Michael Feldbrügge

Heinrich-Heine-Universität Düsseldorf, Düsseldorf, North Rhine-Westphalia, Germany

Are you Michael Feldbrügge?

Claim your profile

Publications (50)285.99 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Active transport and local translation of mRNAs ensure the appropriate spatial organization of proteins within cells. Recent work has shown that this process is intricately connected to membrane trafficking. Here, we focus on new findings obtained in fungal model systems. Important highlights are that RNA-binding proteins recognize cargo mRNA synergistically and that mRNAs are co-transported with membranous compartments such as the endoplasmic reticulum (ER) and endosomes. We further discuss a novel concept of endosome-coupled translation that loads shuttling endosomes with septin cargo, a process important for correct septin filamentation. Interestingly, evidence is accumulating that RNA and membrane trafficking are also tightly interwoven in higher eukaryotes, suggesting that this phenomenon is a common theme and not an exception restricted to fungi.
    Trends in genetics : TIG. 08/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: To produce the full repertoire of biopharmaceutical proteins, alternative expression platforms are required. Systems that enable secretion of the target protein are favored because this facilitates downstream processing. Ustilago maydis is a promising fungal model organism for future applications in protein expression. Recently, we described the exploitation of a novel unconventional secretion mechanism for the export of heterologous proteins. In this mode of secretion, the endochitinase Cts1 functions as a carrier for export with the main advantage of avoiding potentially harmful N-glycosylation. The major limitation until now was a low yield of secreted full-length protein. For optimization, we identified two bottlenecks: mRNA amount and extracellular proteolytic activity. By generating novel expression vectors harboring a strong constitutive promoter as well as eliminating harmful proteases, yields were increased significantly. A scFv antibody fragment against the cMyc epitope served as proof-of-principle and could be purified in its active, full-length form from the culture supernatant. Thus, we improved the novel expression system in U. maydis such that it can now be investigated with respect to other targets with potential applications for instance in diagnostics and medicine.
    Journal of biotechnology. 07/2014;
  • [Show abstract] [Hide abstract]
    ABSTRACT: CalB of Pseudozyma aphidis (formerly named Candida antarctica) is one of the most widely applied enzymes in industrial biocatalysis. Here, we describe a protein with 66 % sequence identity to CalB, designated Ustilago maydis lipase 2 (Uml2), which was identified as the product of gene um01422 of the corn smut fungus U. maydis. Sequence analysis of Uml2 revealed the presence of a typical lipase catalytic triad, Ser-His-Asp with Ser125 located in a Thr-Xaa-Ser-Xaa-Gly pentapeptide. Deletion of the uml2 gene in U. maydis diminished the ability of cells to hydrolyse fatty acids from tributyrin or Tween 20/80 substrates, thus demonstrating that Uml2 functions as a lipase that may contribute to nutrition of this fungal pathogen. Uml2 was heterologously produced in Pichia pastoris and recombinant N-glycosylated Uml2 protein was purified from the culture medium. Purified Uml2 released short- and long-chain fatty acids from p-nitrophenyl esters and Tween 20/80 substrates. Furthermore, phosphatidylcholine substrates containing long-chain saturated or unsaturated fatty acids were effectively hydrolysed. Both esterase and phospholipase A activity of Uml2 depended on the Ser125 catalytic residue. These results indicate that Uml2, in contrast to CalB, exhibits not only esterase and lipase activity but also phospholipase A activity. Thus, by genome mining, we identified a novel CalB-like lipase with different substrate specificities.
    Applied Microbiology and Biotechnology 01/2014; · 3.69 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Endosomes transport lipids and proteins over long distances by shuttling along microtubules. They also carry mRNAs on their surface, but the precise molecular function of this trafficking process is unknown. By live cell imaging of polarized fungal hyphae, we show microtubule-dependent transport of septin mRNA and encoded septin protein on the same shuttling endosomes. Consistent with the hypothesis that septin mRNA is translated on endosomes, the accumulation of septin protein on endosomes requires the recruitment of septin mRNA. Furthermore, ribosomal proteins co-localise with shuttling endosomes, but only if mRNA is present. Importantly, endosomal trafficking is essential for an efficient delivery of septin protein to filaments at growth poles, a process necessary to establish unipolar growth. Thus, we propose that local mRNA translation loads endosomes with septins for assembly and efficient delivery to septin filaments.
