Using simulated fluorescence cell micrographs for the evaluation of cell image segmentation algorithms

The zip archive contains real images showing macrophages. (ZIP 28979 kb

Measurement of TLR-Induced Macrophage Spreading by Automated Image Analysis: Differential Role of Myd88 and MAPK in Early and Late Responses

Sensing of infectious danger by toll-like receptors (TLRs) on macrophages causes not only a reprogramming of the transcriptome but also changes in the cytoskeleton important for cell spreading and motility. Since manual determination of cell contact areas from fluorescence micrographs is very time-consuming and prone to bias, we have developed and tested algorithms for automated measurement of macrophage spreading. The two-step method combines identification of cells by nuclear staining with DAPI and cell surface staining of the integrin CD11b. Automated image analysis correlated very well with manual annotation in resting macrophages and early after stimulation, whereas at later time points the automated cell segmentation algorithm and manual annotation showed slightly larger variation. The method was applied to investigate the impact of genetic or pharmacological inhibition of known TLR signaling components. Deficiency in the adapter protein Myd88 strongly reduced spreading activity at the late time points, but had no impact early after LPS-stimulation. A similar effect was observed upon pharmacological inhibition of MEK1, the kinase activating the mitogen-activated protein kinases (MAPK) ERK1/2, indicating that ERK1/2 mediates Myd88-dependent macrophages spreading. In contrast, macrophages lacking the MAPK p38 were impaired in the initial spreading response but responded normally 8–24 h after stimulation. The dichotomy of p38 and ERK1/2 MAPK effects on early and late macrophage spreading raises the question which of the respective substrate proteins mediate(s) cytoskeletal remodeling and spreading. The automated measurement of cell spreading described here increases the objectivity and greatly reduces the time required for such investigations and is therefore expected to facilitate larger throughput analysis of macrophage spreading, e.g., in siRNA knockdown screens

Endosomal accumulation of APP in wobbler motor neurons reflects impaired vesicle trafficking: Implications for human motor neuron disease

Palmisano R, Golfi P, Heimann P, et al. Endosomal accumulation of APP in wobbler motor neurons reflects impaired vesicle trafficking: Implications for human motor neuron disease. BMC Neuroscience. 2011;12(1): 24.Background: The cause of sporadic amyotrophic lateral sclerosis (ALS) is largely unknown but hypotheses about disease mechanisms include oxidative stress, defective axonal transport, mitochondrial dysfunction and disrupted RNA processing. Whereas familial ALS is well represented by transgenic mutant SOD1 mouse models, the mouse mutant wobbler (WR) develops progressive motor neuron degeneration due to a point mutation in the Vps54 gene, and provides an animal model for sporadic ALS. VPS54 protein as a component of a protein complex is involved in vesicular Golgi trafficking; impaired vesicle trafficking might also be mechanistic in the pathogenesis of human ALS. Results: In motor neurons of homozygous symptomatic WR mice, a massive number of endosomal vesicles significantly enlarged (up to 3 mu m in diameter) were subjected to ultrastructural analysis and immunohistochemistry for the endosome-specific small GTPase protein Rab7 and for amyloid precursor protein (APP). Enlarged vesicles were neither detected in heterozygous WR nor in transgenic SOD1(G93A) mice; in WR motor neurons, numerous APP/Rab7-positive vesicles were observed which were mostly LC3-negative, suggesting they are not autophagosomes. Conclusions: We conclude that endosomal APP/Rab7 staining reflects impaired vesicle trafficking in WR mouse motor neurons. Based on these findings human ALS tissues were analysed for APP in enlarged vesicles and were detected in spinal cord motor neurons in six out of fourteen sporadic ALS cases. These enlarged vesicles were not detected in any of the familial ALS cases. Thus our study provides the first evidence for wobbler-like aetiologies in human ALS and suggests that the genes encoding proteins involved in vesicle trafficking should be screened for pathogenic mutations

