Publications by authors named "Ralf Dahm"

45 Publications

Historic nucleic acids isolated by Friedrich Miescher contain RNA besides DNA.

Biol Chem 2021 09 23;402(10):1179-1185. Epub 2021 Aug 23.

Department of Biology, University of Padova, I-35131 Padua, Italy.

One hundred fifty years ago, Friedrich Miescher discovered DNA when he isolated "Nuclein"-as he named it-from nuclei of human pus cells. Miescher recognized his isolate as a new type of molecule equal in importance to proteins. He realised that it is an acid of large molecular weight and high phosphorus content. Subsequently, he discovered Nuclein also in the nuclei of other cell types, realised that it chemically defines the nucleus, and speculated on its role in proliferation, heredity and fertilisation. While now universally recognised as the discoverer of DNA, whether Miescher also discovered RNA has not yet been addressed. To determine whether his isolation also yielded RNA, we first reproduced his historic protocols. Our resulting modern Nuclein contained a significant percentage of RNA. Encouraged by this result, we then analysed a sample of Nuclein isolated by Miescher from salmon sperm. Assuming that the RNA present in this sample had degraded to nucleobases, we tested for the presence of uracil in the historic Nuclein. Detection of significant levels of uracil by LC-UV-MS demonstrates that Miescher isolated both forms of nucleic acid-DNA and RNA-and underlines the fundamental nature of his discovery for the field of molecular genetics.
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http://dx.doi.org/10.1515/hsz-2021-0226DOI Listing
September 2021

How research institutions can foster innovation.

Bioessays 2021 09 14;43(9):e2100107. Epub 2021 Jul 14.

Centre for Transformative Learning, University of Limerick, Limerick, Ireland.

Carrying out research means being innovative, which requires novelty. Novelty is an important source of scientific breakthroughs and has great technological impact. Research institutions stand to benefit from fostering innovation. Here, we outline what academic institutions can do to help their scientists become more innovative.
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http://dx.doi.org/10.1002/bies.202100107DOI Listing
September 2021

Interdisciplinary Communication Needs to Become a Core Scientific Skill.

Bioessays 2019 09 29;41(9):e1900101. Epub 2019 Jul 29.

Academic Practice and eLearning, Trinity College Dublin, 3-4 Foster Place, Dublin 2, Republic of Ireland.

As scientific research has advanced so too has the complexity of the questions addressed. Cross-disciplinary collaborations are often the most efficient route to managing that complexity and require effective communication across boundaries. To continue driving science forward and be able to tackle global challenges, the art of good interdisciplinary communication needs to become a core skill in a scientist's portfolio.
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http://dx.doi.org/10.1002/bies.201900101DOI Listing
September 2019

How We Forgot Who Discovered DNA: Why It Matters How You Communicate Your Results.

Bioessays 2019 04;41(4):e1900029

Department of English and Linguistics, Obama Institute of Transnational American Studies, Johannes Gutenberg University Mainz, Welderweg 18, Mainz, 55118, Germany.

One hundred and fifty years ago, a hopeful young researcher reported a recent discovery he had made. Working in the bowels of a medieval castle in the German city of Tübingen, he had isolated a then entirely new type of molecule. This was the birth of a field that would fundamentally change the course of biology, medicine, and beyond. His discovery: DNA. His name: Friedrich Miescher. In this article, the authors try to find answers to the question why-despite the fact that virtually everyone nowadays knows DNA-hardly anyone remembers the man who discovered it. In the history of science, the discovery of DNA was a seminal moment. Why then did it not enter into public memory? Ground-breaking discoveries can occur in a historical context that is not ready to appreciate them. But that's not all that decides who is remembered and who is forgotten. Scientific pioneers sometimes fail to publicize their findings in a way that ensures that they receive the attention they merit. As discussed here, their personalities and habits may cause discoveries to be "overwritten" by more recent researchers, resulting in distorted cultural memories no longer reflecting the initial event.
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http://dx.doi.org/10.1002/bies.201900029DOI Listing
April 2019

Living autobiographically: Concepts of aging and artistic expression in painting and modern dance.

J Aging Stud 2017 Jan 22;40:8-15. Epub 2016 Dec 22.

Johannes Gutenberg University Mainz, Institute of Molecular Biology GmbH (IMB), Ackermannweg 4, 55128 Mainz, Germany.