    EMBO Reports 12/2013; · 7.19 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The corn pathogen Ustilago maydis is a well-studied fungal model organism. Along with a broad set of experimental tools, versatile strategies for the generation of gene replacement mutants by homologous recombination in U. maydis have been developed. Nevertheless, the production of corresponding linear DNA constructs still constitutes a time-limiting step. To overcome this bottleneck, various resistance cassette modules were adopted for use with the so-called Golden Gate cloning strategy. These modules not only allow simple gene deletions but also more sophisticated genetic manipulations like inserting sequences for C-terminal protein tagging. The type IIs restriction enzyme BsaI was selected for this novel approach as its recognition sites are comparatively rare in the U. maydis genome. To test the efficiency of the new strategy it was used to test the influence of varying flank lengths as well as the effect of non-homologous flank ends on homologous recombination. Importantly, to proof a broad applicability in other fungi the same strategy was used to generate mutants in the filamentous ascomycete Aspergillus nidulans. Hence, we present a highly efficient and economic cloning strategy that speeds up reverse genetic approaches in fungi.
    Fungal Genetics and Biology 11/2013; · 3.26 Impact Factor
  • Source
    Vera Göhre, Carl Haag, Michael Feldbrügge
    PLoS Pathogens 10/2013; 9(10):e1003617. · 8.14 Impact Factor
  • Michael Feldbrügge, Ronny Kellner, Kerstin Schipper
    [Show abstract] [Hide abstract]
    ABSTRACT: Plant pathogens of the family Ustilaginaceae parasitise mainly on grasses and cause smut disease. Among the best characterised members of this family are the covered smut fungus Ustilago hordei colonising barley and oat as well as the head smut Sporisorium reilianum and the corn smut Ustilago maydis, both infecting maize. Over the past years, U. maydis in particular has matured into a model system for diverse topics like plant-pathogen interaction, cellular transport processes or DNA repair. Consequently, a broad set of genetic, molecular and system biological methods has been established. This set currently serves as a strong foundation to improve existing and establish novel biotechnological applications. Here, we review four promising aspects covering different fields of applied science: (1) synthesis of secondary metabolites produced at fermenter level. (2) Lipases and other hydrolytic enzymes with potential roles in biocatalytic processes. (3) Degradation of ligno-cellulosic plant materials for biomass conversion. (4) Protein expression based on unconventional secretion, a novel approach inspired by basic research on mRNA transport. Thus, plant pathogenic Ustilaginaceae offer a great potential for future biotechnological applications by combining basic research and applied science.
    Applied Microbiology and Biotechnology 03/2013; · 3.69 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Long-distance trafficking of membranous structures along the cytoskeleton is crucial for secretion and endocytosis in eukaryotes. Molecular motors are transporting both secretory and endocytic vesicles along polarized microtubules. Here, we review the transport mechanism and biological function of a distinct subset of large vesicles marked by the G-protein Rab5a in the model microorganism Ustilago maydis. These Rab5a-positive endosomes shuttle bi-directionally along microtubules mediated by the Unc104/KIF1A-related motor Kin3 and dynein Dyn1/2. Rab5a-positive endosomes exhibit diverse functions during the life cycle of U. maydis. In haploid budding cells they are involved in cytokinesis and pheromone signaling. During filamentous growth endosomes are used for long-distance transport of mRNA, a prerequisite to maintain polarity most likely via local translation of specific proteins at both the apical and distal ends of filaments. Endosomal co-transport of mRNA constitutes a novel function of these membrane compartments supporting the view that endosomes function as multipurpose platforms.
