Rapid production of gene replacement constructs and generation of a green fluorescent protein-tagged centromeric marker in Aspergillus nidulans

A method to rapidly generate gene replacement constructs by fusion PCR is described for Aspergillus nidulans. The utility of the approach is demonstrated by green fluorescent protein (GFP) tagging of A. nidulans ndc80 to visualize centromeres through the cell cycle. The methodology makes possible large-scale GFP tagging, promoter swapping, and deletion analysis of A. nidulans.     

Functional characterization of a maize purine transporter by expression in Aspergillus nidulans

We have characterized the function of Leaf Permease1 (LPE1), a protein that is necessary for proper chloroplast development in maize, by functional expression in the filamentous fungus Aspergillus nidulans. The choice of this ascomycete was dictated by the similarity of its endogenous purine transporters to LPE1 and by particular genetic and physiological features of purine transport and metabolism in A. nidulans. When Lpe1 was expressed in a purine transport-deficient A. nidulans strain, the capacity for uric acid and xanthine transport was acquired. This capacity was directly dependent on Lpe1 copy number and expression level. Interestingly, overexpression of LPE1 from >10 gene copies resulted in transformants with pleiotropically reduced growth rates on various nitrogen sources and the absolute inability to transport purines. Kinetic analysis established that LPE1 is a high-affinity (K(m) = 30 +/- 2.5 microM), high-capacity transporter specific for the oxidized purines xanthine and uric acid. Competition studies showed that high concentrations of ascorbic acid (>30 mM) competitively inhibit LPE1-mediated purine transport. This work defines the biochemical function of LPE1, a plant representative of a large and ubiquitous transporter family. In addition, A. nidulans is introduced as a novel model system for the cloning and/or functional characterization of transporter genes.     

Protein expression and subcellular localization of the general purine transporter UapC from Aspergillus nidulans

The uapC gene of Aspergillus nidulans belongs to a family of nucleobase-specific transporters conserved in prokaryotic and eucaryotic organisms. We report the use of immunological and green fluorescent protein based strategies to study protein expression and subcellular distribution of UapC. A chimeric protein containing a plant-adapted green fluorescent protein (sGFP) fused to the C-terminus of UapC was shown to be functional in vivo, as it complements a triple mutant (i.e., uapC(-) uapA(-) azgA(-)) unable to grow on uric acid as the sole nitrogen source. UapC-GFP is located in the plasma membrane and, secondarily, in internal structures observed as fluorescent dots. A strong correlation was found between cellular levels of UapC-GFP fluorescence and known patterns of uapC gene expression. This work represents the first in vivo study of protein expression and subcellular localization of a filamentous fungal nucleobase transporter.     

Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans

Green fluorescent protein (GFP) is a useful reporter to follow the in vivo behaviour of proteins, but the wild-type gfp gene does not function in many organisms, including many plants and filamentous fungi. We show that codon-modified forms of gfp , produced for use in plants, function effectively in Aspergillus nidulans both as gene expression reporters and as vital reporters for protein location. To demonstrate the use of these modified gfp s as reporter genes we have used fluorescence to follow ethanol-induced GFP expression from the alcA promoter. Translational fusions with the modified gfp were used to follow protein location in living cells; plant ER-retention signals targeted GFP to the endoplasmic reticulum, whereas fusion to the GAL4 DNA-binding domain targeted it to the nucleus. Nuclear-targeted GFP allowed real-time observation of nuclear movement and division. These modified gfp genes should provide useful markers to follow gene expression, organelle behaviour and protein trafficking in real time.     

Mutational analysis of the major proline transporter (PrnB) of Aspergillus nidulans

PrnB, the l-proline transporter of Aspergillus nidulans, belongs to the Amino acid Polyamine Organocation (APC) transporter family conserved in prokaryotes and eukaryotes. In silico analysis and limited biochemical evidence suggest that APC transporters comprise 12 transmembrane segments (TMS) connected with relatively short hydrophilic loops (L). However, very little is known on the structure-function relationships in APC transporters. This work makes use of the A. nidulans PrnB transporter to address structure-function relationships by selecting, constructing and analysing several prnB mutations. In the sample, most isolated missense mutations affecting PrnB function map in the borders of cytoplasmic loops with transmembrane domains. These are I119N and G120W in L2-TMS3, F278V in L6-TMS7, NRT378NRTNRT and PY382PYPY in L8-TMS9 and T456N in L10-TMS11. A single mutation (G403E) causing, however, a very weak phenotype, maps in the borders of an extracellular loop (L9-TMS10). An important role of helix TMS6 for proline binding and transport is supported by mutations K245L and, especially, F248L that clearly affect PrnB uptake kinetics. The critical role of these residues in proline binding and transport is further shown by constructing and analysing isogenic strains expressing selected prnB alleles fused to the gene encoding the Green Fluorescent Protein (GFP). It is shown that, while some prnB mutations affect proper translocation of PrnB in the membrane, at least two mutants, K245E and F248L, exhibit physiological cellular expression of PrnB and, thus, the corresponding mutations can be classified as mutations directly affecting proline binding and/or transport. Finally, comparison of these results with analogous studies strengthens conclusions concerning amino acid residues critical for function in APC transporters.     

