20 research outputs found

    Function and organisation of actin and septins in Neurospora crassa

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    This thesis deals with the organisation and function of actin and septins in the model filamentous fungus, Neurospora crassa. Firstly, study demonstrates the utility of the Lifeact peptide probe for the investigation of actin dynamics in N. crassa. Lifeact fused to fluorescent proteins allowed live-cell imaging of actin patches, cables and rings without interfering with cellular functions. Actin cables and patches localised to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organisation was markedly different between the tip regions of mature hyphae and germ tubes. Only mature hyphae displayed a sub-apical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Proper organisation of actin cables required the class-V myosin, MYO-5, and the frequency of rapid transport of actin patches was reduced in its absence, suggesting that MYO-5 participates in actin patch translocation. Deletion of myo-5 caused gross morphological and polarity defects, demonstrating the importance of this motor for normal cell function. GFP-tagged MYO-5 localised as a crescent at germ tube tips and to the core of the Spitzenkörper in mature hyphae. Secondly, analysis of septin null mutants demonstrated that septins limit the emergence of germ tubes and are important for septation and conidiation in N. crassa. Septins showed different patterns of localisation at hyphal tips, with GFP-CDC-10 and CDC- 11-GFP organised as a collar with lower signal intensity at the tip apex, CDC-3-GFP and CDC-12-GFP constituted as a cap at the tip apex and GFP-SPN-1 forming an extended collar. Septins formed a range of different higher-order structures in N. crassa – rings, loops, fibres, bar-like structures, and caps – which can co-exist within the same cell. Purification of the septin complex and mass spectrometry of isolated proteins revealed that the septin complex consists predominantly of CDC-3, CDC-10, CDC-11 and CDC-12. Immunoprecipitation of SPN-1 revealed that this septin interacts with the core septin complex

    F-Actin Dynamics in Neurospora crassa

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    This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Coexpression of Lifeact-TagRFP and β-tubulin–GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth

    Septins Are Important for Cell Polarity, Septation and Asexual Spore Formation in <i>Neurospora crassa</i> and Show Different Patterns of Localisation at Germ Tube Tips

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    <div><p>Septins are GTP-binding cytoskeletal proteins that contribute to cell polarity, vesicle trafficking, cytokinesis and cell morphogenesis. Here we have characterised the six septins encoded by the genome of the model filamentous fungus <i>Neurospora crassa</i>. Analysis of septin null mutants demonstrated that septins limit the sites of emergence of germ tubes and are important for septation and conidiation in <i>N. crassa</i>. Septins constituted a range of different higher-order structures in <i>N. crassa</i> – rings, loops, fibres, bands, and caps – which can co-exist within the same cell. They showed different patterns of localisation at germ tube tips, with GFP-CDC-10 and CDC-11-GFP forming a subapical collar with lower signal intensity at the tip apex, CDC-3-GFP and CDC-12-GFP organized as a cap at the tip apex and GFP-ASP-1 forming an extended subapical collar. Purification of the septin complex and mass spectrometry of isolated proteins revealed that the septin complex consists predominantly of CDC-3, CDC-10, CDC-11 and CDC-12. Immunoprecipitation of the putative septin ASP-1 revealed that this protein interacts with the core septin complex.</p></div

    Rational engineering of photosynthetic electron flux enhances light-powered cytochrome P450 activity

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    In this study we exploited a modified photosynthetic electron-transfer chain (PET) in the model cyanobacterium Synechococcus PCC 7002, where electrons derived from water-splitting are used to power reactions catalyzed by a heterologous cytochrome P450 (CYP1A1). A simple in vivo fluorescent assay for CYP1A1 activity was employed to determine the impact of rationally engineering of photosynthetic electron flow. This showed that knocking out a subunit of the type I NADH dehydrogenase complex (NDH-1), suggested to be involved in cyclic photosynthetic electron flow (ΔndhD2), can double the activity of CYP1A1, with a concomitant increase in the flux of electrons from photosynthesis. This also resulted in an increase in cellular ATP and the ATP/NADPH ratio, suggesting that expression of a heterologous electron sink in photosynthetic organisms can be used to modify the bioenergetic landscape of the cell. We therefore demonstrate that CYP1A1 is limited by electron supply and that photosynthesis can be re-engineered to increase heterologous P450 activity for the production of high-value bioproducts. The increase in cellular ATP achieved could be harnessed to support metabolically demanding heterologous processes. Furthermore, this experimental system could provide valuable insights into the mechanisms of photosynthesis