    Communicative & integrative biology 09/2012; 5(5):485-90.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The demand on the biotechnological production of proteins for pharmaceutical, medical and industrial applications is steadily growing. For the production of challenging proteins, we aim to establish a novel expression platform in the well characterized eukaryotic microorganism Ustilago maydis. In filaments of this fungus, secretion of the endochitinase Cts1 depends on mRNA transport along microtubules, which is mediated by the key RNA-binding protein Rrm4. Here, we report two important findings: (i) Cts1 secretion occurs via a novel unconventional route and (ii) this secretory mechanism can be exploited for the export of active heterologous proteins. Initially, we used β-glucuronidase (Gus) as a reporter for unconventional secretion. This bacterial enzyme is inactivated by N-glycosylation during its passage through the conventional eukaryotic secretory pathway. By contrast, in our system Gus was exported in its active form by fusion to Cts1 confirming its secretion by an unconventional route. As a proof-of-principle for economically important biopharmaceuticals we expressed an active single-chain antibody. Importantly, the novel protein export pathway circumvents N-glycosylation which is advantageous in many applications, e.g., to avoid undesired immune reactions in humans. Thus, the unconventional Cts1 secretion machinery has a high potential for the production of biotechnologically relevant proteins.
    Journal of Biotechnology 03/2012; 161(2):80-91. · 3.18 Impact Factor
  • Source
    Evelyn Vollmeister, Kerstin Schipper, Michael Feldbrügge
    [Show abstract] [Hide abstract]
    ABSTRACT: Microtubule-dependent trafficking is essential in moving mRNAs over long distances. This transport mechanism regulates important cellular events such as determining polarity and local protein secretion. Key examples are developmental and neuronal processes studied in Drosophila melanogaster, Xenopus laevis as well as in mammalian cells. A simple eukaryotic system to uncover basic mechanisms was missing. Fungal models are generally well suited for this purpose, since transgenic strains can be generated easily by homologous recombination allowing in vivo studies at native expression levels. Substantial advances in understanding Ustilago maydis showed that this fungus fulfils important criteria to serve as model for microtubule-dependent mRNA trafficking. Here, we summarize progress focusing on target mRNAs, RNA localization elements, RNA-binding proteins, mRNPs, molecular motors and microtubule organization. This serves as the basis to discuss the novel mechanism of mRNP hitchhiking on endosomes as well as an unexpected link to unconventional secretion with its implications for applied sciences.
    RNA biology 03/2012; 9(3):261-8. · 5.56 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Long-distance transport of mRNAs is important in determining polarity in eukaryotes. Molecular motors shuttle large ribonucleoprotein complexes (mRNPs) containing RNA-binding proteins and associated factors along microtubules. However, precise mechanisms including the interplay of molecular motors and a potential connection to membrane trafficking remain elusive. Here, we solve the motor composition of transported mRNPs containing the RNA-binding protein Rrm4 of the pathogen Ustilago maydis. The underlying transport process determines the axis of polarity in infectious filaments. Plus-end-directed Kin3, a kinesin-3 type motor, mediates anterograde transport of mRNPs and is also present in transport units moving retrogradely. Split dynein Dyn1-Dyn2 functions in retrograde movement of mRNPs. Plus-end-directed conventional kinesin Kin1 is indirectly involved by transporting minus-end-directed dynein back to plus ends. Importantly, we additionally demonstrate that Rrm4-containing mRNPs colocalise with the t-SNARE Yup1 on shuttling endosomes and that functional endosomes are essential for mRNP movement. Either loss of Kin3 or removal of its lipid-binding pleckstrin-homology domain abolishes Rrm4-dependent movement without preventing colocalisation of Rrm4 and Yup1-positive endosomes. In summary, we uncovered the combination of motors required for mRNP shuttling along microtubules. Furthermore, intimately linked co-transport of endosomes and mRNPs suggests vesicle hitchhiking as mode of mRNP transport.