This article discusses the ways in which artists have incorporated or failed to incorporate the aging process of their bodies into their art. Using Russian ballet dancer Mikhail Baryshnikov and the French painter Claude Monet as cases in point, we explore situations in which physical changes brought about by aging compromises artists' ability to engage with their artistic medium. Connecting Monet's oeuvre and Baryshnikov's dance performances to life writing accounts, we draw on John Paul Eakin's concept of "living autobiographically": In this vein, life writing research does not only have to take into account concepts of identity as they emerge from life writing narratives, but it also needs to explore the somatic, corporeal and material dimensions of these narratives.
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http://dx.doi.org/10.1016/j.jaging.2016.12.002DOI Listing
January 2017

Evolution of the vertebrate beaded filament protein, Bfsp2; comparing the in vitro assembly properties of a "tailed" zebrafish Bfsp2 to its "tailless" human orthologue.

Exp Eye Res 2012 Jan 11;94(1):192-202. Epub 2011 Dec 11.

In bony fishes, Bfsp2 orthologues are predicted to possess a C-terminal tail domain, which is absent from avian, amphibian and mammalian Bfsp2 sequences. These sequences, are however, not conserved between fish species and therefore questions whether they have a functional role. For other intermediate filament proteins, the C-terminal tail domain is important for both filament assembly and regulating interactions between filaments. We confirm that zebrafish has a single Bfsp2 gene by radiation mapping. Two transcripts (bfsp2α and bfsp2β) are produced by alternative splicing of the last exon. Using a polyclonal antibody specific to a tridecameric peptide in the C-terminal tail domain common to both zebrafish Bfsp2 splice variants, we have confirmed its expression in zebrafish lens fibre cells. We have also determined the in vitro assembly properties of zebrafish Bfsp2α and conclude that the C-terminal sequences are required to regulate not only the diameter and uniformity of the in vitro assembly filaments, but also their filament-filament associations in vitro. Therefore we conclude zebrafish Bfsp2α is a functional orthologue conforming more closely to the conventional domain structure of intermediate filament proteins. Data mining of the genome databases suggest that the loss of this tail domain could occur in several stages leading eventually to completely tailless orthologues, such as human BFSP2.
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http://dx.doi.org/10.1016/j.exer.2011.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593068PMC
January 2012

Homeostasis in the vertebrate lens: mechanisms of solute exchange.

Philos Trans R Soc Lond B Biol Sci 2011 Apr;366(1568):1265-77

Center for Brain Research, Medical University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria.

The eye lens is avascular, deriving nutrients from the aqueous and vitreous humours. It is, however, unclear which mechanisms mediate the transfer of solutes between these humours and the lens' fibre cells (FCs). In this review, we integrate the published data with the previously unpublished ultrastructural, dye loading and magnetic resonance imaging results. The picture emerging is that solute transfer between the humours and the fibre mass is determined by four processes: (i) paracellular transport of ions, water and small molecules along the intercellular spaces between epithelial and FCs, driven by Na(+)-leak conductance; (ii) membrane transport of such solutes from the intercellular spaces into the fibre cytoplasm by specific carriers and transporters; (iii) gap-junctional coupling mediating solute flux between superficial and deeper fibres, Na(+)/K(+)-ATPase-driven efflux of waste products in the equator, and electrical coupling of fibres; and (iv) transcellular transfer via caveoli and coated vesicles for the uptake of macromolecules and cholesterol. There is evidence that the Na(+)-driven influx of solutes occurs via paracellular and membrane transport and the Na(+)/K(+)-ATPase-driven efflux of waste products via gap junctions. This micro-circulation is likely restricted to the superficial cortex and nearly absent beyond the zone of organelle loss, forming a solute exchange barrier in the lens.
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http://dx.doi.org/10.1098/rstb.2010.0299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3061106PMC
April 2011

Investigating the genetics of visual processing, function and behaviour in zebrafish.

Neurogenetics 2011 May 26;12(2):97-116. Epub 2011 Jan 26.

Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.

Over the past three decades, the zebrafish has been proven to be an excellent model to investigate the genetic control of vertebrate embryonic development, and it is now also increasingly used to study behaviour and adult physiology. Moreover, mutagenesis approaches have resulted in large collections of mutants with phenotypes that resemble human pathologies, suggesting that these lines can be used to model diseases and screen drug candidates. With the recent development of new methods for gene targeting and manipulating or monitoring gene expression, the range of genetic modifications now possible in zebrafish is increasing rapidly. Combined with the classical strengths of the zebrafish as a model organism, these advances are set to substantially expand the type of biological questions that can be addressed in this species. In this review, we outline how the potential of the zebrafish can be harvested in the context of eye development and visual function. We review recent technological advances used to study the formation of the eyes and visual areas of the brain, visual processing on the cellular, subcellular and molecular level, and the genetics of visual behaviour in vertebrates.
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http://dx.doi.org/10.1007/s10048-011-0273-xDOI Listing
May 2011

A slip in the date of DNA's discovery.

