Name: investigating the function of coronin a in the early starvation response of dictyostelium discoideum by aggregation assays
Uploaded: 2016-06-18T15:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays at JoVE.com

We thank the Dictyostelium Stock Center for strains and reagents. This study was financed by grants from the Swiss National Science Foundation and the Canton of Basel.

<ol><li> Beachten Sie die frühen Hunger Reaktion von Dictyostelium discoideum durch Zeitraffer-Mikroskopie. <ol><li> Wachsen DH1.10 Zellen oder CORA - defizienten Zellen in einem Erlenmeyerkolben, der HL-5 - Medium (für 1 L: 5 g Proteosepepton, 5 g thiotone E Pepton, 10 g Glucose, 5 g Hefeextrakt, 0,35 g Na 2 HPO 4 * 7H 2 O, 0,35 g KH 2 PO 4, 0,05 g Dihydrostreptomycin-Sulfat, pH 6,6) bei 22 ° C in einem Schüttelinkubator mit einer Rotation von 160 Umdrehungen pro Minute. Halten Zellen mit einer Dichte zwischen 0,01 x 10 6 Zellen / ml und 2 x 10 6 Zellen / ml. </li><li> Untersuchen Zellaggregation durch Ernte log-Phase wachsende Zellen DH1.10 21 oder CORA - defizienten Zellen 10 im Hintergrund DH1.10 erzeugt wird , bei 22 ° C in Schüttelkultur mit HL-5 - Medium gezüchtet. Um dies zu tun, nehmen Sie entsprechende Menge an Zellen (in der Regel zwischen 10 und 50 ml), Zentrifuge 3 min bei 400 xg und waschen Sie die zweimal Zellen in BSS (10 mM NaCl, 10 mM KCl, 2,5 mM CaCl 2, pH 6.5). </li><li> Zählen von Zellen mit einer Zählkammer. Anschließend Platten Zellen bei einer Dichte von (5, 10, 20 oder 40) x 10 4 Zellen / cm 2 in eine 24-Well - Platte. Damit sie für 1 h bei 22 ° C in BSS zu haften. </li><li> Visualisieren Sie die Aggregation von Zeitraffer-Mikroskopie wie vor 10 beschrieben, Bilder alle 135 sec nehmen. unter Verwendung der entsprechenden Software automatisiert eine Live - Cell - Imaging eingerichtet , ausgestattet mit 5X Objektiv und einer Elektronenvervielfachungs charge-coupled device - Kamera (siehe Materialien Tabelle). </li></ol></li><li> cAMP Pulsieren von Dictyostelium discoideum Zellen während der Hungertod. <ol><li> Untersuchen die Wirkung von außen aufgebrachten cAMP Impulse auf die Entwicklung von Zellen DH1.10 21 oder DH1.10 CORA - defizienten Zellen 10, durch die Zellen zu ernten. Um dies zu tun, nehmen Sie entsprechende Menge an Zellen (in der Regel zwischen 10 und 50 ml), Zentrifuge 3 min bei 400 xg und waschen zweimal in BSS. </li><li> count - Zellen unter Verwendung eines Hämozytometers und Resuspendieren der Zellen zu einer Dichte von 1 x 10 7 Zellen / ml in BSS. Schütteln der Kulturen (160 rpm) bei 22 ° C für 2 Stunden vor der Anwendung von Impulsen. In cAMP Impulse mit einer Zeitschaltuhr gesteuert peristaltische Pumpe. alle 6,5 Minuten von 15 ul 50 nM cAMP (Endkonzentration) über einen Zeitraum von 5 Stunden zu liefern Programmieren Sie die Pumpe mit einem 5 s Puls. </li><li> Zählen von Zellen mit einer Zählkammer. Anschließend wird die Platte , die Zellen in einer Dichte von (5, 10, 20 oder 40) x 10 4 Zellen / cm 2 in eine 24-Well - Platte. Lassen Sie für 1 Stunde in BSS einzuhalten. </li><li> Visualisieren Aggregation nach 16 Stunden bei 22 ° C von Hellfeld-Mikroskopie ein 5fach Ziel verwenden. </li></ol></li><li> Die Induktion von Dictyostelium discoideum Entwicklung durch den Zugang zu konditionierten Medium. <ol><li> Bereiten Sie frisch konditioniertes Medium als 22 beschrieben. Sammeln Sie log-Phase DH1.10 Zellen 21 oder DH1.10 CORA - defizienten Zellen 10, von Kult Schüttelnmen mit HL-5 - Medium bei 22 ° C mit einer Pipette, Zentrifuge für 3 min bei 400 xg und wasche die Zellen dreimal in PBM (0,02 M Kaliumphosphat, 10 &amp; mgr; M CaCl 2 und 1 mM MgCl 2, pH 6.1) verwendet wird . </li><li> Zählen von Zellen unter Verwendung eines Hämozytometers resuspendieren diese in PBM in einer Dichte von 1 x 10 7 Zellen / ml und schüttle 20 Stunden bei 110 rpm / 22 ° C. </li><li> Sammeln konditionierten Medium nach der Zentrifugation bei 400 × g für 3 min und Klärung durch Zentrifugation bei 8.000 × g für 15 min bei 4 ° C. </li><li> Filter konditionierte Medium durch ein 0,45 - um - Filter (siehe Materialien Tabelle) und dreifach in PBM verdünnen. </li><li> Zählen von Zellen unter Verwendung eines Hämozytometers und Platte bei einer Dichte von (5, 10, 20 oder 40) x 10 4 Zellen / cm 2 in eine 24-Well - Platte. Damit sie für 1 h bei 22 ° C zu halten. </li><li> Wechselüberstand der Zellen mit dem vorher konditionierten Medium hergestellt. </li><li> Visualisieren Aggregation einN ach 16 Stunden von Hellfeld-Mikroskopie ein 5fach Ziel verwenden. </li></ol></li></ol>

