NRAS Q61R immunohistochemical staining in thyroid pathology: sensitivity, specificity and utility.

AIMS: The diagnosis of thyroid neoplasms relies upon the demonstration of histological parameters that can be focal and prone to subjective interpretation. We evaluated the utility of NRAS Q61R immunohistochemistry (IHC) in the diagnosis of thyroid lesions after determining its specificity and sensitivity as a surrogate marker for RAS Q61R mutation. METHOD AND RESULTS: NRAS Q61R IHC was performed on 282 primary or metastatic thyroid lesions from 256 patients. RAS mutation status was collected from patients' charts. Sensitivity and specificity of NRAS Q61R IHC for detecting a RAS Q61R mutation was calculated. IHC-positive cases were reviewed to determine the diagnostic utility of NRAS Q61R IHC. NRAS Q61R immunopositivity was seen in non-neoplastic, benign and malignant thyroid lesions. NRAS Q61R antibody cross-reactivity led to the detection of NRAS Q61R, KRAS Q61R and HRAS Q61R proteins. Among primary thyroid carcinomas, immunopositivity was most frequent in papillary thyroid carcinomas, follicular variant (48.0%). The sensitivity and specificity of NRAS Q61R IHC in detecting RAS Q61R mutation was 90.6% and 92.3%, respectively. When positive, the NRAS Q61R stain was determined to be helpful in demonstrating infiltration, tumour size, capsular and/or vascular invasion and multifocality. CONCLUSION: NRAS Q61R IHC is highly sensitive and specific for the detection of RAS Q61R mutations in thyroid pathology and is particularly relevant in follicular-patterned neoplasms. When evaluated alongside histological features, NRAS Q61R immunoreactivity can be instrumental in the diagnosis and classification of thyroid nodules.

MITOL regulates endoplasmic reticulum-mitochondria contacts via Mitofusin2.

The mitochondrial ubiquitin ligase MITOL regulates mitochondrial dynamics. We report here that MITOL regulates mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) domain formation through mitofusin2 (Mfn2). MITOL interacts with and ubiquitinates mitochondrial Mfn2, but not ER-associated Mfn2. Mutation analysis identified a specific interaction between MITOL C-terminal domain and Mfn2 HR1 domain. MITOL mediated lysine-63-linked polyubiquitin chain addition to Mfn2, but not its proteasomal degradation. MITOL knockdown inhibited Mfn2 complex formation and caused Mfn2 mislocalization and MAM dysfunction. Sucrose-density gradient centrifugation and blue native PAGE retardation assay demonstrated that MITOL is required for GTP-dependent Mfn2 oligomerization. MITOL knockdown reduced Mfn2 GTP binding, resulting in reduced GTP hydrolysis. We identified K192 in the GTPase domain of Mfn2 as a major ubiquitination site for MITOL. A K192R mutation blocked oligomerization even in the presence of GTP. Taken together, these results suggested that MITOL regulates ER tethering to mitochondria by activating Mfn2 via K192 ubiquitination.

Targeted Proteomic Analysis of Small GTPases in Murine Adipogenesis.

Small GTPases are essential signaling molecules for regulating glucose uptake in adipose tissues upon insulin stimulation, and this regulation maintains an appropriate range of glycemia. The involvement of small GTPases in adipogenesis, however, has not been systemically investigated. In this study, we applied a high-throughput scheduled multiple-reaction monitoring (MRM) method, along with the use of synthetic stable isotope-labeled peptides, to identify differentially expressed small GTPase proteins during adipogenesis of cultured murine cells. We were able to quantify the relative levels of expression of 55 and 49 small GTPases accompanied by adipogenic differentiation in 3T3-L1 and C3H10T1/2 cells, respectively. When compared with analysis conducted in the data-dependent acquisition (DDA) mode, the MRM-based proteomic platform substantially increased the coverage of the small GTPase proteome. Western blot analysis further corroborated the MRM quantification results for selected small GTPases. Interestingly, overall a significant number of small GTPases were down-regulated during adipogenesis. Among them, the expression levels of Rab32 protein were consistently lower in differentiated adipocytes than the corresponding undifferentiated precursors in both cell lines. Overexpression of Rab32 in 3T3-L1 and C3H10T1/2 cells prior to adipogenesis induction suppressed their differentiation. Together, this is the first comprehensive analysis of the alterations in small GTPase proteome during adipogenesis, and we reveal a previously unrecognized role of Rab32 in adipogenic differentiation.