    Journal of Cell Science 02/2012; 125(Pt 11):2740-52. · 5.88 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The grass smuts comprise a speciose group of biotrophic plant parasites, so-called Ustilaginaceae, which are specifically adapted to hosts of sweet grasses, the Poaceae family. Mating takes a central role in their life cycle, as it initiates parasitism by a morphological and physiological transition from saprobic yeast cells to pathogenic filaments. As in other fungi, sexual identity is determined by specific genomic regions encoding allelic variants of a pheromone-receptor (PR) system and heterodimerising transcription factors. Both operate in a biphasic mating process that starts with PR-triggered recognition, directed growth of conjugation hyphae, and plasmogamy of compatible mating partners. So far, studies on the PR system of grass smuts revealed diverse interspecific compatibility and mating type determination. However, many questions concerning the specificity and evolutionary origin of the PR system remain unanswered. Combining comparative genetics and biological approaches, we report on the specificity of the PR system and its genetic diversity in 10 species spanning about 100 million years of mating type evolution. We show that three highly syntenic PR alleles are prevalent among members of the Ustilaginaceae, favouring a triallelic determination as the plesiomorphic characteristic of this group. Furthermore, the analysis of PR loci revealed increased genetic diversity of single PR locus genes compared to genes of flanking regions. Performing interspecies sex tests, we detected a high potential for hybridisation that is directly linked to pheromone signalling as known from intraspecies sex. Although the PR system seems to be optimised for intraspecific compatibility, the observed functional plasticity of the PR system increases the potential for interspecific sex, which might allow the hybrid-based genesis of newly combined host specificities.
    PLoS Genetics 12/2011; 7(12):e1002436. · 8.52 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The grass smuts comprise a speciose group of biotrophic plant parasites, so-called Ustilaginaceae, which are specifically adapted to hosts of sweet grasses, the Poaceae family. Mating takes a central role in their life cycle as it initiates parasitism by a morphological and physiological transition from saprobic yeast cells to pathogenic filaments. As in other fungi sexual identity is determined by specific genomic regions encoding allelic variants of a pheromone-receptor (PR) system and heterodimerising transcription factors. Both operate in a biphasic mating process that starts with PR-triggered recognition, directed growth of conjugation hyphae and plasmogamy of compatible mating partners. So far, studies on the PR system of grass smuts revealed diverse interspecific compatibility and mating type determination. However, many questions concerning the specificity and evolutionary origin of the PR system remain unanswered. Combining comparative genetics and biological approaches we report on the specificity of the PR system and its genetic diversity in 10 species spanning about 100 million years of mating type evolution. We show that three highly syntenic PR alleles are prevalent among members of the Ustilaginaceae favouring a triallelic determination as the plesiomorphic characteristic of this group. Furthermore, the analysis of PR loci revealed increased genetic diversity of single PR locus genes compared to genes of flanking regions. Performing interspecies sex tests we detected a high potential for hybridisation that is directly linked to pheromone signalling as known from intraspecies sex. Although the PR system seems to be optimised for intraspecific compatibility, the observed functional plasticity of the PR system increases the potential for interspecific sex, which might allow the hybrid-based genesis of newly combined host specificities.
    PLoS Genetics 12/2011; 7:e1002436. · 8.52 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Long-distance transport of mRNAs is crucial in determining spatio-temporal gene expression in eukaryotes. The RNA-binding protein Rrm4 constitutes a key component of microtubule-dependent mRNA transport in filaments of Ustilago maydis. Although a number of potential target mRNAs could be identified, cellular processes that depend on Rrm4-mediated transport remain largely unknown. Here, we used differential proteomics to show that ribosomal, mitochondrial, and cell wall-remodeling proteins, including the bacterial-type endochitinase Cts1, are differentially regulated in rrm4Δ filaments. In vivo UV crosslinking and immunoprecipitation and fluorescence in situ hybridization revealed that cts1 mRNA represents a direct target of Rrm4. Filaments of cts1Δ mutants aggregate in liquid culture suggesting an altered cell surface. In wild type cells Cts1 localizes predominantly at the growth cone, whereas it accumulates at both poles in rrm4Δ filaments. The endochitinase is secreted and associates most likely with the cell wall of filaments. Secretion is drastically impaired in filaments lacking Rrm4 or conventional kinesin Kin1 as well as in filaments with disrupted microtubules. Thus, Rrm4-mediated mRNA transport appears to be essential for efficient export of active Cts1, uncovering a novel molecular link between mRNA transport and the mechanism of secretion.