How enhancers regulate wavelike gene expression patterns

A key problem in development is to understand how genes turn on or off at the right place and right time during embryogenesis. Such decisions are made by non-coding sequences called ‘enhancers.’ Much of our models of how enhancers work rely on the assumption that genes are activated de novo as stable domains across embryonic tissues. Such a view has been strengthened by the intensive landmark studies of the early patterning of the anterior-posterior (AP) axis of the Drosophila embryo, where indeed gene expression domains seem to arise more or less stably. However, careful analysis of gene expression patterns in other model systems (including the AP patterning in vertebrates and short-germ insects like the beetle Tribolium castaneum) painted a different, very dynamic view of gene regulation, where genes are oftentimes expressed in a wavelike fashion. How such gene expression waves are mediated at the enhancer level is so far unclear. Here, we establish the AP patterning of the short-germ beetle Tribolium as a model system to study dynamic and temporal pattern formation at the enhancer level. To that end, we established an enhancer prediction system in Tribolium based on time- and tissue-specific ATAC-seq and an enhancer live reporter system based on MS2 tagging. Using this experimental framework, we discovered several Tribolium enhancers, and assessed the spatiotemporal activities of some of them in live embryos. We found our data consistent with a model in which the timing of gene expression during embryonic pattern formation is mediated by a balancing act between enhancers that induce rapid changes in gene expression patterns (that we call ‘dynamic enhancers’) and enhancers that stabilize gene expression patterns (that we call ‘static enhancers’). However, more data is needed for a strong support for this or any other alternative models

Fluoreszenz-Resonanz-Energie-Transfer-basierter spezifischer Nachweis von mRNA in vitro und in situ

Palmisano R. Fluoreszenz-Resonanz-Energie-Transfer-basierter spezifischer Nachweis von mRNA in vitro und in situ. Bielefeld (Germany): Bielefeld University; 2003.In den letzten Jahren sind große Fortschritte auf dem Gebiet der molekularen Diagnostik erzielt worden. Die molekulargenetischen Grundlagen vieler de novo oder erblich auftretender Erkrankungen bei Föten konnten eingegrenzt oder aufgeklärt werden. Dies hat zu verbesserter und aussagekräftigerer vorgeburtlicher Befundung in der Folge nicht-invasiver und invasiver Diagnostik geführt. Unbefriedigend ist die derzeitige Situation, dass keine risikofreie nicht-invasive Routine zur Erlangung fötalen Zellmaterials zur Verfügung steht. Bei der Separation humaner Zelltypen spielt die Bindung von fluoreszenzmarkierten Antikörpern oder Liganden und anschließende Fluoreszenz-aktivierte Zellsortierung eine wichtige Rolle. Diese Markierungen sind jedoch nicht in jedem Falle von ausreichender Spezifität und damit Selektivität hinsichtlich der gesuchten Zellen. Eine andere Möglichkeit der selektiven Identifikation einer Zelle bestimmter Herkunft kann der Expressionsstatus ausgewählter Gene sein. Die Korrelation der Aktivität eines spezifischen Gens mit Herkunft und Typ der Zelle muss zweifelsfrei und einzigartig sein. Wenn sich dieses Transkriptionsprodukt in situ eindeutig nachweisen ließe, hätte man eine Methode, die hochspezifisch wäre, wenn gleichzeitig falsch positive Signale ausgeschlossen werden können. Eine Methode, die aufgrund ihrer Wirkungsweise dafür geeignet scheint, ist die Transfektion mit fluoreszenzmarkierten Antisense-Oligonucleotiden, welche nach Hybridisierung an die Ziel mRNA einen Fluoreszenz Resonanz Energie Transfer (FRET) erzeugen können. In dieser Arbeit wird untersucht, ob durch eine solche in situ Hybridisierung von DNA-Oligonucleotiden an mRNA ein FRET-Signal erzeugt werden kann, das außerhalb der Zelle detektierbar ist. Daher teilt sich die Arbeit in zwei Schwerpunkte auf. In dem einen Teil wurden fluoreszenzmarkierte DNA-Oligonucleotide hinsichtlich der optimalen Parameter für einen FRET untersucht, der im sichtbaren Wellenlängenbereich detektierbar sein muss. Diese Untersuchungen wurden mit Hilfe der Anregungs-Emissions-Spektroskopie (AES) durchgeführt, die eine zweidimensionale Aufnahme von Fluoreszenzspektren ermöglicht. Hierbei wurde gezeigt, dass die Kombination der beiden Fluorophore Bodipy493/503 und Texas-Red den stärksten FRET aus einer untersuchten Gruppe von jeweils fünf verschiedenen Donor- und Akzeptor-Fluorophoren erzeugt. Es wurde auch gezeigt, dass geeignete DNA-Oligonucleotide jeweils eine Länge von mindestens 25 Nucleotiden und einen hohen GC-Gehalt aufweisen sollten, um die Stabilität der Hybridisierung zu gewährleisten als auch Sequenzhomologien, die zu Fehlpaarungen führen, auszuschließen. Es wurde des weiteren gezeigt, dass sich Phosphothioat-modifizierte DNA-Oligonucleotide in ihren spektroskopischen Eigenschaften nicht von unmodifizierten unterscheiden. Im zweiten Teil der Arbeit wurden die Möglichkeiten der Transfektion von Zellen mit Antisense-Oligonucleotiden untersucht. Es wurden verschiedene physikalische und chemische Transfektionsmethoden verglichen und nach einer geeigneten Fixierungsmethode gesucht, welche die Zellen für weitere Untersuchungen stabilisiert. Als Modellsystem wurde die chronisch myelogene Leukämie-Zelllinie K-562 ausgewählt, die nach Induktion stabil die fötalen [epsilon]- und [gamma]-Globin Gene exprimiert. Als beste Methode hat sich die Transfektion der Zellen mit Phosphothioat-modifizierten Oligonucleotiden und dem kationischen Transfektionsmittel Oligofectamine sowie nachfolgender Paraformaldehyd/Eisessig Fixierung herausgestellt. Die Zellen wurden mit einem Confokalen Laser Scanning Mikroskop untersucht und der FRET in den Zellen durch "Akzeptor-Bleichung" nachgewiesen und quantifiziert. Als Negativkontrollen dienten zum einen Sense-Oligonucleotide der jeweiligen Sequenzen und eine zweite Zelllinie, die keine Globin Gene exprimiert und mit den Antisense-Oligonucleotiden transfiziert wurde