    Live cell imaging of β-tubulin mRNA reveals spatiotemporal expression dynamics in the filamentous fungus Aspergillus oryzae

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    Abstract In filamentous fungi, microtubules are important for polar growth and morphological maintenance and serve as rails for intracellular trafficking. The molecular mechanisms associated with microtubules have been analyzed. However, little is known about when and where tubulin, a component of microtubules, is biosynthesized in multinuclear and multicellular filamentous fungi. In this study, we visualized microtubules based on the enhanced green fluorescence protein (EGFP)-labeled α-tubulin and β-tubulin mRNA tagged by the EGFP-mediated MS2 system in living yellow Koji mold Aspergillus oryzae cells in order to understand the spatiotemporal production mechanism of tubulin. We found that mRNA of btuA, encoding for β-tubulin, localized at dot-like structures through the apical, middle and basal regions of the hyphal cells. In addition, some btuA mRNA dots showed microtubule-dependent motor protein-like dynamics in the cells. Furthermore, it was found that btuA mRNA dots were decreased in the cytoplasm just before mitosis but increased immediately after mitosis, followed by a gradual decrease. In summary, the localization and abundance of β-tubulin mRNA is spatiotemporally regulated in living A. oryzae hyphal cells

    F-Actin Dynamics in Neurospora crassa

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    This study demonstrates the utility of Lifeact for the investigation of actin dynamics in Neurospora crassa and also represents the first report of simultaneous live-cell imaging of the actin and microtubule cytoskeletons in filamentous fungi. Lifeact is a 17-amino-acid peptide derived from the nonessential Saccharomyces cerevisiae actin-binding protein Abp140p. Fused to green fluorescent protein (GFP) or red fluorescent protein (TagRFP), Lifeact allowed live-cell imaging of actin patches, cables, and rings in N. crassa without interfering with cellular functions. Actin cables and patches localized to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 mu m/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organization was markedly different in the tip regions of mature hyphae and in germ tubes. Only mature hyphae displayed a subapical collar of actin patches and a concentration of F-actin within the core of the Spitzenkorper. Coexpression of Lifeact-TagRFP and beta-tubulin-GFP revealed distinct but interrelated localization patterns of F-actin and microtubules during the initiation and maintenance of tip growth.</p

    Horizontal Gene Transfer from Bacteria Has Enabled the Plant-Parasitic Nematode Globodera pallida to Feed on Host-Derived Sucrose

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    The evolution of plant-parasitic nematodes (PPN) is unusual in that these organisms have acquired a range of genes from bacteria via horizontal gene transfer (HGT). The proteins encoded by most of these genes are involved in metabolism of various components of the plant cell wall during invasion of the host. Recent genome sequencing projects for PPN have shown that Glycosyl Hydrolase Family 32 (GH32) sequences are present in several PPN species. These sequences are absent from almost all other animals. Here, we show that the GH32 sequences from an economically important cyst nematode species, Globodera pallida are functional invertases, are expressed during feeding and are restricted in expression to the nematode digestive system. These data are consistent with a role in metabolizing host-derived sucrose. In addition, a detailed phylogenetic analysis shows that the GH32 sequences from PPN and those present in some insect species have distinct bacterial origins and do not therefore derive from a gene present in the last common ancestor of ecdysozoan species. HGT has therefore played at least two critical roles in the evolution of PPN, enabling both invasion of the host and feeding on the main translocation carbohydrate of the plant.</p

