the result of post-transcriptional events that limit either synthesis or stability of the proteins in vivo.

Plant and mammalian sorting signals for protein retention in the endoplasmic reticulum contain a conserved epitope.

We studied protein sorting signals which are responsible for the retention of reticuloplasmins in the lumen of the plant endoplasmic reticulum (ER). A non-specific passenger protein, previously shown to be secreted by default, was used as a carrier for such signals. Tagging with C-terminal tetrapeptide sequences of mammalian (KDEL) and yeast (HDEL) reticuloplasmins led to effective accumulation of the protein chimeras in the lumen of the plant ER. Some single amino acid substitutions within the tetrapeptide tag (-SDEL, -KDDL, -KDEI and -KDEV) can cause a complete loss of its function as a retention signal, demonstrating the high specificity of the retention machinery. However, other modifications confer efficient (-RDEL) or partial (-KEEL) retention. It is also shown that the efficiency of protein retention is not significantly impaired by an increased ligand concentration in plants. The efficiently retained chimeras (-KDEL, -HDEL and -RDEL) were shown to be recognized by a monoclonal antibody directed against the C-terminus of the mammalian reticuloplasmin protein disulfide isomerase (PDI). The recognized epitope is also present in several putative reticuloplasmins in microsomal fractions of plant and mammalian cells, suggesting that the antibodies recognize an important structural determinant of the retention signal. In addition, data are discussed which support the view that upstream sequences beyond the C-terminal tetrapeptide can influence or may be part of the structure of reticuloplasmin retention signals.

Diagnosis of N-acetylglutamate synthase deficiency by use of cultured fibroblasts and avoidance of nonsense-mediated mRNA decay.

Molecular diagnosis of N-acetylglutamate synthase deficiency (NAGSD) has become possible now that the corresponding gene has been identified. We describe the genetic analysis of a patient with NAGSD using low-level transcripts derived from cultured fibroblasts. One defective allele (c.1306-1307insT) was detected by PCR amplification. However, the transcript from a second mutation (IVS3 - 2A>T), causing aberrant splicing with the generation of a premature termination codon, was not detected until interference of nonsense-mediated mRNA decay was abrogated by the translation inhibitor cycloheximide. We demonstrate that low-level transcripts in cells that do not express significant enzyme activity are a valuable tool for molecular studies of genetic alterations, and suggest routine abrogation of nonsense-mediated mRNA decay using cycloheximide when transcript analysis is performed.

In situ localization and in vitro induction of plant COPI-coated vesicles.

Coat protein (COP)-coated vesicles have been shown to mediate protein transport through early steps of the secretory pathway in yeast and mammalian cells. Here, we attempt to elucidate their role in vesicular trafficking of plant cells, using a combined biochemical and ultrastructural approach. Immunogold labeling of cryosections revealed that COPI proteins are localized to microvesicles surrounding or budding from the Golgi apparatus. COPI-coated buds primarily reside on the cis-face of the Golgi stack. In addition, COPI and Arf1p show predominant labeling of the cis-Golgi stack, gradually diminishing toward the trans-Golgi stack. In vitro COPI-coated vesicle induction experiments demonstrated that Arf1p as well as coatomer could be recruited from cauliflower cytosol onto mixed endoplasmic reticulum (ER)/Golgi membranes. Binding of Arf1p and coatomer is inhibited by brefeldin A, underlining the specificity of the recruitment mechanism. In vitro vesicle budding was confirmed by identification of COPI-coated vesicles through immunogold negative staining in a fraction purified from isopycnic sucrose gradient centrifugation. Similar in vitro induction experiments with tobacco ER/Golgi membranes prepared from transgenic plants overproducing barley alpha-amylase-HDEL yielded a COPI-coated vesicle fraction that contained alpha-amylase as well as calreticulin.

Neurofibromatosis type 1: motor and cognitive function and T2-weighted MRI hyperintensities.

BACKGROUND: Cognitive impairment and fine motor deficits are frequent manifestations in patients with neurofibromatosis type 1 (NF1). More than 50% of patients with NF1 show focal areas of high signal intensity (T2H) on T2-weighted MRI of the brain. It has been hypothesized that T2H may be associated with the cognitive and motor problems. METHODS: The authors investigated 100 patients with NF1 and 100 healthy control subjects matched for age, sex, and socioeconomic status for their IQ (Wechsler Intelligence Scale for Children-Revised [WISC-R] and Wechsler Adult Intelligence Scale-Revised [WAIS-R]), fine motor abilities (Motorische Leistungs-Serie [motor performance task]), and T2H (MRI). RESULTS: As a group, the 100 patients performed within normal limits of WISC-R and WAIS-R scores. However, the scores for the NF1 patients with normal MRI were at the mean, whereas those for the patients who had T2H (n = 58) were significantly depressed. On measures of fine motor skills, patients with T2H performed poorer than patients with normal MRI. CONCLUSION: Hyperintensities on T2-weighted MRI represent a biological marker for impaired cognitive and fine motor performance in patients with NF1.

Regression of neuropsychological deficits in early-treated phenylketonurics during adolescence.

Even early-treated phenylketonuric patients suffer from phenylalanine-associated (mild) neuropsychological impairment. To date it is still unclear whether patients' deficits show a progression on ageing. This unsolved question seems to be an important aspect in the still ongoing debate about how long and how strictly the patients should be maintained on diet. Twenty early-treated (20 +/- 10, 9-30 days) adolescent phenylketonurics (10 boys, 10 girls) and 20 healthy controls, matched for age, sex and IQ, were investigated twice at a mean ages of 11 and 14 years for their IQ (Culture Fair Intelligence Test-Scale 2; CFT-20), fine motor abilities (Motor Performance Task), sustained (Test d2) and selective attention (Stroop-Task). At the first test, examinations revealed significant blood phenylalanine-correlated neuropsychological deficits in PKU patients. In spite of raised blood phenylalanine concentrations during the following 3 years and significantly elevated concurrent blood phenylalanine concentrations, the repeated measurements revealed a significant decrease of patients' deficits compared to controls. Clinical-neurological status of patients and controls was normal at both test times. The results indicate a decreased vulnerability of PKU-patients with respect to their neuropsychological functioning against elevated blood phenylalanine levels on ageing.