Immobilized metal affinity cryogel-based high-throughput platform for screening bioprocess and chromatographic parameters of His6-GTPase.

Among various tools of product monitoring, chromatography is of vital importance as it also extends to the purification of product. Immobilized metal affinity cryogel (Cu(II)-iminodiacetic acid- and Ni(II)-nitrilotriacetic acid-polyacrylamide) minicolumns (diameter 8 mm, height 4 mm, void volume 250 µl) were inserted in open-ended 96-well plate and different chromatographic parameters and bioprocess conditions were analysed. The platform was first validated with lysozyme. Optimum binding of lysozyme (∼90%) was achieved when 50 µg of protein in 20 mM Tris, pH 8.0 was applied to the minicolumns with maximum recovery (∼90%) upon elution with 300 mM imidazole. Thereafter, the platform was screened for chromatographic conditions of His6-GTPase. Since cryogels have large pore size, they can easily process non-clarified samples containing debris and particulate matters. The bound enzymes on the gel retain its activity and therefore can be assayed on-column by adding substrate and then displacing the product. Highest binding of His6-GTPase was achieved when 50 µl of non-clarified cell lysate was applied to the cryogel and subsequently washed with 50 mM Tris, 150 mM NaCl, 5 mM MgCl2, 10 mM imidazole, pH 8.0 with dynamic and static binding capacities of ∼1.5 and 3 activity units. Maximum recovery was obtained upon elution with 300 mM imidazole with a purification fold of ∼10; the purity was also analysed by SDS-PAGE. The platform showed reproducible results which were validated by Bland-Altman plot. The minicolumn was also scaled up for chromatographic capture and recovery of His6-GTPase. The bioprocess conditions were monitored which displayed that optimum production of His6-GTPase was attained by induction with 200 µM isopropyl-ß-D-thiogalactoside at 25 °C for 12 h. It was concluded that immobilized metal affinity cryogel-based platform can be successfully used as a high-throughput platform for screening of bioprocess and chromatographic parameters. Graphical abstract Capture and on-column analysis of bound enzyme from non-clarified cell lysate on immobilized metal affinity cryogel minicolumn-based high-throughput platform.

Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection.

BACKGROUND: The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse. RESULTS: We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family). In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes. Another GMS protein, Irgm3, is localized to endoplasmic reticulum (ER) membranes; in the Irgm3-deficient mouse, activated GKS proteins are found at the ER. The Irgm3-deficient mouse does not show the drastic phenotype of the Irgm1 mouse. In the Irgm1/Irgm3 double knock-out mouse, activated GKS proteins associate with lipid droplets, but not with lysosomes, and the Irgm1/Irgm3(-/-) does not have the generalized immunodeficiency phenotype expected from its Irgm1 deficiency. CONCLUSIONS: The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles. Our data suggest that the generalized lymphomyeloid collapse that occurs in Irgm1(-/-) mice upon infection with a variety of pathogens may be due to lysosomal damage caused by off-target activation of GKS proteins on lysosomal membranes and consequent failure of autophagosomal processing.

Interferon-inducible GTPase: a novel viral response protein involved in rabies virus infection.

Rabies virus infection is a major public health concern because of its wide host-interference spectrum and nearly 100 % lethality. However, the interactions between host and virus remain unclear. To decipher the authentic response in the central nervous system after rabies virus infection, a dynamic analysis of brain proteome alteration was performed. In this study, 104 significantly differentially expressed proteins were identified, and intermediate filament, interferon-inducible GTPases, and leucine-rich repeat-containing protein 16C were the three outstanding groups among these proteins. Interferon-inducible GTPases were prominent because of their strong upregulation. Moreover, quantitative real-time PCR showed distinct upregulation of interferon-inducible GTPases at the level of transcription. Several studies have shown that interferon-inducible GTPases are involved in many biological processes, such as viral infection, endoplasmic reticulum stress response, and autophagy. These findings indicate that interferon-inducible GTPases are likely to be a potential target involved in rabies pathogenesis or the antiviral process.