Functional characterization and localization of the Aspergillus nidulans formin SEPA

Formins are a family of multidomain scaffold proteins involved in actin-dependent morphogenetic events. In Aspergillus nidulans, the formin SEPA participates in two actin-mediated processes, septum formation and polarized growth. In this study, we use a new null mutant to demonstrate that SEPA is required for the formation of actin rings at septation sites. In addition, we find that a functional SEPA::GFP fusion protein localizes simultaneously to septation sites and hyphal tips, and that SEPA colocalizes with actin at each site. Using live imaging, we show that SEPA localization at septation sites and hyphal tips is dynamic. Notably, at septation sites, SEPA forms a ring that constricts as the septum is deposited. Moreover, we demonstrate that actin filaments are required to maintain the proper localization pattern of SEPA, and that the amino-terminal half of SEPA is sufficient for localization at septation sites and hyphal tips. In contrast, only localization at septation sites is affected by loss of the sepH gene product. We propose that specific morphological cues activate common molecular pathways to direct SEPA localization to the appropriate morphogenetic site.     

Live-cell analysis of a green fluorescent protein-tagged herpes simplex virus infection

Many stages of the herpes simplex virus maturation pathway have not yet been defined. In particular, little is known about the assembly of the virion tegument compartment and its subsequent incorporation into maturing virus particles. Here we describe the construction of a herpes simplex virus type 1 (HSV-1) recombinant in which we have replaced the gene encoding a major tegument protein, VP22, with a gene expressing a green fluorescent protein (GFP)-VP22 fusion protein (GFP-22). We show that this virus has growth properties identical to those of the parental virus and that newly synthesized GFP-22 is detectable in live cells as early as 3 h postinfection. Moreover, we show that GFP-22 is incorporated into the HSV-1 virion as efficiently as VP22, resulting in particles which are visible by fluorescence microscopy. Consequently, we have used time lapse confocal microscopy to monitor GFP-22 in live-cell infection, and we present time lapse animations of GFP-22 localization throughout the virus life cycle. These animations demonstrate that GFP-22 is present in a diffuse cytoplasmic location when it is initially expressed but evolves into particulate material which travels through an exclusively cytoplasmic pathway to the cell periphery. In this way, we have for the first time visualized the trafficking of a herpesvirus structural component within live, infected cells.     

Trafficking of green fluorescent protein-tagged SNARE proteins in HSY cells

SNARE proteins are widely accepted to be involved in the docking and fusion process of intracellular vesicle trafficking. VAMP-2, syntaxin-4, and SNAP-23 are plausible candidate SNARE proteins for non-neuronal exocytosis. Thus, we examined the localization, protein-protein interaction, and intracellular trafficking of these proteins by expressing them as green fluorescent protein (GFP)- and FLAG-tagged fusion proteins in various cells, including HSY cells, a human parotid epithelial cell line. GFP-VAMP-2 was ex-pressed strongly in the Golgi area and weakly on the plasma membrane. Although GFP-SNAP-23 seemed to be expressed universally in the cytosol, the GFP signal was clearly seen on the plasma membrane, when soluble GFP-SNAP-23 was removed by treatment with saponin. GFP-syntaxin-4 was undetectable on the plasma membrane but was strongly expressed on unidentified unusually large vesicles. GFP-syntaxin-4 without its transmembrane domain was still incompletely soluble and observed as aggregates. When syntaxin-4 and munc18c were coexpressed, syntaxin-4 was translocated at least in part to the plasma membrane. The protein-protein interaction between syntaxin-4 and VAMP-2 with their transmembrane domains was markedly inhibited on coexpression of munc18c. These results suggest that munc18c plays an important role in the trafficking of syntaxin-4 to its proper destination by preventing premature interactions with other proteins, including SNARE proteins.     