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No conflicts of interest declared.

Die Coronin Proteine ​​werden in den meisten Taxa der eukaryotischen Klade. Dictyostelium discoideum Coronin A, das Homolog von Säuger Coronin 1, gefunden wird im frühen Verhungern Antwort beteiligt, da Coronin A-defizienten Zellen Aggregationsstellen nicht in der Lage sind , während der frühen Entwicklungs zu bilden Zyklus 10. Um in der Lage sein, um quantitativ und genau die Verzögerung in der Entwicklung zwischen den Stämmen beurteilen zu können, ein Mikroskop Live Cell Imaging Set-up m...

Die Coronin Familie von Proteinen ist stark in ganz Eukaryoten konserviert. Diese Proteine ​​werden durch die Gegenwart eines Amino-terminalen Tryptophan-aspartat (WD) wiederholen haltigen Bereich , gefolgt von einer einzigartigen Region verbunden mit einem carboxy-terminalen coiled-coil Domäne 13,14 (Figur 1) charakterisiert. Coronine wurden in einer Vielzahl von zellulären Funktionen beteiligt, einschließlich der Zytoskelett - Regulation und Signaltransduktion 12. Bei Säugetieren bis zu sechs kurze Coronin Moleküle (Coronin 1-6) sowie ein &quot;Tandem&quot; Coronin 7 können coexprimiert 12,15 werden. Coronin 1 ist das am intensivsten untersuchten Familienmitglied und wurde pathogen Zerstörung, T-Zell-Überleben und die neuronalen Signalübertragung beteiligt werden gezeigt. Wie genau Coronin 1 trägt diese Aktivitäten aus, bleibt unklar. Während Coronin 1 wurde gezeigt , Ca 2+ und cAMP-abhängige Signalgebung sowie F-Aktin - Zytoskelett Modulation 16-18, das Potential co zu regulieren-Ausdruck von bis zu 7 Familienmitglieder in Säugetieren hat es schwierig, das Molekular Funktion Coronine in diesen Systemen zu untersuchen, aufgrund potentieller Redundanzen. Im Gegensatz zu Säugetierorganismen exprimiert die niederen Eukaryoten Dictyostelium discoideum nur zwei Coronin Familienmitglieder (Coronin A, das Ortholog von Säugetier Coronin 1 und Coronin B, das Ortholog von Säugetier Coronin 7) mit scheinbar nicht-redundante Funktionen 15,19,20. Diese Tatsache macht Dictyostelium discoideum ein starkes Modell , um die Funktion von Coronine zu studieren. Um die Rolle von Coronin A in Dictyostelium discoideum studieren, induziert wir den Entwicklungszyklus durch Verhungern in Gewebekulturplatten mit ausgeglichener Salzlösung (BSS) Puffer entweder Wildtyp - Zellen oder Zellen mit fehlenden A 10 Coronin. Wir fanden heraus, dass Zellen ohne Coronin A waren nicht in der Lage mehrzelligen Aggregate auf Hunger zu bilden. Für eine genaue quantitative Beurteilung dieser Phänotyp derautomatisierte Live Cell Imaging in diesem Protokoll beschrieben ist ein wichtiges Instrument. Der Defekt in der Einleitung des frühen Verhungern Reaktion in Zellen Coronin A fehlt, kann durch Zuführen von Pulsen von cAMP gerettet werden, was darauf hindeutet, dass eine Akte vor der cAMP-Kaskade Coronin. Die exogene Applikation von cAMP Impulse für die Einleitung der Entwicklung zu simulieren wurde von mehreren Laboratorien in der Vergangenheit 8,9 verwendet worden. Jedoch ist dieses Verfahren auch in hohem Maße von Zelldichten und den Zeitpunkt bekannt sein. Daher beschrieben das Protokoll hier zum Ziel, diese Variabilitäten zu reduzieren, um ein hohes Maß an Reproduzierbarkeit zu gewährleisten. Zusammengenommen sind die verwendeten Techniken in diesen Studien liefern robuste Werkzeuge Funktionen von Proteinen während der frühen Phasen des Entwicklungszyklus von Dictyostelium discoideum zu untersuchen und haben das Potenzial , zu identifizieren Up- sowie Downstream - Effektoren einer Funktion Coronin.