Solid-state 31P NMR investigation on the status of guanine nucleotides in paclitaxel-stabilized microtubules.

Microtubule dynamics is a target for many chemotherapeutic drugs. In order to understand the biochemical effects of paclitaxel on the GTPase activity of tubulin, the status of guanine nucleotides in microtubules was investigated by (31)P cross-polarization magic angle spinning (CPMAS) NMR. Microtubules were freshly prepared in vitro in the presence of paclitaxel and then lyophilized in sucrose buffer for solid-state NMR experiments. A (31)P CPMAS NMR spectrum with the SNR of 25 was successfully acquired from the lyophilized microtubule sample. The broadness of the (31)P spectral lines in the spectrum indicates that the molecular environments around the guanine nucleotides inside tubulin may not be as crystalline as reported by many diffraction studies. Deconvolution of the spectrum into four spectral components was carried out in comparison with the (31)P NMR spectra obtained from five control samples. The spectral analysis suggested that about 13% of the nucleotides were present as GTP and 37% as GDP in the ß-tubulin (E-site) of the microtubules. It was found that most of the GDPs were present as GDP-Pi complex in the microtubules, which seems to be one of the effects of paclitaxel binding.

In astrocytes the accumulation of the immunity-related GTPases Irga6 and Irgb6 at the vacuole of Toxoplasma gondii is dependent on the parasite virulence.

Toxoplasma gondii is an obligate intracellular protozoan parasite responsible for a common infection of the central nervous system. Interferon (IFN) γ is the key cytokine of host defence against T. gondii. However, T. gondii strains differ in virulence and T. gondii factors determining virulence are still poorly understood. In astrocytes IFN γ primarily induces immunity-related GTPases (IRGs), providing a cell-autonomous resistance system. Here, we demonstrate that astrocytes prestimulated with IFN γ inhibit the proliferation of various avirulent, but not virulent, T. gondii strains. The two analyzed immunity-related GTPases Irga6 and Irgb6 accumulate at the PV only of avirulent T. gondii strains, whereas in virulent strains this accumulation is only detectable at very low levels. Both IRG proteins could temporarily be found at the same PV, but did only partially colocalize. Coinfection of avirulent and virulent parasites confirmed that the accumulation of the two analyzed IRGs was a characteristic of the individual PV and not determined by the presence of other strains of T. gondii in the same host cell. Thus, in astrocytes the accumulation of Irga6 and Irgb6 significantly differs between avirulent and virulent T. gondii strains correlating with the toxoplasmacidal properties suggesting a role for this process in parasite virulence.

Identification of a novel alternatively spliced isoform of the ribosomal uL10 protein.

Alternative splicing is one of the key mechanisms extending the complexity of genetic information and at the same time adaptability of higher eukaryotes. As a result, the broad spectrum of isoforms produced by alternative splicing allows organisms to fine-tune their proteome; however, the functions of the majority of alternatively spliced protein isoforms are largely unknown. Ribosomal protein isoforms are one of the groups for which data are limited. Here we report characterization of an alternatively spliced isoform of the ribosomal uL10 protein, named uL10ß. The uL10 protein constitutes the core element of the ribosomal stalk structure within the GTPase associated center, which represents the landing platform for translational GTPases - trGTPases. The stalk plays an important role in the ribosome-dependent stimulation of GTP by trGTPases, which confer unidirectional trajectory for the ribosome, allosterically contributing to the speed and accuracy of translation. We have shown that the newly identified uL10ß protein is stably expressed in mammalian cells and is primarily located within the nuclear compartment with a minor signal within the cytoplasm. Importantly, uL10ß is able to bind to the ribosomal particle, but is mainly associated with 60S and 80S particles; additionally, the uL10ß undergoes re-localization into the mitochondria upon endoplasmic reticulum stress induction. Our results suggest a specific stress-related dual role of uL10ß, supporting the idea of existence of specialized ribosomes with an altered GTPase associated center.

