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>Observe the early starvation response of Dictyostelium discoideum by time-lapse microscopy.
	<ol>
		<li>Grow DH1.10 cells or corA-deficient cells in an Erlenmayer flask containing HL-5 medium (for 1 L: 5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose, 5 g yeast extract, 0.35 g Na2HPO4*7H2O, 0.35 g KH2PO4, 0.05 g dihydrostreptomycin-sulfate, pH 6.6) at 22 &#176;C in a shaking incubator with a rotation of 160 rpm. Keep cells at a density between 0.01 x 10...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+regulation+of+early+gene+expression+in+Dictyostelium+discoideum:+mediation+via+the+cell+surface+cyclic+AMP+receptor.">Cyclic AMP regulation of early gene expression in Dictyostelium discoideum: mediation via the cell surface cyclic AMP receptor.</a> Mol Cell Biol. 7, 458-469 (1987).</li><li>Vinet, A. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+multicellular+differentiation+in+Dictyostelium+discoideum+is+regulated+by+coronin+A.">Initiation of multicellular differentiation in Dictyostelium discoideum is regulated by coronin A.</a> Mol Biol Cell. 25, 688-701 (2014).</li><li>Eckert, C., Hammesfahr, B., Kollmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+holistic+phylogeny+of+the+coronin+gene+family+reveals+an+ancient+origin+of+the+tandem-coronin,+defines+a+new+subfamily,+and+predicts+protein+function.">A holistic phylogeny of the coronin gene family reveals an ancient origin of the tandem-coronin, defines a new subfamily, and predicts protein function.</a> BMC Evol Biol. 11, 268 (2011).</li><li>Pieters, J., Muller, P., Jayachandran, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+guard:+coronin+proteins+in+innate+and+adaptive+immunity.">On guard: coronin proteins in innate and adaptive immunity.</a> Nat Rev Immunol. 13, 510-518 (2013).</li><li>Gatfield, J., Albrecht, I., Zanolari, B., Steinmetz, M. O., Pieters, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+the+Leukocyte+Plasma+Membrane+with+the+Actin+Cytoskeleton+through+Coiled+Coil-mediated+Trimeric+Coronin+1+Molecules.">Association of the Leukocyte Plasma Membrane with the Actin Cytoskeleton through Coiled Coil-mediated Trimeric Coronin 1 Molecules.</a> Mol Biol Cell. 16, 2786-2798 (2005).</li><li>Kammerer, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+trimerization+motif+controls+the+topology+of+short+coiled+coils.">A conserved trimerization motif controls the topology of short coiled coils.</a> Proc Natl Acad Sci U S A. 102, 13891-13896 (2005).</li><li>Uetrecht, A. C., Bear, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronins:+the+return+of+the+crown.">Coronins: the return of the crown.</a> Trends Cell Biol. 16, 421-426 (2006).</li><li>Jayachandran, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronin+1+Regulates+Cognition+and+Behavior+through+Modulation+of+cAMP/Protein+Kinase+A+Signaling.">Coronin 1 Regulates Cognition and Behavior through Modulation of cAMP/Protein Kinase A Signaling.</a> PLoS Biol. 12, e1001820 (2014).</li><li>Suo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronin-1+is+a+neurotrophin+endosomal+effector+that+is+required+for+developmental+competition+for+survival.">Coronin-1 is a neurotrophin endosomal effector that is required for developmental competition for survival.</a> Nat Neurosci. 17, 36-45 (2014).</li><li>Mueller, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+T+cell+survival+through+coronin-1-mediated+generation+of+inositol-1,4,5-trisphosphate+and+calcium+mobilization+after+T+cell+receptor+triggering.">Regulation of T cell survival through coronin-1-mediated generation of inositol-1,4,5-trisphosphate and calcium mobilization after T cell receptor triggering.</a> Nat Immunol. 9, 424-431 (2008).</li><li>de Hostos, E. L., Bradtke, B., Lottspeich, F., Guggenheim, R., Gerisch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronin,+an+actin+binding+protein+of+Dictyostelium+discoideum+localized+to+cell+surface+projections,+has+sequence+similarities+to+G+protein+beta+subunits.">Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits.</a> EMBO J. 10, 4097-4104 (1991).</li><li>Shina, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redundant+and+unique+roles+of+coronin+proteins+in+Dictyostelium.">Redundant and unique roles of coronin proteins in Dictyostelium.</a> Cell Mol Life Sci. 68, 303-313 (2011).</li><li>Cornillon, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phg1p+is+a+nine-transmembrane+protein+superfamily+member+involved+in+dictyostelium+adhesion+and+phagocytosis.">Phg1p is a nine-transmembrane protein superfamily member involved in dictyostelium adhesion and phagocytosis.</a> J Biol Chem. 275, 34287-34292 (2000).</li><li>Gomer, R. H., Yuen, I. S., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+secreted+80+x+10(3)+Mr+protein+mediates+sensing+of+cell+density+and+the+onset+of+development+in+Dictyostelium.">A secreted 80 x 10(3) Mr protein mediates sensing of cell density and the onset of development in Dictyostelium.</a> Development. (112), 269-278 (1991).</li><li>Iijima, N., Takagi, T., Maeda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proteinous+factor+mediating+intercellular+communication+during+the+transition+of+Dictyostelium+cells+from+growth+to+differentiation.">A proteinous factor mediating intercellular communication during the transition of Dictyostelium cells from growth to differentiation.</a> Zoological science. 12, 61-69 (1995).</li><li>Mehdy, M. C., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+secreted+factor+and+cyclic+AMP+jointly+regulate+cell-type-specific+gene+expression+in+Dictyostelium+discoideum.">A secreted factor and cyclic AMP jointly regulate cell-type-specific gene expression in Dictyostelium discoideum.</a> Mol Cell Biol. 5, 705-713 (1985).</li><li>Jain, R., Yuen, I. S., Taphouse, C. R., Gomer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+density-sensing+factor+controls+development+in+Dictyostelium.">A density-sensing factor controls development in Dictyostelium.</a> Genes Dev. 6, 390-400 (1992).</li><li>Loomis, W. 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No conflicts of interest declared.