    Epidemiology analysis of bovine acute mastitis

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    從83年7月至85年6月自台灣中,南部地區奶牛場,收集罹患急性乳房炎 牛隻之乳樣進行細菌分離並分析發生情形(臨床症狀,乳房之肉眼病變,乳 汁之肉眼變化等),結果:由收集108 頭發病乳牛採到 117 個分房乳樣,分 離出 110 株細菌( 9 個分房未分離出細菌,2 個分房分離出2 種細 菌),110 株病原菌的分佈上以Coliform bacteria 53 株(48.2%)和 Staphlococci 27 株 (24.5%) 佔絕大多數,其次為 Streptococci 9 株 (8.2%),Bacillus spp 6株(5.5%),Actinomyces pyogenes 4株(3.6%), Pseudomona aeruginosa3 株(2.7%)其他病原菌 8 株(7.3). 在53 株 Coliform bacteria(包括E.coli 37 株, Klebsiella spp15 株及 Enterobacter spp 1 株)取22種抗生素進行抗生素感受性試驗,結果抗藥 性菌株出現率以Amoxicillin 98.11%(52/53),Ampicillin98.11%(52/53), Oxacillin98.11%(52/53)及Penicillin98.11(52/53)等四種為最高,只有 Ceftazidime及Netilmicin無抗藥性菌株出現;在9 株Streptococci (包括 Streptococcus uberis 2 株, Streptococcus bovis 1 株Streptococcus pyogenes 4 株,Enterococcus faecalis 2 株) 則以Streptomycin 100%(9/9)為最高,其次是Colistin和 Oxytetracycline 88.9%(8/9),Amoxicillin,Bacitracin,Ceftazidime,Netilmicin, Oxacillin 和 Vancomycin 則均無抗藥性菌株出現;在 3 株 Staphylococcus aureus 以 Colistin 100%(3/3) 最高,對Amikacin, Amoxicillin,Cephalothin,Enrofloxacin,Erythromycin,Gentamicin, Novobiotin,Oxacillin, Penicillin 和 Vacomycin 均無抗藥性菌株的出 現;在24株 CNS ( Coagulase Negative Staphylococcus) 以Penicillin 79.17%(19/24) 最高,Colistin70.83% (17/24) 次之,對 Amikacin, Amoxicillin,Cephalothin,Enrofloxacin,Erythromycin,Gentamicin, Novobiotin,Oxacillin,Penicillin 和 Vancomycin 均無抗藥性菌株的出 現. 急性乳房炎發生率在濕熱季節(5-10月)比乾冷季節(11-4月)有較 高之發生率,兩者呈極顯著差異(p<0.01) ;乳牛年齡愈大急性乳房炎發生 率愈高,急性乳房炎發生率在不同年齡間呈極顯著差異(p<0.01),經產牛比 初產牛急性乳房炎之發生率為高且兩者間呈極顯著差異(p<0.01),乳牛後 分房比前分房有較高之急性乳房炎發生率;兩者間呈極顯著差異(p<0.01). 急性乳房炎發生分房各類局部變化之出現率依次是腫88.9% (104/117),痛81.2% (95/117),熱53.9%(63/117),紅46.2%(54/117).罹患 急性乳房炎乳牛其乳房附著形態以搾乳機式乳房(milking machine udder)佔23.08%(27/117)比例最高,腿間乳房(udder in the thinghs)oyr 17.09%(20/117)為最低.在乳頭形態方面以短乳頭(short teat)發生急性 乳房炎比例最高42.74%(50/117),圓錐狀乳頭(conical teat)發生率最 少4.27%(5/117).急性乳房炎牛隻其體溫高於39.5 C 佔67.3%(68/101)大 部份急性乳房炎發生牛隻有較高之體溫反應. 急性乳房炎乳汁特性已改變 佔 89.7%(105/117),CMT 反應呈3 級佔91.5%(107/117),乳汁呈鹼性反應 佔77.8%(91/117). 為找出可鑑別Coliform bacteria 和 Non- coliform bacteria 急性乳房炎之臨床資料,將41頭Coliform 急性乳房炎 臨床資料及53頭Non-coliform 急性乳房炎臨床資料進行區分性分析( Discriminant analysis ) 結果發現有下述10個指標最為有效可用;包括 乳汁已失去原有特性,乳汁變成黃色,乳汁呈水樣變化,發病分房有熱感,發 病分房皮膚潮紅,病牛體溫大於40.5 C,發病分房曾得過乳房炎,在分娩後 四週內發生,乳汁有白色結塊,乳房附著情形是搾乳機式乳房等10項.結果 在區別Coliform bacteria 與Non-coliform bacteria所引起之急性乳房 炎,預測之正確率達88.29%(sensitivity = 0.85 ,specificity = 0.90).A survey was conducted from July,1996 to June,1996. Milk sample and clinicaldata of cows suffered from acute mastitis were collected from dairy farm in the middle and southern parts of Taiwan.108 cows and 117 quarter samples were analyzed in total. 110 strains were isolated from these samples. No pathogen wasisolated from 9 quarter samples and 2 isolates in the same sample were found in 2 cases. The distribution of important pathogens of bovine acute mastitis were as followed:Coliform bacteria 42.8%(53/110),Staphylococci 24.5%(24/110),Streptococci8.2%(9/110),Bacillus spp.5.5%(6/110), Actinomyces pyogenes 3.6%(4/110),Pseudomonas aeruginosa 2.7%(3/110)and other pathogens 7.3%(8/110). 