Clathrin localization and dynamics in Aspergillus nidulans

Cell growth necessitates extensive membrane remodeling events including vesicle fusion or fission, processes that are regulated by coat proteins. The hyphal cells of filamentous fungi concentrate both exocytosis and endocytosis at the apex. This investigation focuses on clathrin in Aspergillus nidulans, with the aim of understanding its role in membrane remodeling in growing hyphae. We examined clathrin heavy chain (ClaH‐GFP) which localized to three distinct subcellular structures: late Golgi (trans‐Golgi equivalents of filamentous fungi), which are concentrated just behind the hyphal tip but are intermittently present throughout all hyphal cells; the region of concentrated endocytosis just behind the hyphal apex (the “endocytic collar”); and small, rapidly moving puncta that were seen trafficking long distances in nearly all hyphal compartments. ClaH localized to distinct domains on late Golgi, and these clathrin “hubs” dispersed in synchrony after the late Golgi marker PH^OSBP. Although clathrin was essential for growth, ClaH did not colocalize well with the endocytic patch marker fimbrin. Tests of FM4‐64 internalization and repression of ClaH corroborated the observation that clathrin does not play an important role in endocytosis in A. nidulans. A minor portion of ClaH puncta exhibited bidirectional movement, likely along microtubules, but were generally distinct from early endosomes.     

带GFP标记嗜肺军团菌的构建和在细胞感染中的应用

【目的】为能实时直观了解嗜肺军团菌感染细胞的过程,研究细菌在细胞内的变化及其与宿主细胞间的相互作用关系。【方法】通过基因敲除、克隆回补等重组构建绿色荧光蛋白(GFP)稳定高表达的嗜肺军团菌株,利用该菌株建立小鼠巨噬细胞Raw264.7的感染模型。【结果】通过荧光显微镜可实时观察细菌感染细胞的全过程,包括细菌在细胞内的形态变化、增殖和裂解宿主细胞等。【结论】重组菌可替代野生菌株在细胞感染中应用,为直观研究嗜肺军团菌与被感染细胞之间的相互作用关系,以及进行相关药物模型的制备、药物筛选、耐药机制研究等提供了新的手段。     

The proline permease of Aspergillus nidulans: functional replacement of the native cysteine residues and properties of a cysteine-less transporter

The major proline transporter (PrnB) of Aspergillus nidulans belongs to the Amino acid Polyamine Organocation (APC) transporter superfamily. Members of this family have not been subjected to systematic structure-function relationship studies. In this report, we examine the functional replacement of the three native Cys residues (Cys54, Cys352 and Cys530) of PrnB and the properties of an engineered Cys-less PrnB protein, as background for employing a Cys-scanning mutagenesis approach. We show that simultaneous replacement of Cys54 with Ala, Cys352 with Ala and Cys530 with Ser results in a functional Cys-less PrnB transporter. We also introduce the use of a biotin-acceptor domain tag to quantitate protein levels of the engineered PrnB mutants by Western blot analysis. Finally, by using the background of the Cys-less PrnB transporter, we evaluate the functional importance of amino acids Q219, K245 and F248 of PrnB, which our previous data had suggested to be involved in the mechanism of PrnB-mediated proline uptake. In the current study, we show that K245 and F248 but not Q219 are critical for PrnB-mediated proline uptake.     

Kinase activity and subcellular distribution of a chimeric green fluorescent protein-tagged Janus kinase 2

Summary Janus kinase 2 (JAK2) is an essential intracellular signal transducer for numerous cytokines and hormones. To examine how JAK2 structural modifications can affect cellular physiology, we created expression vectors for chimeric proteins containing an enhanced green fluorescent protein (EGFP) fused to rat JAK2 (EGFP/rJAK2), and a kinase-inactive variant, EGFP/rJAK2(K882E). The properties of EGFP/rJAK2 were examined following transient transfection of COS-7 cells. EGFP/rJAK2 was expressed throughout the cell, and was found in subcellular membrane, cytosolic and nuclear fractions. Interestingly, EGFP/rJAK2 phosphorylated other proteins in situ without additional cytokine stimulation. Furthermore, despite a much higher level of tyrosine phosphorylation arising from in situ autophosphorylation, the in vitro radiolabelling autokinase activity of EGFP/rJAK2 was significantly less than that of the endogenous JAK2. These results reveal a technical limitation of the application of the “conventional” in vitro radiolabelling autokinase assay to hyperphosphorylated forms of the enzyme and illustrate the potential weaknesses in individual assays commonly used to determine JAK2’s enzymatic activity and subcellular distribution. We also suggest that the EGFP/rJAK2 model can be very useful in studying JAK2-related cancers, because its ubiquitous distribution and abnormal constitutive hyperphosphorylation may distinguish it from the cytokine-regulated, membrane-proximal form of JAK2 associated with normal physiology.