Eisosome organization in the filamentous ascomycete Aspergillus nidulans

Eisosomes are subcortical organelles implicated in endocytosis and have hitherto been described only in Saccharomyces cerevisiae. They comprise two homologue proteins, Pil1 and Lsp1, which colocalize with the transmembrane protein Sur7. These proteins are universally conserved in the ascomycetes. We identify in Aspergillus nidulans (and in all members of the subphylum Pezizomycotina) two homologues of Pil1/Lsp1, PilA and PilB, originating from a duplication independent from that extant in the subphylum Saccharomycotina. In the aspergilli there are several Sur7-like proteins in each species, including one strict Sur7 orthologue (SurG in A. nidulans). In A. nidulans conidiospores, but not in hyphae, the three proteins colocalize at the cell cortex and form tightly packed punctate structures that appear different from the clearly distinct eisosome patches observed in S. cerevisiae. These structures are assembled late during the maturation of conidia. In mycelia, punctate structures are present, but they are composed only of PilA, while PilB is diffused in the cytoplasm and SurG is located in vacuoles and endosomes. Deletion of each of the genes does not lead to any obvious growth phenotype, except for moderate resistance to itraconazole. We could not find any obvious association between mycelial (PilA) eisosome-like structures and endocytosis. PilA and SurG are necessary for conidial eisosome organization in ways that differ from those for their S. cerevisiae homologues. These data illustrate that conservation of eisosomal proteins within the ascomycetes is accompanied by a striking functional divergence. © 2010, American Society for Microbiology.This work was supported by research grants from the NCSR Demokritos to I.V. and IKY to C.G.Peer Reviewe

EglD, a putative endoglucanase, with an expansin like domain is localized in the conidial cell wall of Aspergillus nidulans

a b s t r a c t Although the process of conidial germination in filamentous fungi has been extensively studied, many aspects remain to be elucidated since the asexual spore or conidium is vital in their life cycle. Breakage and reformation of cell wall polymer bonds along with the maintenance of cell wall plasticity during conidia germination depend upon a range of hydrolytic enzymes whose activity is analogous to that of expansins, a highly conserved group of plant cell wall proteins with characteristic wall loosening activity. In the current study, we identified and characterized the eglD gene in Aspergillus nidulans, an expansinlike gene the product of which shows strong similarities with bacterial and fungal endo-b1,4-glucanases. However, we failed to show such activity in vitro. The eglD gene is constitutively expressed in all developmental stages and compartments of A. nidulans asexual life cycle. However, the EglD protein is exclusively present in conidial cell walls. The role of the EglD protein in morphogenesis, growth and germination rate of conidia was investigated. Our results show that EglD is a conidial cell wall localized expansin-like protein, which could be involved in cell wall remodeling during germination

Genes that encode protein factors participating in protein trafficking and membrane targeting of amino acids transporters