    Molecular &amp Cellular Proteomics 08/2011; 10(12):M111.011213. · 7.25 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The maize pathogen Ustilago maydis has to undergo various morphological transitions for the completion of its sexual life cycle. For example, haploid cells respond to pheromone by forming conjugation tubes that fuse at their tips. The resulting dikaryon grows filamentously, expanding rapidly at the apex and inserting retraction septa at the basal pole. In this review, we present progress on the underlying mechanisms regulating such defined developmental programmes. The key findings of the postgenomic era are as follows: (1) endosomes function not only during receptor recycling, but also as multifunctional transport platforms; (2) a new transcriptional master regulator for pathogenicity is part of an intricate transcriptional network; (3) determinants for uniparental mitochondrial inheritance are encoded at the a2 mating-type locus; (4) microtubule-dependent mRNA transport is important in determining the axis of polarity; and (5) a battery of fungal effectors encoded in gene clusters is crucial for plant infection. Importantly, most processes are tightly controlled at the transcriptional, post-transcriptional and post-translational levels, resulting in a complex regulatory network. This intricate system is crucial for the timing of the correct order of developmental phases. Thus, new insights from all layers of regulation have substantially advanced our understanding of fungal development.
    FEMS microbiology reviews 07/2011; 36(1):59-77. · 10.96 Impact Factor
  • Evelyn Vollmeister, Michael Feldbrügge
    [Show abstract] [Hide abstract]
    ABSTRACT: The corn pathogen Ustilago maydis relies on defined developmental programs to regulate morphological switches during the infection process. Research on RNA-binding proteins has demonstrated the substantial contribution of posttranscriptional control in regulating growth, morphology and pathogenicity. In particular, functional analysis of the two sequence-specific RNA-binding proteins Khd4 and Rrm4 revealed the importance of regulated mRNA stability and transport, respectively. For example, in infectious filaments microtubule-dependent transport of mRNAs encoding polarity factors is key in establishing the axis of polarity. These findings probably represent only a few of the emerging discoveries related to posttranscriptional control in filamentous fungi. Research progress may also prove informative compared to similar regulatory mechanisms in operation during developmental and neuronal processes in vertebrates.
    Current opinion in microbiology 09/2010; 13(6):693-9. · 7.87 Impact Factor
  • Source
    Kathi Zarnack, Michael Feldbrügge
    [Show abstract] [Hide abstract]
    ABSTRACT: The localization and local translation of mRNAs constitute an important mechanism to promote the correct subcellular targeting of proteins. mRNA localization is mediated by the active transport of mRNPs, large assemblies consisting of mRNAs and associated factors such as RNA-binding proteins. Molecular motors move mRNPs along the actin or microtubule cytoskeleton for short-distance or long-distance trafficking, respectively. In filamentous fungi, microtubule-based long-distance transport of vesicles, which are involved in membrane and cell wall expansion, supports efficient hyphal growth. Recently, we discovered that the microtubule-mediated transport of mRNAs is essential for the fast polar growth of infectious filaments in the corn pathogen Ustilago maydis. Combining in vivo UV cross-linking and RNA live imaging revealed that the RNA-binding protein Rrm4, which constitutes an integral part of the mRNP transport machinery, mediates the transport of distinct mRNAs encoding polarity factors, protein synthesis factors, and mitochondrial proteins. Moreover, our results indicate that microtubule-dependent mRNA transport is evolutionarily conserved from fungi to higher eukaryotes. This raises the exciting possibility of U. maydis as a model system to uncover basic concepts of long-distance mRNA transport.