The coronin proteins are found in most taxa of the eukaryotic clade. Dictyostelium discoideum coronin A, the homologue of mammalian coronin 1, is involved in the early starvation response, since coronin A-deficient cells are not able to form aggregation centers during the early developmental cycle10. To be able to quantitatively and accurately assess the delay in development between the strains, a microscope live cell imaging set-up with the automated stage controller is an essential tool.
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The coronin family of proteins is highly conserved throughout eukaryotes. These proteins are characterized by the presence of an amino-terminal tryptophan-aspartate (WD) repeat-containing region followed by a unique region connected to a carboxy-terminal coiled-coil domain13,14 (Figure 1). Coronins have been implicated in a variety of cellular functions, including cytoskeletal regulation and signal transduction12. In mammals, up to six short coronin molecules (coronin 1-6) as well as a &#39;tandem&#39; coronin 7, can be co-expressed12,15. Coronin 1 is the most extensively studied family member, and was shown to be involved in pathogen destruction, T cell survival and neuronal signaling. How, exactly, coronin 1 carries out these activities remains unclear. While coronin 1 was shown to regulate Ca2+ and cAMP-dependent signaling as well as F-actin cytoskeleton modulation 16-18, the potential co-expression of up to 7 family members in mammals has made it challenging to study the molecular function of coronins in these systems, due to potential redundancies. Unlike mammalian organisms, the lower eukaryote Dictyostelium discoideum expresses only two coronin family members (coronin A, the ortholog of mammalian coronin 1 and coronin B, the ortholog of mammalian coronin 7) with apparently non-redundant functions15,19,20. This fact makes Dictyostelium discoideum a potent model to study the function of coronins.
To study the role of coronin A in Dictyostelium discoideum, we induced the developmental cycle by starvation in tissue-culture plates containing balanced salt solution (BSS) buffer using either wild type cells or cells lacking coronin A10. We found that cells lacking coronin A were unable to form multicellular aggregates upon starvation. For an accurate quantitative assessment of this phenotype the automated live cell imaging described in this protocol is a vital tool. The defect in the initiation of the early starvation response in cells lacking coronin A can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade. The exogenous application of cAMP pulses to simulate the initiation of development has been utilized by several laboratories in the past8,9. However, this procedure is also known to be highly dependent on cell densities and timing. Therefore, the protocol described here aims to reduce these variabilities in order to guarantee a high degree of reproducibility. Taken together, the techniques utilized in these studies provide robust tools to investigate functions of proteins during early stages of the developmental cycle of Dictyostelium discoideum and have the potential to identify up- as well as downstream effectors of coronin A function.