Fluorescence detection of GDP in real time with the reagentless biosensor rhodamine-ParM.

The development of novel fluorescence methods for the detection of key biomolecules is of great interest, both in basic research and in drug discovery. Particularly relevant and widespread molecules in cells are ADP and GDP, which are the products of a large number of cellular reactions, including reactions catalysed by nucleoside triphosphatases and kinases. Previously, biosensors for ADP were developed in this laboratory, based on fluorophore adducts with the bacterial actin homologue ParM. It is shown in the present study that one of these biosensors, tetramethylrhodamine-ParM, can also monitor GDP. The biosensor can be used to measure micromolar concentrations of GDP on the background of millimolar concentrations of GTP. The fluorescence response of the biosensor is fast, the response time being <0.2 s. Thus the biosensor allows real-time measurements of GTPase and GTP-dependent kinase reactions. Applications of the GDP biosensor are exemplified with two different GTPases, measuring the rates of GTP hydrolysis and nucleotide exchange.

Septins enforce morphogenetic events during sexual reproduction and contribute to virulence of Cryptococcus neoformans.

Septins are conserved, cytoskeletal GTPases that contribute to cytokinesis, exocytosis, cell surface organization and vesicle fusion by mechanisms that are poorly understood. Roles of septins in morphogenesis and virulence of a human pathogen and basidiomycetous yeast Cryptococcus neoformans were investigated. In contrast to a well-established paradigm in S. cerevisiae, Cdc3 and Cdc12 septin homologues are dispensable for growth in C. neoformans yeast cells at 24 degrees C but are essential at 37 degrees C. In a bilateral cross between septin mutants, cells fuse but the resulting hyphae exhibit morphological abnormalities, including lack of properly fused specialized clamp cells and failure to produce spores. Interestingly, post-mating hyphae of the septin mutants have a defect in nuclear distribution. Thus, septins are essential for the development of spores, clamp cell fusion and also play a specific role in nuclear dynamics in hyphae. In the post-mating hyphae the septins localize to discrete sites in clamp connections, to the septa and the bases of the initial emerging spores. Strains lacking CDC3 or CDC12 exhibit significantly reduced virulence in a Galleria mellonella model of infection. Thus, C. neoformans septins are vital to morphology of the hyphae and contribute to virulence.

Speckled-like pattern in the germinal center (SLIP-GC), a nuclear GTPase expressed in activation-induced deaminase-expressing lymphomas and germinal center B cells.

We identified a novel GTPase, SLIP-GC, with expression limited to a few tissues, in particular germinal center B cells. It lacks homology to any known proteins, indicating that it may belong to a novel family of GTPases. SLIP-GC is expressed in germinal center B cells and in lymphomas derived from germinal center B cells such as large diffuse B cell lymphomas. In cell lines, SLIP-GC is expressed in lymphomas that express activation-induced deaminase (AID) and that likely undergo somatic hypermutation. SLIP-GC is a nuclear protein, and it localizes to replication factories. Reduction of SLIP-GC levels in the Burkitt lymphoma cell line Raji and in non-Hodgkin lymphoma cell lines resulted in an increase in DNA breaks and apoptosis that was AID-dependent, as simultaneous reduction of AID abrogated the deleterious effects of SLIP-GC reduction. These results strongly suggest that SLIP-GC is a replication-related protein in germinal center B cells whose reduction is toxic to cells through an AID-dependent mechanism.

Diagnostic accuracy of molecular testing with three molecular markers on thyroid fine-needle aspiration cytology with abnormal category.