    Eukaryotic Cell 07/2010; 9(7):982-90. · 3.59 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: RNA-binding proteins constitute key factors of the post-transcriptional machinery. These regulatory proteins recognize specific elements within target transcripts to promote, for example, maturation, translation, or stability of mRNAs. In Ustilago maydis, evidence is accumulating that post-transcriptional processes are important to determine pathogenicity. Deletion of khd4, encoding a predicted RNA-binding protein with five K homology (KH) domains, causes aberrant cell morphology and reduced virulence. Here, we demonstrate that Khd4 recognizes the sequence AUACCC in vivo via its tandem KH domains 3 and 4. This sequence most likely functions as a regulatory RNA element in U. maydis, since it accumulates in 3' untranslated regions. Consistently, an independent mRNA expression profiling approach revealed that the binding motif is significantly enriched in transcripts showing altered expression levels in khd4Delta strains. Since the vast majority of potential Khd4 target mRNAs exhibit increased amounts in deletion mutants, Khd4 might promote mRNA instability. Mutants that fail to bind AUACCC resemble deletion mutants, which exhibit altered cell morphology, disturbed filamentous growth, and severely reduced virulence. Hence, RNA binding is essential for function of Khd4, stressing the importance of post-transcriptional control in regulating morphology and pathogenicity.
    RNA 10/2009; 15(12):2206-18. · 5.09 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cytoskeletal transport promotes polar growth in filamentous fungi. In Ustilago maydis, the RNA-binding protein Rrm4 shuttles along microtubules and is crucial for polarity in infectious filaments. Mutations in the RNA-binding domain cause loss of function. However, it was unclear which RNAs are bound and transported. Here, we applied in vivo RNA binding studies and live imaging to determine the molecular function of Rrm4. This new combination revealed that Rrm4 mediates microtubule-dependent transport of distinct mRNAs encoding, for example, the ubiquitin fusion protein Ubi1 and the small G protein Rho3. These transcripts accumulate in ribonucleoprotein particles (mRNPs) that move bidirectionally along microtubules and co-localise with Rrm4. Importantly, the 3' untranslated region of ubi1 containing a CA-rich binding site functions as zipcode during mRNA transport. Furthermore, motile mRNPs are not formed when the RNA-binding domain of Rrm4 is deleted, although the protein is still shuttling. Thus, Rrm4 constitutes an integral component of the transport machinery. We propose that microtubule-dependent mRNP trafficking is crucial for hyphal growth introducing U. maydis as attractive model for studying mRNA transport in higher eukaryotes.
    The EMBO Journal 07/2009; 28(13):1855-66. · 9.82 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Pheromone signalling during mating is essential for pathogenicity of Ustilago maydis. The activity of the key transcription factor Prf1 is controlled at the transcriptional level and post-translationally by mitogen-activated protein kinase (MAPK) and protein kinase A (PKA) phosphorylation. However, the precise contribution of these regulatory mechanisms to the transcriptional output is unknown. Here, we genetically dissected the three levels of Prf1 regulation. We performed transcriptional profiling of respective mutants to identify and classify targets. This approach revealed that transcriptional regulation of prf1 had only minor influence on target gene expression stressing the importance of post-translational control. PKA regulation of Prf1 was sufficient to control expression of nine pheromone-responsive genes including the major transcription factor regulating pathogenicity. MAPK regulation was necessary for the pheromone response of a set of 57 genes. In 35 cases, pheromone responsiveness was completely lost, while in the remaining 22 cases regulation was alleviated. This indicated a novel level of complexity in MAPK signalling suggesting that target genes respond differentially to MAPK phosphorylation of the respective transcription factors.
    Molecular Microbiology 08/2008; 69(4):1041-53. · 4.96 Impact Factor

Publication Stats

1k Citations
285.99 Total Impact Points

Institutions

  • 2010–2014
    • Heinrich-Heine-Universität Düsseldorf
      • • Institute of Microbiology
      • • Institut für Medizinische Mikrobiologie und Krankenhaushygiene
      Düsseldorf, North Rhine-Westphalia, Germany
  • 2003–2010
    • Max Planck Institute for Terrestrial Microbiology
      • Department of Organismic Interactions
      Marburg, Hesse, Germany
  • 2002
    • Michigan State University
      • MSU-DOE Plant Research Laboratory
      East Lansing, MI, United States
    • Ludwig-Maximilian-University of Munich
      • Division of Genetics
      München, Bavaria, Germany
  • 1999–2001
    • University Hospital München
      München, Bavaria, Germany
  • 1993–1997
    • Max Planck Institute of Biochemistry
      München, Bavaria, Germany