Approaches to automatic parameter fitting in a microscopy image segmentation pipeline: An exploratory parameter space analysis

Held C, Nattkemper TW, Palmisano R, Wittenberg T. Approaches to automatic parameter fitting in a microscopy image segmentation pipeline: An exploratory parameter space analysis. Journal of Pathology Informatics. 2013;4(2): 5

Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time

Martini J, Schmied K, Palmisano R, Tönsing K, Anselmetti D, Merkle T. Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time. Journal of Structural Biology. 2007;158(3):401-409.We used multifocal two-photon laser scanning microscopy for local and selective protein activation and quantitative investigation of intracellular protein dynamics. The localized activation was realized with photo-activatable green-fluorescent-proteins (pa-GFP) and optical two-photon excitation in order to investigate the real-time intracellular dynamics in vivo. Such processes are of crucial importance for a deep understanding and modelling of regulatory and metabolic processes in living cells. Exemplarily, the intracellular dynamics of the Arabidopsis MYB transcription factor LHY/CCA1-like 1 (LCL1) that contains both a nuclear import and a nuclear export signal was quantitatively investigated. We used tobacco BY-2 protoplasts co-transfected with plasmids encoding photo-activatable green fluorescent protein (pa-GFP) fusion proteins and a red fluorescing transfection marker and measured the rapid nuclear export of pa-GFP-LCL1 I after its photo-activation in the nucleus. In contrast, an export-negative mutant of LCL1 remained trapped inside the nucleus. We detemined average time constants of 51s and 125s for the decrease of fluorescence in the nucleus due to active bi-directional nuclear transport of pa-GFP-LCL1 and diffusion of pa-GFP, respectively. (c) 2007 Elsevier Inc. All rights reserved

Analysis of Subcellular Surface Structure, Function and Dynamics

Anselmetti D, Hansmeier N, Kalinowski J, et al. Analysis of Subcellular Surface Structure, Function and Dynamics. Analytical and Bioanalytical Chemistry. 2007;387(1):83-89.Analytics of single biological cells allows quantitative investigation from a structural, functional and dynamical point of view and opens novel possibilities to an unamplified subcellular analysis. In this article, we report on three different experimental methods and their applications to single cellular systems with a subcellular sensitivity down to the single molecule level. First, the subcellular surface structure of living bacteria (Corynebacterium glutamicum) was investigated with atomic force microscopy (AFM) at the resolution of individual surface layer (S-layer) proteins; discrimination of bacterial strains that lack the expression of hexagonally packed surface layer proteins was possible. Second, quantitative measurement of individual recognition events of membrane-bound receptors on living B-cells was achieved in single cell manipulation and probing experiments with optical tweezers (OT) force spectroscopy. And third, intracellular dynamics of translocating photoactivatable GFP in plant protoplasts (Nicotiana tabacum BY-2) was quantitatively monitored by two-photon laser scanning microscopy (2PLSM)