BACKGROUND: Cases with abnormal category, determined by thyroid fine-needle aspiration (FNA), frequently undergo surgical resection, despite the majority of cases being identified as benign after resection. Additional diagnostic markers are needed to guide the management of patients with abnormal thyroid nodules. MATERIALS AND METHODS: The retrospective study enrolled 150 cases diagnosed abnormal by FNA cytology that had undergone molecular testing with three markers (BRAF V600E, NRAS, and KRAS) on the cell block. Seventy-one cases had a surgical follow-up. RESULTS: When NIFTP is not considered as malignant, positive predictive values (PPVs) of cytology and combined cytology and molecular testing (CC-MT) were 67.6% (95% CI: 0.555-0.782) and 89.2% (95% CI: 0.746-0.970) (P = .004), respectively. The sensitivity of the CC-MT was 68.8%, specificity was 82.5%, and the false-positive rate was 17.4%. When NIFTP is considered as malignant, PPVs of cytology and CC-MT were 83.1% (95% CI: 0.743-0.918) and 94.6% (95% CI: 0.873-1.018) (P = .047), respectively. The sensitivity of the CC-MT was 59.3%, specificity was 83.3%, and the false-positive rate was 16.7%. CONCLUSION: The addition of molecular testing with a small panel to FNA cytology may increase the PPV of cytology in abnormal categories. Small panel (BRAF V600E, KRAS, and NRAS) with high specificity and high PPVs may be used particularly for the detection of thyroid malignancy. Cell blocks can be an especially useful and straightforward method for molecular diagnostic studies.

In Vivo Visualization of Moving Synaptic Cargo Complexes within Drosophila Larval Segmental Axons.

Identifying moving synaptic vesicle complexes and isolating specific proteins present within such complexes in vivo is challenging. Here we detail a protocol that we have developed that is designed to simultaneously visualize the axonal transport of two fluorescently tagged synaptic vesicle proteins in living Drosophila larval segmental nerves in real time. Using a beam-splitter and split view software, larvae expressing GFP-tagged Synaptobrevin (Syb) and mRFP-tagged Rab4-GTPase or YFP-tagged Amyloid Precursor protein (APP) and mRFP-tagged Rab4-GTPase are imaged simultaneously using separate wavelengths. Merged kymographs from the two wavelengths are evaluated for colocalization analysis. Vesicle velocity analysis can also be done. Such analysis enables us to visualize the motility behaviors of two synaptic proteins present on a single vesicle complex and identify candidate proteins moving on synaptic vesicles in vivo, under physiological conditions.

Induction of mutant dynamin specifically blocks endocytic coated vesicle formation.

Dynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline-inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.

A novel dynamin-like protein associates with cytoplasmic vesicles and tubules of the endoplasmic reticulum in mammalian cells.

Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.

Guanine nucleotide dissociation inhibitor is essential for Rab1 function in budding from the endoplasmic reticulum and transport through the Golgi stack.

The small GTPase Rab1 is required for vesicular traffic from the ER to the cis-Golgi compartment, and for transport between the cis and medial compartments of the Golgi stack. In the present study, we examine the role of guanine nucleotide dissociation inhibitor (GDI) in regulating the function of Rab1 in the transport of vesicular stomatitis virus glycoprotein (VSV-G) in vitro. Incubation in the presence of excess GDI rapidly (t1/2 < 30 s) extracted Rab1 from membranes, inhibiting vesicle budding from the ER and sequential transport between the cis-, medial-, and trans-Golgi cisternae. These results demonstrate a direct role for GDI in the recycling of Rab proteins. Analysis of rat liver cytosol by gel filtration revealed that a major pool of Rab1 fractionates with a molecular mass of approximately 80 kD in the form of a GDI-Rab1 complex. When the GDI-Rab1 complex was depleted from cytosol by use of a Rab1-specific antibody, VSV-G failed to exit the ER. However, supplementation of depleted cytosol with a GDI-Rab1 complex prepared in vitro from recombinant forms of Rab1 and GDI efficiently restored export from the ER, and transport through the Golgi stack. These results provide evidence that a cytosolic GDI-Rab1 complex is required for the formation of non-clathrin-coated vesicles mediating transport through the secretory pathway.

Dynamin-mediated internalization of caveolae.

The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.