53 Coliform strains isolated during the whole program were selected to analyzethe antibiotic resistant patterns among 22 antibiotics, the highest resistant percentage were found on the following antibiotic:Amoxicillin 98.11%(52/53),Ampicillin 98.11%(52/53),Oxacillin 98.11%(52/53) and Penicillin 98.11%(52/53).No resistant strains were found to be against Ceftazidime and Netilmicin .The same test were performed on 9 isolated streptococci. The highest resistent percentage was found in Streptomycin 100%(9/9) and than Colistin 88.9%(8/9)and Oxytetracycline 88.9%(8/9). No resistant strains were found to be against Amoxicillin,Bacitracin,Ceftazidime,Netilmicin, Oxacillin and vancomycin. Test also conducted on the isolated Staphylococcus aureus, the highest resistent percentage was found in Colistin 100%(3/3), and no resistant strains were found to be against Amikacin, Amoxicillin, Cephalothin, Enrofloxacin, Erythromycin, Gentamicin,Novobiotin, OXacillin, Penicillin and Vacomycin.On 24 isolated coagulase negative staphylococci, highest resistent percentage was found in Penicillin 79.17%(19/24),then Colistin 70.83%(19/24). No resistant strains were found to be againstAmikacin, Cephalothin, Enrofloxacin, Erythromycin, Gentamycin, Novobiotin, Oxacillin, Penicillin and Vancomycin. On 24 isolated coagulase negative staphylococci, highest resistent percentage was found in Penicillin 79.17%(19/24). No resistant strains were found to be ahainst Amikacin, Amoxixillin,Cephalothin,Enrofloxacin,Erythromycin, Gentamycin,Novobiotin,Oxacillin,Penicillin and Vancomycin. In the aspect of clinical investigation, higher percentage of occurrence was found in hot and humid season than in cold and dry one, and the differnet was significant (p<0.01). The perscntage of occurrence raise with cow age. significant difference was detected among among cohorts(p<0.01). Posterior quarters have higher percentage of acute mastitis than anterior quarters(p<0.01). The percentage of local changes in actue mastitis were as follows:swelling 88.9%(104/117), pain 81.2%(95/117), hot 53.9%(63/117) and redness 46.2% (54/117). The forms of quarter in acute mastitis was highest in milking machine udder 23.08% (27/117), lowest in udder in the thighs 17.09%(20/117). In the forms of teat, highest was found in short teat 42.74%(50/117), lowest was in conical teat 4.27%(5/117). 67.3%(68/101) of affected cows have body temperatures higher than 39.5 C, thus most cows in acute mastitis have higher body temperature. In 89.7%(105/117) of the cases the change of milk appearance, 91.5%(107/117) with third grade of CMT reaction,and 77.8%(91/117) with basic milk. Data from 41 coliform and 53 non- coliformacute mastitis cases were analyzed using the method of discriminate analysis.Among them 10 indicators are related to coliform acute mastitism including change in milk appearance, milk color yellow, a watery consistency, hot of the affected quarter, redness of the affected quarter, elevated rectal temperature,previous mastitis in quarter, mastitis occurs within 4 weeks after calving,white flake in milk, milking-machine shape udder. Using the 10 indicators,35 out of 41 cases of acute mastitis causing from coliform pathogens can be grouped correctly, and 48 out of 53 cases of non-coliform caused ones can also be grouped corectly, the accuracy percentage is 88.29%(sensitivity=0.85,specificity==0.90
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