<table><tbody><tr><td>HL-5 media (for 1 L: 5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose, 5 g yeast extract, 0.35 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O, 0.35 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 0.05 g dihydrostreptomycin-sulfate, pH 6.6)</td><td></td><td></td><td></td></tr><tr><td>Proteose peptone</td><td>BD Bioscience</td><td>211693</td><td></td></tr><tr><td>Thiotone E peptone</td><td>BD Bioscience</td><td>211684</td><td></td></tr><tr><td>Yeast extract</td><td>BD Bioscience</td><td>212750</td><td></td></tr><tr><td>Glucose</td><td>AppliChem</td><td>A3666</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fluka</td><td>71643</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>AppliChem</td><td>A1043</td><td></td></tr><tr><td>dihydrostreptomycin-sulfate</td><td>Sigma-Aldrich</td><td>D1954000</td><td></td></tr><tr><td>PBM (0.02 M potassium phosphate, 10 &amp;mu;M CaCl&lt;sub&gt;2&lt;/sub&gt;, and l mM MgCl&lt;sub&gt;2&lt;/sub&gt;, pH 6.1)</td><td></td><td></td><td>self made</td></tr><tr><td>BSS (10 mM NaCl, 10 mM KCl, 2.5 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, pH 6.5)</td><td></td><td></td><td>self made</td></tr><tr><td>0.45-&amp;mu;m Filtropure S filter</td><td>Sarstedt</td><td>83.1826</td><td></td></tr><tr><td>Falcon 24-well Tissue culture plate</td><td>Fisher Scientific</td><td>08-772-1H</td><td></td></tr><tr><td>Cellobserver microscope</td><td>Zeiss</td><td></td><td>custom built</td></tr><tr><td>AxioVision software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>IPC Microprocessor&amp;ndash;controlled dispensing pump</td><td>ISMATEC</td><td>ISM 931</td><td></td></tr><tr><td>Axiovert 135M microscope</td><td>Zeiss</td><td>491237-0001-000</td><td></td></tr><tr><td>Incubation Shaker</td><td>Inforst HT Minitron</td><td></td><td></td></tr></tbody></table>

investigating the function of coronin a in the early starvation response of dictyostelium discoideum by aggregation assays

Dictyostelium discoideum Amöbe sind im Boden und ernähren sich von Bakterien gefunden. Wenn Nahrungsquellen knapp werden, sie Faktoren absondern einen vielzelligen Entwicklungsprogramm zu initiieren, in denen 1-4 einzelne Zellen chemotax zur Aggregation Zentren. Dieser Prozess ist abhängig von der Freisetzung von zyklischem Adenosinmonophosphat (cAMP) 5. cAMP wird in Wellen durch die konzertierte Aktion der Adenylatcyclase und Phosphodiesterasen produziert und bindet an G - Protein-gekoppelten Rezeptoren cAMP 6,7. Ein weit verbreiteter Test die Mechanismen in dem Entwicklungszyklus des niederen Eukaryoten Dictyostelium discoideum beteiligt zu analysieren ist in submersen Bedingungen 8,9 auf der Beobachtung der Zellaggregation basiert. Dieses Protokoll beschreibt die Analyse der Rolle von Coronin A im Entwicklungszyklus durch Verhungern in Gewebekulturplatten unter Wasser in ausgewogener Salzlösung (BSS) 10. Coronin A ist ein Mitglied der weitgehend konservierten protein Familie von Coronine , die 11,12 in einer Vielzahl von Aktivitäten eine Rolle spielen . Dictyostelium Zellen ohne A Coronin sind unfähig mehrzelligen Aggregate zu bilden, und dieser Fehler kann durch Zuführen von Pulsen von cAMP gerettet werden, was darauf hindeutet , dass eine Akte stromaufwärts von der Coronin cAMP Kaskade 10. Die Techniken , die in diesen Studien beschriebenen liefern robuste Werkzeuge Funktionen von Proteinen während der Anfangsstadien des Entwicklungszyklus von Dictyostelium discoideum stromauf der cAMP - Kaskade zu untersuchen. Daher erlauben die weitere Untersuchung der Coronin Eine Funktion und unser Verständnis der Biologie Coronin kann diese Aggregation Assay verwendet.