<table>
	<tbody>
		<tr>
			<td>HL-5 media (for 1L: 5 g proteose peptone, 5 g thiotone E peptone, 10 g glucose, 5 g yeast extract, 0.35 g Na2HPO4&#8727;2H2O, 0.35 g KH2PO4, 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>Na2HPO4&#8727;2H2O</td>
			<td>Fluka</td>
			<td>71643</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</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 &#956;M CaCl2, and l mM MgCl2, 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 CaCl2, pH 6.5)</td>
			<td></td>
			<td></td>
			<td>self made</td>
		</tr>
		<tr>
			<td>0.45-&#956;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><a href="https://www.fishersci.com/shop/products/falcon-tissue-culture-plates-23/087721h">08-772-1H</a></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&#8211;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 amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular development program, during which single cells chemotax towards aggregation centers1-4. This process is dependent on the release of cyclic adenosine monophosphate (cAMP)5. cAMP is produced in waves through the concerted action of adenylate cyclase and phosphodiesterases, and binds to G protein-coupled cAMP receptors6,7. A widely used assay to analyze the mechanisms involved in the developmental cycle of the lower eukaryote Dictyostelium discoideum is based on the observation of cell aggregation in submerged conditions8,9. This protocol describes the analysis of the role of coronin A in the developmental cycle by starvation in tissue-culture plates submerged in balanced salt solution (BSS)10. Coronin A is a member of the widely conserved protein family of coronins that have been implicated in a wide variety of activities11,12. Dictyostelium cells lacking coronin A are unable to form multicellular aggregates, and this defect can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade10. The techniques described in these studies provide robust tools to investigate functions of proteins during the initial stages of the developmental cycle of Dictyostelium discoideum upstream of the cAMP cascade. Therefore, utilizing this aggregation assay may allow the further study of coronin A function and advance our understanding of coronin biology.

Cells deficient in coronin A show a defect in early development (Figure 2). In the absence of coronin A cells are unable to form multicellular aggregates, which is the initial step during the developmental cycle of Dictyostelium discoideum. Therefore, coronin A appears to play a role during the early starvation response and/or cAMP signaling. Indeed, the lack of multicellular aggregate formation in the absence of coronin A is accompanied by reduced cAMP signaling...

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.