Eukayotic microorganisms use amino acids not only for protein synthesis but also as sole nitrogen and/or carbon sources. These organisms are capable of amino acids biosynthesis in general, but they also can uptake them from their environment via specific transmembrane proteins called amino acids transporters. Until nowadays, study of amino acids transporters has been mainly performed in microbial systems, such as Saccharomyces cerevisiae and Aspergillus nidulans, whom genetic, molecular and biochemical manipulations are easier than of higher eukaryotes. Fungal amino acids transporters belong to APC (Amino acid/Polyamine/organoCation) superfamily, which is conserved in all kingdoms. In A. nidulans three amino acids transporters have been extensively studied at the molecular and functional level: the major proline transporter PrnB, the γ-amino-n-butyrate (GABA) transporter GabA and the acidic amino acid transporter ΑgtA. A limited number of information exists concerning targeting (topogenesis) of these transporters to the plasma membrane, as well as concerning the environmental conditions, cis-regulating elements and trans-protein factors that regulate this process. Recent studies in A. nidulans revealed that shrA and aauΖ gene products affect AgtA targeting to the plasma membrane. Cloning and functional characterization of the shrA gene showed that its product localized in the endoplasmic reticulum and is necessary for targeting to the plasma membrane of PrnB and AgtA transporters. Cloning and functional characterization of the aauZ gene, via genetic complementation of the aauΖ102 mutation, revealed that its product is a casein kinase type I (CKI) that is also necessary for targeting to the plasma membrane of PrnB and AgtA transporters. aauΖ102 mutation is one of a significant number of mutations that have been collected recently, the aau mutations (amino acid uptake), affecting growth capacity of fungal strains on a large number of amino acids as sole nitrogen source. In this thesis, fbaA1013 mutation has been studied in an attempt to identify novel protein factors that are involved in regulation and function of AgtA, as well as of amino acids transporters in general. This mutation affects A. nidulans growth capacity on amino acids as sole nitrogen sources. Additionally, fbaA1013 mutation also affects A. nidulans growth capacity on different carbon sources independently of the nitrogen source present (amino acid or not). Particularly, fbaA1013 mutant strains showed reduced growth on glycolytic carbon sources compared to a wild type strain, while their growth is completely inhibited on gluconeogenenic carbon sources. The growth phenotype of the fbaA1013 mutant strain was found to be complemented by two genes. The fbaA gene, coding for a fructose 1,6-biphosphate aldolase of Class IIA, (FBAIIA), which complements the fbaA1013 mutation in single copy and the ΑΝID 02876.1 gene, coding for a hypothetical major facilitator permease (similar to Atg22A1 protein of S. cerevisiae), which acts as a multi-copy suppressor of the growth phenotype of the fbaA1013 mutant strain. FbaA protein shows 69% identity with Fba1p of S. serevisiae and is localized in the entire cytoplasm except the vacuoles. It is also localized in distinct sites of cell nuclei. The expression of the fbaA gene is constitutive and depends on the presence of a carbon source. Deletion of fbaA gene results in fungal lethality. fbaA1013 mutation is a big chromosome rearrangement mapped at the 5΄regulatory region of the fbaA gene resulting in dramatic reduction and complete elimination of fbaA mRNA steadystate levels in the presence of glycolytic and gluconeogenic carbon sources respectively, It is thus possible that fbaA gene expression is regulated by a dual mechanism depending on the carbon source present. Additionally, it was shown that chimeric AgtA-sGFP molecules in an fbaA1013 genetic background are not targeted to the plasma membrane but they remain stacked in intracellular compartments. fbaA1013 mutation also affects functional expression of secondary proline transporter. Taken together all the above results suggest that study of FbaA protein-protein interactions could lead to the identification of novel proteins that regulate transmembrane amino acids transporters in A. nidulans.Οι ευκαρυωτικοί µικροοργανισµοί χρησιµοποιούν τα αµινοξέα για τη σύνθεση πεπτιδίων και πρωτεϊνών, αλλά και ως µοναδικές πηγές αζώτου ή/και άνθρακα. Οι οργανισµοί αυτοί είναι γενικά ικανοί να βιοσυνθέσουν τα αµινοξέα που χρειάζονται, αλλά µπορούν να τα προσλάβουν και από το περιβάλλον τους µέσω της δράσης εξειδικευµένων πολυτοπικών διαµεµβρανικών πρωτεϊνών της πλασµατικής µεµβράνης, τους µεταφορείς αµινοξέων. Μέχρι σήµερα, η µελέτη των µεταφορέων αµινοξέων έχει επικεντρωθεί κυρίως σε πρότυπα µικροβιακά συστήµατα, όπως οι ασκοµύκητες Saccharomyces cerevisiae και Aspergillus nidulans, όπου οι γενετικοί, µοριακοί και βιοχηµικοί χειρισµοί είναι ευκολότεροι σε σχέση µε τους ανώτερους ευκαρυωτικούς οργανισµούς. Οι µεταφορείς αµινοξέων των µυκήτων είναι µέλη της συντηρηµένης σε όλα τα βασίλεια υπεροικογένειας APC (Amino acid/Polyamine/organoCation). Στον A. nidulans τρεις µεταφορείς αµινοξέων έχουν χαρακτηριστεί λεπτοµερειακά σε µοριακό και λειτουργικό επίπεδο: ο κύριος