Authors:
Ralf Dahm

Nature 2010 Dec;468(7326):897

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http://dx.doi.org/10.1038/468897dDOI Listing
December 2010

Transfection techniques for neuronal cells.

J Neurosci 2010 May;30(18):6171-7

Institute for Neuropathology, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany.

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http://dx.doi.org/10.1523/JNEUROSCI.0183-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6632737PMC
May 2010

From discovering to understanding. Friedrich Miescher's attempts to uncover the function of DNA.

Authors:
Ralf Dahm

EMBO Rep 2010 Mar 19;11(3):153-60. Epub 2010 Feb 19.

Department of Biology, University of Padua, Padua, Italy.

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http://dx.doi.org/10.1038/embor.2010.14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838690PMC
March 2010

The zebrafish mutant bumper shows a hyperproliferation of lens epithelial cells and fibre cell degeneration leading to functional blindness.

Mech Dev 2010 Apr 1;127(3-4):203-19. Epub 2010 Feb 1.

Max Planck Institute for Developmental Biology, Department of Genetics, Spemannstr. 35, D-72076 Tübingen, Germany.

The development of the eye lens is one of the classical paradigms of induction during embryonic development in vertebrates. But while there have been numerous studies aimed at discovering the genetic networks controlling early lens development, comparatively little is known about later stages, including the differentiation of secondary lens fibre cells. The analysis of mutant zebrafish isolated in forward genetic screens is an important way to investigate the roles of genes in embryogenesis. In this study we describe the zebrafish mutant bumper (bum), which shows a transient, tumour-like hyperproliferation of the lens epithelium as well as a progressively stronger defect in secondary fibre cell differentiation, which results in a significantly reduced lens size and ectopic location of the lens within the neural retina. Interestingly, the initial hyperproliferation of the lens epithelium in bum spontaneously regresses, suggesting this mutant as a valuable model to study the molecular control of tumour progression/suppression. Behavioural analyses demonstrate that, despite a morphologically normal retina, larval and adult bum(-/-) zebrafish are functionally blind. We further show that these fish have defects in their craniofacial skeleton with normal but delayed formation of the scleral ossicles within the eye, several reduced craniofacial bones resulting in an abnormal skull shape, and asymmetric ectopic bone formation within the mandible. Genetic mapping located the mutation in bum to a 4cM interval on chromosome 7 with the closest markers located at 0.2 and 0cM, respectively.
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http://dx.doi.org/10.1016/j.mod.2010.01.005DOI Listing
April 2010

Functions of the intermediate filament cytoskeleton in the eye lens.

J Clin Invest 2009 Jul 1;119(7):1837-48. Epub 2009 Jul 1.

Center for Ophthalmic Research/Surgery, Brigham and Women's Hospital, and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA.

Intermediate filaments (IFs) are a key component of the cytoskeleton in virtually all vertebrate cells, including those of the lens of the eye. IFs help integrate individual cells into their respective tissues. This Review focuses on the lens-specific IF proteins beaded filament structural proteins 1 and 2 (BFSP1 and BFSP2) and their role in lens physiology and disease. Evidence generated in studies in both mice and humans suggests a critical role for these proteins and their filamentous polymers in establishing the optical properties of the eye lens and in maintaining its transparency. For instance, mutations in both BFSP1 and BFSP2 cause cataract in humans. We also explore the potential role of BFSP1 and BFSP2 in aging processes in the lens.
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http://dx.doi.org/10.1172/JCI38277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2701874PMC
July 2009

Transfection of cultured primary neurons via nucleofection.

Curr Protoc Neurosci 2009 Apr;Chapter 4:Unit4.32

Medical University of Vienna, Center for Brain Research, Vienna, Austria.

Despite the development of various transfection methods, the transfection of post-mitotic cells, including neurons, poses a challenging task. Nucleofection, a specialized form of electroporation described in this unit, achieves high transfection efficiencies in primary mammalian neurons, such as hippocampal neurons, while simultaneously maintaining high cell viability. Therefore, it allows for biochemical analyses that rely on large numbers of transfected cells. The recently developed 96-well shuttle system described in this unit further permits the transfection of up to 96 different constructs in a single experiment. This opens up the possibility for large-scale experiments in primary neurons, such as shRNA-mediated knock-down of a wide range of target genes.
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http://dx.doi.org/10.1002/0471142301.ns0432s47DOI Listing
April 2009

Perplexing bodies: The putative roles of P-bodies in neurons.