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

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

The overall goal of this experimental procedure, is to investigate the Coronin-A dependent early starvation response of Dictyostelium discoideum. This method can help answer key questions in the field of Developmental Biology, such as the identification of signaling pathways and factors involved in the early development of Dictyostelium discoideum. The main advantage of this technique is that it has the potential to provide a tool for automated and high-throughput screening of development inducing factors and proteins involved in early developmental signaling.Generally, individuals new to this method, will struggle because of multiple variables inherent to the assay, such as cell densities, time of adherence, and time of development. To begin this procedure, grow DH1.10 cells or corA-deficient cells in an Erlenmeyer flask containing HL-5 medium, at 22 degrees Celsius, in a shaking incubator at 160 rpm. Keep the cells at a density between one times 10 to the four, and two times 10 to the six cells per milliliter.To examine cell aggregation, harvest 10 to 50 milliliters of log-phase-growing DH1.10 cells or corA-deficient cells generated in the DH1.10 background. Centrifuge them for three minutes at 400 times g. Then was the cells twice in BSS.Afterward count the cells using a hemocytometer. Subsequently, plate the cells in a 24 well-plate. Allow them to adhere for one hour at 22 degrees Celsuis in BSS.Then, visualize the cell aggregation with time-lapse microscopy, and take images every 135 seconds. To examine the affect of the externally applied cyclic AMP pulses on the development of DH1.10 cells, or DH1.10 corA-deficient cells, harvest 10 to 50 milliliters of the cells and centrifuge them for three minutes at 400 times g. After that, wash them twice in BSS.Count the cells using a hemocytometer. Subsequently, re-suspend the cells to a density of one times 10 to the seven cells per milliliter in BSS. Then, shake the cultures at 22 degrees Celsius for two hours at 160 rpm before applying pulses.Next, apply cyclic AMP pulses using a timer controlled peristaltic pump. Program the pump to deliver a five second pulse of cyclic AMP every six and a half minutes over a period of five hours. A critical step is to ensure that the tip of the cyclic AMP injection is not continuously submerged in the solution, but only submerged periodically in the waves generated by the shaker.After cyclic AMP application, count the cells again using a hemocytometer. Then, plate the cells in a 24 well-plate and allow them to adhere for one hour in BSS. After 16 hours, visualize the cell aggregation at 22 degrees Celsius, with bright-field microscopy using a five x subjective.In this procedure, prepare fresh conditioned medium, by collecting log phase DH1.10 cells or DH1.10 corA-deficient cells from the shaking cultures with HL5 medium, at 22 degrees Celsius. Centrifuge the cells for three minutes at 400 times g. After that, wash them three times in PBM.Subsequently, count the cells using a hemocytometer, Then, re-suspend them in PBM at a density of one times 10 to the seven cells per milliliter and shake them for 20 hours at 22 degrees Celsius at 110 rpm. Next, centrifuge the cells at 400 times g for three minutes, followed by collecting the conditioned medium. Afterward, clarify the medium by centrifugation at 8, 000 times g for 15 minutes at four degrees Celsuis.Then, filter the conditioned medium through a 0.45 micrometer filter and dilute it three-fold in PBM. Count the cells using a hemocytometer, plate them in a 24 well-plate, and allow them to adhere for one hour at 22 degrees Celsius. Next, exchange the supernatant of the cells with a previously prepared conditioned medium.After 16 hours, visualize the cell aggregration with bright-field microscopy using a five x subjective. Here, the cells deficient in Coronin-A show a defect in early development, and were unable to form multi-cellular aggregates, which is the initial step during the developmental cycle of Dictyostelium discoideum. In this figure, the vegetative Wild type, and corA-deficient cells, were washed and starved for two hours before being pulsed with, or without, 15 animal or cyclic AMP, every six and a half minutes for five hours in suspension.The application of exogenous cyclic AMP pulses, fully restored the Wild type phenotype. In this figure, exponentially growing Wild type and corA-deficient cells, were washed in PBM, seeded into multi well-plates, and incubated in conditioned medium obtained from Wild type, or corA-deficient starving cells. The images were taken 16 hours after seeding the cells.corA-deficient Dictyostelium discoideum, were able to produce all required factors for the developmental induction, but were unable to respond to them. Once mastered, this technique can be done in 20 hours, including incubation time, if it is performed properly. While attempting this procedure, it's important to remember, that for different strains an adjustment of multiple variables of the assay, such as cell densities, time of adherence, time of development, will be required.Following this procedure, biochemical fractionation and subsequent proteomic analysis, can be performed in order to identify factors involved in the early starvation response of Dictyostelium discoideum. After watching this video, one should have a good understanding of how to induce early development in Dictyostelium discoideum under submerged conditions, using either conditioned media, as well as cyclic AMP pulses.

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7.2K Görüntüleme.  University of Basel. The overall goal of this experimental procedure, is to investigate the Coronin-A dependent early starvation response of Dictyostelium discoideum. This method can help answer key questions in the field of Developmental Biology, such as the identification of signaling pathways and factors involved in the early development of Dictyostelium discoideum. The main advantage of this technique is that it has the potential to provide a tool for automated and high-throughput screening of development ind...