Die Zellen einen Mangel an einer Show einen Defekt in der frühen Entwicklung Coronin (Abbildung 2). In Abwesenheit von A - Zellen nicht in der Lage Coronin mehrzellige Aggregate zu bilden, die der erste Schritt während des Entwicklungszyklus von Dictyostelium discoideum ist. Daher erscheint Coronin A eine Rolle während der frühen Hungerantwort und / oder cAMP-Signalisierung zu spielen. Tatsächlich ist das Fehlen von mehrzelligen Aggregatbildung in Abwesenhe...

Watch this Scientific Journal Video about Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays at JoVE.com

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

Die Untersuchung der Funktion von Coronin A in der frühen Starvation Reaktion von<em&gt; Dictyostelium discoideum</em&gt; Durch Aggregationstests

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular ...

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Research

JoVE Journal

Biology

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

7.2K Aufrufe.  University of Basel. The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

Serie: Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Gentechnische Veränderung von Dictyostelium Discoideum Zellen basierend auf Auswahl und das Wachstum von Bakterien

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Forschung

Genetik

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

Überwachung der Proteinaggregationskinetik in vivo mittels automatisierter Einschlusszählung bei Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

Biochemie

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

Imaging G-Protein-gekoppelten Rezeptor (GPCR)-vermittelten Signaltransduktion, die Steuerung der Chemotaxis Dictyostelium discoideum</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Biologie

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Here, we present an optimized protocol to rapidly and semiquantitatively measure ligand-receptor interactions in trans in a heterologous cell system using fluorescence microscopy.

Messung transzellulärer Wechselwirkungen durch Proteinaggregation in einem heterologen Zellsystem

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse ...

Neurowissenschaften

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

Die Untersuchung der Proteinqualitätskontrolle System der<em&gt; D. discoideum</em&gt; Verwendung von Temperaturgesteuerte Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

Bewertung von Dictyostelium Discoideum Reaktion auf akute mechanische Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

Methoden zur Untersuchung Veränderungen inhärenten Proteinaggregation mit dem Alter in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion and the spread of neurodegenerative diseases. The commonly used laboratory strains of the amoeba Dictyostelium discoideum have extremely high fluid uptake rates when grown in nutrient medium, over 90% of which is due to macropinocytosis. In addition, many of the known core components of mammalian macropinocytosis are also present, making it an excellent model system for studying macropinocytosis. Here, the standard technique to measure internalized fluid using fluorescent dextran as a label is adapted to a 96-well plate format, with the samples analyzed by flow cytometry using a high-throughput sampling (HTS) attachment.
Cells are fed non-quenchable fluorescent dextran for a pre-determined length of time, washed by immersion in ice-cold buffer and detached using 5 mM sodium azide, which also stops exocytosis. Cells in each well are then analyzed by flow cytometry. The method can also be adapted to measure membrane uptake and phagocytosis of fluorescent beads or bacteria.
This method was designed to allow measurement of fluid uptake by Dictyostelium in a high-throughput, labor and resource efficient manner. It allows simultaneous comparison of multiple strains (e.g. knockout mutants of a gene) and conditions (e.g. cells in different media or treated with different concentrations of inhibitor) in parallel and simplifies time-courses.

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, large-scale non-specific fluid uptake, is important in many areas of clinical biology including immunology, infection, cancer, and neurodegenerative diseases. Here, existing techniques have been adapted to allow high-throughput, single-cell resolution measurement of macropinocytosis in the macropinocytosis model organism Dictyostelium discoideum using flow cytometry.

Hochdurchsatz-Messung von Dictyostelium Discoideum Macropinocytosis von Flow Cytometry

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

Nachweis der Proteinaggregation mittels Fluoreszenzkorrelationsspektroskopie

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

Die Untersuchung der Funktion von Coronin A in der frühen Starvation Reaktion von

Zusammenfassung

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Imaging G-Protein-gekoppelten Rezeptor (GPCR)-vermittelten Signaltransduktion, die Steuerung der Chemotaxis Dictyostelium discoideum

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Messung transzellulärer Wechselwirkungen durch Proteinaggregation in einem heterologen Zellsystem

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Bewertung von Dictyostelium Discoideum Reaktion auf akute mechanische Stimulation

Methoden zur Untersuchung Veränderungen inhärenten Proteinaggregation mit dem Alter in Caenorhabditis elegans

Hochdurchsatz-Messung von Dictyostelium Discoideum Macropinocytosis von Flow Cytometry

Nachweis der Proteinaggregation mittels Fluoreszenzkorrelationsspektroskopie