RNA Biol 2008 Oct-Dec;5(4):244-8. Epub 2008 Oct 8.

Department of Neuronal Cell Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria.

Processing bodies (P-bodies) have recently come to the fore as important cellular sites of mRNA degradation and translational silencing. Despite these central functions in the control of gene expression, the roles of P-bodies have only been characterized in a limited number of cell types and physiological contexts. Neurons are highly plastic cells that undergo dynamic changes as new connections are made or existing ones modified. This neuronal plasticity relies, in part, on the local synthesis of proteins from localized mRNAs. A strict control of the translation and turnover of these localized mRNAs, both in terms of which proteins are synthesized and when and where they are produced, is a key prerequisite for this process to be synapse-specific. Despite recent advances, the molecular mechanisms mediating this control remain largely elusive. The discovery of P-bodies in neuronal dendrites near synapses and their response to stimuli involved in neuronal plasticity raises the interesting hypothesis that P-bodies might be a component of the cellular machinery that controls neuronal plasticity and thereby processes such as learning and memory.
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http://dx.doi.org/10.4161/rna.6948DOI Listing
March 2009

High-efficiency transfection of short hairpin RNAs-encoding plasmids into primary hippocampal neurons.

J Neurosci Res 2009 Jan;87(1):289-300

Department of Neuronal Cell Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria.

The transfection of expression constructs encoding a variety of transgenes is a widely used method to study gene function in cultured cells. Especially when the efficiency of the knock-down of target proteins via small interfering RNAs (siRNAs) is to be determined by quantitative Western blotting, large proportions of untransfected cells compromise the analysis. Achieving high transfection efficiencies in postmitotic cells, such as neurons, poses a particular problem in that these cells cannot be selected for the expression of the transgene following transfection. It is therefore important to develop protocols that allow for the highly efficient transfection of these cells. In the present study, we identify three important parameters that prove especially useful for chronically difficult to transfect short hairpin RNA (shRNA)-encoding plasmids: the amount and quality of the plasmid DNA used and the use of new nucleofection programs. Combining those changes increases the rate of transfected cells from less than 5% to up to approximately 80%. Importantly, these high transfection efficiencies can be obtained while maintaining good cell viability and normal cellular development. Taken together, these improvements allow for a detailed biochemical and phenotypical analysis of neurons that have been nucleoporated with a wide variety of shRNAs.
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http://dx.doi.org/10.1002/jnr.21840DOI Listing
January 2009

Dynamic interaction between P-bodies and transport ribonucleoprotein particles in dendrites of mature hippocampal neurons.

J Neurosci 2008 Jul;28(30):7555-62

Department of Neuronal Cell Biology, Center for Brain Research, Medical University of Vienna, Vienna, Austria.

The dendritic localization of mRNAs and their subsequent translation at stimulated synapses contributes to the experience-dependent remodeling of synapses and thereby to the establishment of long-term memory. Localized mRNAs are transported in a translationally silent manner to distal dendrites in specific ribonucleoprotein particles (RNPs), termed transport RNPs. A recent study suggested that processing bodies (P-bodies), which have recently been identified as sites of RNA degradation and translational control in eukaryotic cells, may participate in the translational control of dendritically localized mRNAs in Drosophila neurons. This study raised the interesting question of whether dendritic transport RNPs are distinct from P-bodies or whether those structures share significant overlap in their molecular composition in mammalian neurons. Here, we show that P-body and transport RNP markers do not colocalize and are not transported together in the same particles in dendrites of mammalian neurons. Detailed time-lapse videomicroscopy analyses reveal, however, that both P-bodies and transport RNPs can interact in a dynamic manner via docking. Docking is a frequent event involving as much as 50% of all dendritic P-bodies. Chemically induced neuronal activity results in a 60% decrease in the number of P-bodies in dendrites, suggesting that P-bodies disassemble after synaptic stimulation. Our data lend support to the exciting hypothesis that dendritically localized mRNAs might be stored in P-bodies and be released and possibly translated when synapses become activated.
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http://dx.doi.org/10.1523/JNEUROSCI.0104-08.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6670838PMC
July 2008

Subfunctionalization of duplicated zebrafish pax6 genes by cis-regulatory divergence.

PLoS Genet 2008 Feb;4(2):e29

Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh, United Kingdom.

Gene duplication is a major driver of evolutionary divergence. In most vertebrates a single PAX6 gene encodes a transcription factor required for eye, brain, olfactory system, and pancreas development. In zebrafish, following a postulated whole-genome duplication event in an ancestral teleost, duplicates pax6a and pax6b jointly fulfill these roles. Mapping of the homozygously viable eye mutant sunrise identified a homeodomain missense change in pax6b, leading to loss of target binding. The mild phenotype emphasizes role-sharing between the co-orthologues. Meticulous mapping of isolated BACs identified perturbed synteny relationships around the duplicates. This highlights the functional conservation of pax6 downstream (3') control sequences, which in most vertebrates reside within the introns of a ubiquitously expressed neighbour gene, ELP4, whose pax6a-linked exons have been lost in zebrafish. Reporter transgenic studies in both mouse and zebrafish, combined with analysis of vertebrate sequence conservation, reveal loss and retention of specific cis-regulatory elements, correlating strongly with the diverged expression of co-orthologues, and providing clear evidence for evolution by subfunctionalization.
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http://dx.doi.org/10.1371/journal.pgen.0040029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2242813PMC
February 2008

Formation of stromal collagen fibrils and proteoglycans in the developing zebrafish cornea.

Acta Ophthalmol 2008 Sep;86(6):655-65

Nuffield Laboratory of Ophthalmology, Oxford, UK.

Purpose: Collagen fibrils and proteoglycans are the main components of the corneal extracellular matrix and corneal transparency depends crucially on their proper arrangement. In the present study, we investigated the formation of collagen fibrils and proteoglycans in the developing cornea of the zebrafish, a model organism used to study vertebrate embryonic development and genetic disease.

Methods: We employed thin-section electron microscopy to investigate the ultrastructure of the zebrafish cornea at different developmental stages.

Results: The layering of the zebrafish cornea into an epithelium, a Bowman's layer, stroma and endothelium was observed starting at 72 hr post-fertilization. At this stage, the stroma contained orthogonally arranged collagen fibrils and small proteoglycans. The density of proteoglycans increased gradually throughout subsequent development of the cornea. In the stroma of 2-week-old larvae, the collagen fibrils were organized into thin lamellae and were separated by very large, randomly distributed proteoglycans. At 4 weeks, a regular arrangement of proteoglycans in relation to the collagen fibrils was observed for the first time and the lamellae were also thickened.

Conclusion: The present study, for the first time, provides ultrastructural details of collagen fibril and proteoglycan development in the zebrafish cornea. Furthermore, it directly correlates the collagen fibril and proteoglycan composition of the zebrafish cornea with that of the human cornea. The similarities between the two species suggest that the zebrafish could serve as a model for investigating the genetics of human corneal development and diseases.
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http://dx.doi.org/10.1111/j.1600-0420.2007.01135.xDOI Listing
September 2008

Visualizing mRNA localization and local protein translation in neurons.

Methods Cell Biol 2008 ;85:293-327

Center for Brain Research, Division of Neuronal Cell Biology, Medical University of Vienna, Spitalgasse 4, A-1090 Vienna, Austria.

Fluorescent proteins (FPs) have been successfully used to study the localization and interactions of proteins in living cells. They have also been instrumental in analyzing the proteins involved in the localization of RNAs in different cell types, including neurons. With the development of methods that also tag RNAs via fluorescent proteins, researchers now have a powerful tool to covisualize RNAs and associated proteins in living neurons. Here, we review the current status of the use of FPs in the study of transport and localization of ribonucleoprotein particles (RNPs) in neurons and provide key protocols used to introduce transgenes into cultured neurons, including calcium-phosphate-based transfection and nucleofection. These methods allow the fast and efficient expression of fluorescently tagged fusion proteins in neurons at different stages of differentiation and form the basis for fluorescent protein-based live cell imaging in neuronal cultures. Additional protocols are given that allow the simultaneous visualization of RNP proteins and cargo RNAs in living neurons and aspects of the visualization of fluorescently tagged proteins in neurons, such as colocalization studies, are discussed. Finally, we review approaches to visualize the local synthesis of proteins in distal dendrites and axons.
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http://dx.doi.org/10.1016/S0091-679X(08)85013-3DOI Listing
February 2008

The zebrafish mutant lbk/vam6 resembles human multisystemic disorders caused by aberrant trafficking of endosomal vesicles.

Development 2008 Jan 12;135(2):387-99. Epub 2007 Dec 12.

Swiss Federal Institute of Technology, Department of Biology, and Brain Research Institute of the University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.

The trafficking of intracellular vesicles is essential for a number of cellular processes and defects in this process have been implicated in a wide range of human diseases. We identify the zebrafish mutant lbk as a novel model for such disorders. lbk displays hypopigmentation of skin melanocytes and the retinal pigment epithelium (RPE), an absence of iridophore reflections, defects in internal organs (liver, intestine) as well as functional defects in vision and the innate immune system (macrophages). Positional cloning, an allele screen, rescue experiments and morpholino knock-down reveal a mutation in the zebrafish orthologue of the vam6/vps39 gene. Vam6p is part of the HOPS complex, which is essential for vesicle tethering and fusion. Affected cells in the lbk RPE, liver, intestine and macrophages display increased numbers and enlarged intracellular vesicles. Physiological and behavioural analyses reveal severe defects in visual ability in lbk mutants. The present study provides the first phenotypic description of a lack of vam6 gene function in a multicellular organism. lbk shares many of the characteristics of human diseases and suggests a novel disease gene for pathologies associated with defective vesicle transport, including the arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome, the Hermansky-Pudlak syndrome, the Chediak-Higashi syndrome and the Griscelli syndrome.
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http://dx.doi.org/10.1242/dev.006098DOI Listing
January 2008

Human pathologies associated with defective RNA transport and localization in the nervous system.

Biol Cell 2007 Nov;99(11):649-61

Medical University of Vienna, Center for Brain Research, Division of Neuronal Cell Biology, Spitalgasse 4, A-1090 Vienna, Austria.

RNA localization is emerging as an important process to restrict certain proteins to specific subcellular domains and thus spatially control the expression of genes within cells. It is used, for instance, to compartmentalize the developing embryo during early embryogenesis. The localization of RNA also plays important roles later during development, such as in asymmetric cell divisions, cell migration and the outgrowth and pathfinding of axons and dendrites. In differentiated cells, it serves to subdivide the cell into functionally distinct compartments. For example, in mature neurons it is believed to contribute to the plastic changes of individual synapses underlying learning and memory. In this review, we highlight the importance of subcellular RNA localization for the function of the nervous system and neurological diseases associated with defective RNA localization and translation. These diseases include fragile X mental retardation syndrome, spinocerebellar ataxia and spinal muscular atrophy.
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http://dx.doi.org/10.1042/BC20070045DOI Listing
November 2007

The GTP-binding protein Septin 7 is critical for dendrite branching and dendritic-spine morphology.

Curr Biol 2007 Oct 11;17(20):1746-51. Epub 2007 Oct 11.

Department of Neural Cell Biology, Center for Brain Research, Medical University of Vienna, Vienna A1090, Austria.

Septins, a highly conserved family of GTP-binding proteins, were originally identified in a genetic screen for S. cerevisiae mutants defective in cytokinesis [1, 2]. In yeast, septins maintain the compartmentalization of the yeast plasma membrane during cell division by forming rings at the cortex of the bud neck, and these rings establish a lateral diffusion barrier. In contrast, very little is known about the functions of septins in mammalian cells [3, 4] including postmitotic neurons [5-7]. Here, we show that Septin 7 (Sept7) localizes at the bases of filopodia and at branch points in developing hippocampal neurons. Upon downregulation of Sept7, dendritic branching is impaired. In mature neurons, Sept7 is found at the bases of dendritic spines where it associates with the plasma membrane. Mature Sept7-deficient neurons display elongated spines. Furthermore, Sept5 and Sept11 colocalize with and coimmunoprecipitate with Sept7, thereby arguing for the existence of a Septin5/7/11 complex. Taken together, our findings show an important role for Sept7 in regulating dendritic branching and dendritic-spine morphology. Our observations concur with data from yeast, in which downregulation of septins yields elongated buds, suggesting a conserved function for septins from yeast to mammals.
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http://dx.doi.org/10.1016/j.cub.2007.08.042DOI Listing
October 2007

Discovering DNA: Friedrich Miescher and the early years of nucleic acid research.

Authors:
Ralf Dahm

Hum Genet 2008 Jan 28;122(6):565-81. Epub 2007 Sep 28.

Center for Brain Research, Medical University of Vienna, Spitalgasse 4, 1090, Vienna, Austria,

In the winter of 1868/9 the young Swiss doctor Friedrich Miescher, working in the laboratory of Felix Hoppe-Seyler at the University of Tübingen, performed experiments on the chemical composition of leukocytes that lead to the discovery of DNA. In his experiments, Miescher noticed a precipitate of an unknown substance, which he characterised further. Its properties during the isolation procedure and its resistance to protease digestion indicated that the novel substance was not a protein or lipid. Analyses of its elementary composition revealed that, unlike proteins, it contained large amounts of phosphorous and, as Miescher confirmed later, lacked sulphur. Miescher recognised that he had discovered a novel molecule. Since he had isolated it from the cells' nuclei he named it nuclein, a name preserved in today's designation deoxyribonucleic acid. In subsequent work Miescher showed that nuclein was a characteristic component of all nuclei and hypothesised that it would prove to be inextricably linked to the function of this organelle. He suggested that its abundance in tissues might be related to their physiological status with increases in "nuclear substances" preceding cell division. Miescher even speculated that it might have a role in the transmission of hereditary traits, but subsequently rejected the idea. This article reviews the events and circumstances leading to Miescher's discovery of DNA and places them within their historic context. It also tries to elucidate why it was Miescher who discovered DNA and why his name is not universally associated with this molecule today.
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http://dx.doi.org/10.1007/s00439-007-0433-0DOI Listing
January 2008

Transition from enhanced T cell infiltration to inflammation in the myelin-degenerative central nervous system.

Neurobiol Dis 2007 Dec 3;28(3):261-75. Epub 2007 Aug 3.

Medical University Vienna, Center for Brain Research, Division of Neuroimmunology, Spitalgasse 4, A-1090 Vienna, Austria.

Myelin degeneration in the central nervous system (CNS) is often associated with elevated numbers of T cells in brain and spinal cord (SC). In some degenerative diseases, this T cell immigration has no clinical relevance, in others, it may precede severe inflammation and tissue damage. We studied T cells in the myelin-degenerative SC of transgenic (tg) Lewis rats overexpressing the proteolipid protein (PLP). These lymphocytes are T(H)1/T(C)1 cells and represent different T cell clones unique to individual animals. The SC-infiltrating CD8(+) T cell pool is more restricted than its CD4(+) counterpart, possibly due to constrictions in the peripheral CD8(+) T cell repertoire. Some SC-infiltrating T cells are highly motile and cover large distances within their target tissue, others are tethered to MHC class II(+) microglia cells. The activation of the tethered cells may trigger the formation of inflammatory foci and could pave the way for inflammation in degenerative CNS disease.
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http://dx.doi.org/10.1016/j.nbd.2007.05.006DOI Listing
December 2007

Reorganization of centrosomal marker proteins coincides with epithelial cell differentiation in the vertebrate lens.

Exp Eye Res 2007 Nov 15;85(5):696-713. Epub 2007 Aug 15.

Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee, DD1 4HN, UK.

The differentiation of epithelial cells in the vertebrate lens involves a series of changes that includes the degradation of all intracellular organelles and a dramatic elongation of the cells. The latter is accompanied by a substantial remodelling of the cytoskeleton and changes in the distribution of the actin, microtubule and intermediate filament cytoskeletons during lens cell differentiation have been well documented. There have, however, been no studies of microtubule organizing centres (MTOCs) and specifically centrosomes during lens cell differentiation. We have investigated the fate of the centrosomal MTOCs during cellular differentiation in the bovine lens using gamma-tubulin, ninein, centrin 2 and centrin 3 as markers. Our studies show that these markers oscillate between a clear centrosome-based association in epithelial cells and a defocused cluster in lens fibre cells. Our data further reveal a transient loss of signal for the typical centrosomal marker gamma-tubulin as the lens epithelial cells begin to differentiate into lens fibre cells. This marker apparently disappears in the most distal epithelial cells at the lens equator, only to reappear in early lens fibre cells. The changes in gamma-tubulin distribution are mirrored by the other centrosomal markers, centrins 2 and 3 and ninein that also show a similar transient loss of their signals and subsequent clustering at the apical ends of differentiating fibre cells. The transient loss of staining for these centrosomal markers in the most posterior epithelial cells is a distinctive feature that precedes lens cell elongation. The dramatic reorganization of MTOC markers coincides with gap junction reorganization as seen by the loss of connexin 43 (alpha1-connexin) in these lens epithelial cells suggesting that these events mark a significant change preceding subsequent cell elongation and differentiation into fibre cells.
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http://dx.doi.org/10.1016/j.exer.2007.07.022DOI Listing
November 2007

High-efficiency transfection of mammalian neurons via nucleofection.

Nat Protoc 2007 ;2(7):1692-704

Division of Neuronal Cell Biology, Center for Brain Research, Medical University of Vienna, A-1090 Vienna, Austria.

Transfection of foreign DNA is widely used to study gene function. However, despite the development of numerous methods, the transfer of DNA into postmitotic cells, such as neurons, remains unsatisfactory with regard to either transfection efficiency or cytotoxicity. Nucleofection overcomes these limitations. Direct electroporation of expression plasmids or oligonucleotides into the nucleus ensures both good cell viability and consistently high transfection rates. This allows biochemical analyses of transfected neurons, for example, western blot analyses of protein levels after RNA interference (RNAi) knockdown or microRNA transfection. We provide comprehensive protocols for performing nucleofection with high efficiency on primary neurons. The focus is on the recently developed 96-well shuttle system, which allows the simultaneous testing of up to 96 different plasmids or experimental conditions. Using this system, reproducible high-throughput expression of various transgenes is now feasible on primary neurons, for example large-scale RNAi analyses to downregulate gene expression. The protocol typically takes between 2 and 3 h.
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http://dx.doi.org/10.1038/nprot.2007.226DOI Listing
September 2007

Development and adult morphology of the eye lens in the zebrafish.

Exp Eye Res 2007 Jul 19;85(1):74-89. Epub 2007 Mar 19.

Max-Planck-Institute for Developmental Biology, Spemannstrasse 35, D-72076 Tübingen, Germany.

The zebrafish has become an important vertebrate model organism to study the development of the visual system. Mutagenesis projects have resulted in the identification of hundreds of eye mutants. Analysis of the phenotypes of these mutants relies on in depth knowledge of the embryogenesis in wild-type animals. While the morphological events leading to the formation of the retina and its connections to the central nervous system have been described in great detail, the characterization of the development of the eye lens is still incomplete. In the present study, we provide a morphological description of embryonic and larval lens development as well as adult lens morphology in the zebrafish. Our analyses show that, in contrast to other vertebrate species, the zebrafish lens delaminates from the surface ectoderm as a solid cluster of cells. Detachment of the prospective lens from the surface ectoderm is facilitated by apoptosis. Primary fibre cell elongation occurs in a circular fashion resulting in an embryonic lens nucleus with concentric shells of fibres. After formation of a monolayer of lens epithelial cells, differentiation and elongation of secondary lens fibres result in a final lens morphology similar to that of other vertebrate species. As in other vertebrates, secondary fibre cell differentiation includes the programmed degradation of nuclei, the interconnection of adjacent fibres via protrusions at the fibre cells' edges and the establishment of gap junctions between lens fibre cells. The very close spacing of the nuclei of the differentiating secondary fibres in a narrow zone close to the equatorial epithelium, however, suggests that secondary fibre cell differentiation deviates from that described for mammalian or avian lenses. In summary, while there are similarities in the development and final morphology of the zebrafish lens with mammalian and avian lenses, there are also significant differences, suggesting caution when extrapolating findings on the zebrafish to, for example, human lens development or function.
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http://dx.doi.org/10.1016/j.exer.2007.02.015DOI Listing
July 2007

RNA localization: new roles for an evolutionarily ancient mechanism.

Semin Cell Dev Biol 2007 Apr 12;18(2):161-2. Epub 2007 Mar 12.

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http://dx.doi.org/10.1016/j.semcdb.2007.03.001DOI Listing
April 2007

RNA localisation in the nervous system.

Semin Cell Dev Biol 2007 Apr 26;18(2):216-23. Epub 2007 Jan 26.

Medical University of Vienna, Center for Brain Research, Division of Neuronal Cell Biology, Spitalgasse 4, A-1090 Vienna, Austria.

The localisation of specific RNAs is a widely employed mechanism to generate asymmetry in various biological systems, e.g. during embryonic development and cellular differentiation. Here, we highlight the importance of RNA localisation in mature neurons. Specific examples of mRNAs localised in neurons are those encoding Arc, beta-actin, CaMKIIalpha and MAP2. Moreover, non-coding RNAs, such as BC1/BC200 and microRNAs (miRNAs), which play important roles in the translational regulation of localised mRNAs, receive increasing attention. The process of RNA localisation, including RNP biogenesis, transport, anchoring and translational control, and the importance of RNA localisation for the function of the nervous system are discussed.
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http://dx.doi.org/10.1016/j.semcdb.2007.01.009DOI Listing
April 2007
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