µεταφορέας προλίνης PrnB, ο µεταφορέας γ-αµινοβουτυρικού οξέως GabA και ο µεταφορέας όξινων αµινοξέων ΑgtA. Λίγα είναι γνωστά όσον αφορά τη στόχευση των πρωτεϊνών αυτών στην πλασµατική µεµβράνη (τοπογένεση), καθώς και τις περιβαλλοντικές συνθήκες, τα cis-ρυθµιστικά στοιχεία και τους trans-πρωτεϊνικούς παράγοντες που τη ρυθµίζουν. Πρόσφατες µελέτες στον A. nidulans έδειξαν ότι τα προϊόντα των γονιδίων shrA και aauΖ επηρεάζουν τη στόχευση στην πλασµατική µεµβράνη του µεταφορέα AgtA. Η πρωτεΐνη ShrA εντοπίζεται στο ενδοπλασµατικό δίκτυο, είναι απαραίτητη για την τοπογενέση των µεταφορέων PrnB και AgtA και το γονίδιο που την κωδικοποιεί, αποµονώθηκε και χαρακτηρίστηκε λειτουργικά. Η πρωτεΐνη ΑauZ είναι µία κινάση καζεΐνης τύπου Ι (casein kinase I, CKI) απαραίτητη για την τοπογενέση των µεταφορέων PrnB και AgtA και το γονίδιο που την κωδικοποιεί, αποµονώθηκε και χαρακτηρίστηκε λειτουργικά έπειτα από τη γενετική συµπλήρωση της µεταλλαγής aauΖ102. Η εν λόγω µεταλλαγή καθώς και ένας αξιοσηµείωτος αριθµός µεταλλαγών που έχει συγκεντρωθεί τα τελευταία χρόνια µε την ονοµασία aau (amino acid uptake) καθιστούν τα στελέχη των µυκήτων που τις φέρουν αδύναµα να αναπτυχθούν στην πλειοψηφία των αµινοξέων ως µοναδικές πηγές αζώτου. Στα πλαίσια της παρούσας διδακτορικής διατριβής µελετήθηκε η µεταλλαγή fbaA1013 προκειµένου να ταυτοποιηθούν νέοι πρωτεϊνικοί παράγοντες που εµπλέκονται στη ρύθµιση της έκφρασης και της λειτουργίας του µεταφορέα AgtA ειδικά, αλλά και µεταφορέων αµινοξέων γενικότερα. Η συγκεκριµένη µεταλλαγή επηρεάζει την ικανότητα ανάπτυξης του µύκητα στα αµινοξέα ως µοναδικές πηγές αζώτου. Επιπλέον η µεταλλαγή fbaA1013 επηρεάζει την ικανότητά του µύκητα να αναπτύσσεται σε διαφορετικές πηγές άνθρακα παρουσία διαφόρων πηγών αζώτου (αµινοξέων και µη). Συγκεκριµένα, τα µεταλλαγµένα στελέχη fbaA1013 παρουσιάζουν µειωµένη ανάπτυξη σε γλυκολυτικές πηγές άνθρακα σε σύγκριση µε το στέλεχος φυσικού τύπου, ενώ δεν αναπτύσσονται καθόλου σε πηγές γλυκονεογένεσης παρουσία διαφόρων πηγών αζώτου. Η µεταλλαγή fbaA1013 συµπληρώνεται από δύο γονίδια. Το γονίδιο fbaA που κωδικοποιεί για µία Τάξης IIA αλδολάση της 1,6-διφωσφορικής φρουκτόζης (Class IIA fructose 1,6-biphosphate aldolase, FBA), µοναδικό αντίγραφο του οποίου συµπληρώνει τη µεταλλαγή και η γονιδιωµατική αλληλουχία ΑΝID 02876.1 που κωδικοποιεί για µία υποθετική περµεάση (παρόµοια µε την πρωτεΐνη Atg22A1 του S. cerevisiae), πολλαπλά αντίγραφα της οποίας συµπληρώνουν τη µεταλλαγή (όσον αφορά τα αµινοξέα ως µοναδικές πηγές αζώτου). Η πρωτεΐνη FbaA εµφανίζει 69% αµινοξική ταυτότητα µε την Fba1p του S. serevisiae και εντοπίζεται στο κυτταρόπλασµα, µε εξαίρεση τα χυµοτόπια. Επιπλέον, εντοπίζεται σε συγκεκριµένες θέσεις του πυρήνα των εκβλαστηµένων υφών. Η έκφραση του γονιδίου είναι συστατική και εξαρτάται από την παρουσία της πηγής άνθρακα. Η εξάλειψη του γονιδίου fbaA από το γονιδίωµα του µύκητα είναι θνησιγόνος σε όλες τις πηγές άνθρακα που εξετάστηκαν. Η µεταλλαγή fbaA1013 είναι µια µεγάλου µήκους χρωµοσωµική αλλαγή που χαρτογραφήθηκε στην 5΄ ρυθµιστική περιοχή του γονιδίου fbaA και έχει σαν αποτέλεσµα τη δραµατική µείωση των επιπέδων δυναµικής ισορροπίας του µηνύµατός του σε γλυκολυτικές πηγές άνθρακα και την εξάλειψή τους σε πηγές γλυκονεογένεσης. Εποµένως, η έκφραση του γονιδίου fbaA υπόκειται πιθανά σε διαφορετική ρύθµιση η οποία εξαρτάται από το είδος της πηγής άνθρακα. Επιπλέον δείχθηκε ότι στο στέλεχος fbaA1013 µεταφορείς AgtA σηµασµένοι µε την πράσινη φθορίζουσα πρωτεΐνη (GFP) δε στοχεύονται στην πλασµατική µεµβράνη αλλά παραµένουν σε ενδοκυτταρικά διαµερίσµατα. Επιπλέον, η µεταλλαγή fbaA1013 επηρεάζει τη λειτουργική έκφραση του δευτερογενούς µεταφορέα προλίνης. Συνεπώς, η πρωτεΐνη FbaA εµπλέκεται στη µετα-µεταφραστική έκφραση του µεταφορέα AgtA, καθώς και στη λειτουργική έκφραση άλλων µεταφορέων αµινοξέων στον A. nidulans. Η διερεύνηση των πρωτεϊνικών αλληλεπιδράσεων στις οποίες συµµετέχει η πρωτεΐνη FbaA µπορεί να οδηγήσει στην ταυτοποίηση νέων παραγόντων που ρυθµίζουν την τοπογένεση µεταφορέων αµινοξέων στον A. nidulans

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed “satellites,” were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL. Key Points: • In a series of 29 856 CLL patients, the incidence of BcR stereotypy peaked at 41%. • Higher-order relations exist between stereotyped subsets, particularly for those from U-CLL, for which satellite subsets were identified

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B cell receptor immunoglobulins (BcR IG). Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR IG stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR IG stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. In order to address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29,856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed 'satellites', were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B cell receptor immunoglobulins (BcR IG). Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR IG stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR IG stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. In order to address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29,856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed 'satellites', were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL