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 106 cells/ml and 2 x 106 cells/ml.</li>
		<li>Examine cell aggregation, by harvesting log-phase-growing DH1.10 cells21 or corA-deficient cells10 generated in the DH1.10 background, grown in shaking culture with HL-5 medium at 22 &#176;C. To do so, take appropriate amount of cells (usually between 10 and 50 ml), centrifuge for 3 min at 400 x g and wash the cells twice in BSS (10 mM NaCl, 10 mM KCl, 2.5 mM CaCl2, pH 6.5).</li>
		<li>Count cells using a hemocytometer. Subsequently, plate cells at a density of (5, 10, 20, or 40) x 104 cells/cm2 in a 24-well plate. Allow them to adhere for 1 hr at 22 &#176;C in BSS.</li>
		<li>Visualize aggregation by time-lapse microscopy as described before10, taking images every 135 sec. using a live cell imaging set up equipped with 5X objective and an electron-multiplying charge-coupled device camera automated by the appropriate software (see Materials Table).</li>
	</ol>
	</li>
	<li>cAMP pulsing of Dictyostelium discoideum cells during starvation.
	<ol>
		<li>Examine the effect of externally applied cAMP pulses on the development of DH1.10 cells21 or DH1.10 corA-deficient cells10, by harvesting cells. To do so, take appropriate amount of cells (usually between 10 and 50 ml), centrifuge for 3 min at 400 x g and wash twice in BSS.</li>
		<li>Count cells using a hemocytometer and resuspend the cells to a density of 1 x 107 cells/ml in BSS. Shake the cultures (160 rpm) at 22 &#176;C for 2 hr before applying pulses. Add cAMP pulses using a timer controlled peristaltic pump. Program the pump to deliver a 5 sec pulse every 6.5 min of 15 &#181;l of 50 nM cAMP (final concentration) over a period of 5 hr.</li>
		<li>Count cells using a hemocytometer. Subsequently, plate the cells at a density of (5, 10, 20, or 40) x 104 cells/cm2 in a 24-well plate. Allow to adhere for 1 hr in BSS.</li>
		<li>Visualize aggregation after 16 hr at 22 &#176;C by bright-field microscopy using a 5X objective.</li>
	</ol>
	</li>
	<li>Induction of Dictyostelium discoideum development through exposure to conditioned medium.
	<ol>
		<li>Prepare fresh conditioned medium as described22. Collect log-phase DH1.10 cells21 or DH1.10 corA-deficient cells10, from shaking cultures with HL-5 medium at 22 &#176;C using a pipette, centrifuge for 3 min at 400 x g and wash cells three times in PBM (0.02 M potassium phosphate, 10 &#181;M CaCl2, and 1 mM MgCl2, pH 6.1).</li>
		<li>Count cells using a hemocytometer, resuspend these in PBM at a density of 1 x 107 cells/ml and shake for 20 hr at 110 rpm/22 &#176;C.</li>
		<li>Collect conditioned medium after centrifugation at 400 &#215; g for 3 min and clarify by centrifugation at 8,000 &#215; g for 15 min at 4&#176;C.</li>
		<li>Filter conditioned medium through a 0.45 &#181;m filter (see Materials Table) and dilute threefold in PBM.</li>
		<li>Count cells using a hemocytometer and plate at a density of (5, 10, 20, or 40) x 104 cells/cm2 in a 24-well plate. Allow them to adhere for 1 hr at 22 &#176;C.</li>
		<li>Exchange supernatant of the cells with the previously prepared conditioned medium.</li>
		<li>Visualize aggregation after 16 hr by bright-field microscopy using a 5X objective.</li>
	</ol>
	</li>
</ol>

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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. 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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.
D. discoideum utilizes cAMP as an essential signaling molecule during several stages of its life cycle1-4. One of the checkpoints during which cAMP is required is the transition from a single cell to the formation of multicellular structures. The onset of cAMP waves after ~6 hr of starvation is sensed by the cells and provides the chemotactic signal to move towards an aggregation center5. These cAMP waves can be mimicked experimentally by pulsing starving D. discoideum cells with cAMP every 6.5 min over a period of 5h8,9. Using this procedure it is important to starve the cells for up to 2h before applying the cAMP pulses, in order to obtain reliable results. Utilizing this experimental set up we were able to show that coronin A is acting upstream of cAMP signaling, as these externally applied pulses rescued the development in corA-deficient cells (Figure 3 and 5). These results also indicate that while coronin A appears to have an essential role in sensing the early starvation response, it is dispensable for processes such as chemotaxis and cell aggregation per se10.
The aggregation assay itself is a robust method for studying the early development of Dictyostelium discoideum. However, there are several pitfalls that can influence the reproducibility of these experiments, and it is therefore important that several factors are taken into account: the age of the cells plays an important role when analyzing corA-deficient cells. These cells tend to compensate the phenotype and re-aggregate approximately after 2 weeks from the point they are thawed. The density of the cells is a crucial factor as well, as cells that show a signaling defect and not a chemotactic defect might tend to aggregate at higher densities due to the increased concentration of early starvation factors. The importance of the precise counting and dilution of cells in these assays cannot be overstated. For different strains, an adjustment of multiple variables of the assay, such as cell densities, time of adherence, time of development, will be required. The aggregation assay as described here is a versatile technique that can be modified to suit different inquiries: the cells can be starved, or development can be stimulated or inhibited using different compounds, allowing for a specific modulation of aggregation. The here described set-up also allows for unbiased approaches to identify strains with early developmental defects, as well as for the identification of genes or factors which are important for early development. This is also the method&#39;s major drawback: the aggregation assay is a reduced version of the complete developmental cycle. Therefore, it is not suitable for the analysis of phenotypes affecting later stages of the developmental cycle. The main advantage is the possibility to adopt the aggregation assay to a large scale and automated format: in 48-well plates and with 16 hr of development recorded during automated imaging, the aggregation assay proves to be more time and resource-efficient than development on agar plates.
In experiments in which we bypassed the early starvation response, using externally applied cAMP pulses, we showed that coronin A is involved in the sensing of secreted factors by Dictyostelium cells upon starvation. The precise mechanism of coronin A-mediated signaling during the early starvation response remains unclear. Several factors involved during this stage of development have been studied, in particular conditioned medium factor (CMF)25,26. However, whether CMF acts on its own or with co-factors remains unclear and its signaling cascade is only partially understood. Using the aggregation capacity of Dictyostelium cells under submerged conditions here described as a positive read-out for development allows to conduct large-scale screening experiments in a time-efficient manner as compared to aggregation assays on agar surfaces. However, aggregation assays under submerged conditions do have the disadvantage of not fulfilling the complete developmental cycle as cells will be arrested in the initial aggregation stage. Therefore, the technique described here is not suitable for the examination of later developmental processes. In summary, the experimental approach documented here may allow further characterization of potential early starvation factor(s) that induce coronin A-dependent entry into the early starvation response.

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 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 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 10. However, the application of exogenous cAMP pulses fully restores the wild-type phenotype (Figure 3). This implicates that coronin A, rather than being directly involved in cAMP signaling, functions upstream of the cAMP cascade (Figure 5). When Dictyostelium cells are exposed to starving conditions they induce the secretion of early starvation factors22,23. One of the best characterized factors involved in the early starvation response is conditioned medium factor (CMF)22. Based on our results, we hypothesized that coronin A either regulates the secretion of these early starvation factors or is involved in the signal transduction induced by these factors. In order to be able to differentiate between these two effects we performed conditioned medium experiments. Wild-type or corA-deficient cells were exposed to supernatants from either starved wild-type or corA-deficient cells10 (Figure 4). These supernatants are potent initiators of multicellular aggregate formation and therefore contain factors that initiate downstream developmental signaling pathways, including cAMP signaling. Our results show that supernatant from corA-deficient cells was equally capable to induce development as was the case for supernatant from wild-type cells (Figure 4). This implicates that corA-deficient cells are able to produce and secrete early starvation factors at similar levels to their wild-type controls. However, corA-deficient cells are neither able to respond to their own supernatant nor to supernatant provided from wild-type cells (Figure 4). Therefore, coronin A appears to be involved in signaling pathways induced by early starvation factors, rather than their expression/secretion10 (Figure 5).
In general, to be able to further investigate the signals secreted by starving Dictyostelium cells that induce the developmental cycle, the aggregation assay under submerged conditions is a potent read-out. Cells seeded at higher densities will spontaneously aggregate due to an abundance of all required signals. However, at lower densities only the addition of conditioned medium will induce cellular aggregation (Figure 4)22,24. Therefore, this procedure can be utilized to fractionate conditioned medium and study distinct components for their activity during the early starvation response.
<img alt="Figure 1" src="/files/ftp_upload/53972/53972fig1.jpg" /> 
Figure 1: Coronin proteins present in mammals and Dictyostelium discoideum. The coronin family is characterized by the presence of N-terminal WD-repeat regions followed by a unique domain and a C-terminal coiled coil region. While mammals express 7 coronin proteins (A), Dictyostelium discoideum only contains two family members (B), making the risk of redundancy less likely when studying their function. Coronin A is the ortholog of mammalian coronin 1 and coronin B shows a similar domain structure as coronin 7. Abbreviations: CC, coiled-coil domain; UD, unique domain; WD repeats, tryptophan-aspartate repeats. Length is given for mouse sequences (A) in amino acids (aa). Modified from Pieters et al., 201312. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53972/53972fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53972/53972fig2.jpg" /> 
Figure 2: corA-Dictyostelium discoideum are unable to aggregate during the early starvation response. Wild-type (A) or corA-deficient (B) Dictyostelium cells were seeded into multiwell plates at a density of 2 x 105 cells/cm2, starved in BSS, and imaged over a period of 20 hr. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53972/53972fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53972/53972fig3.jpg" /> 
Figure 3: Externally applied cAMP pulses rescue the early developmental defect of corA-Dictyostelium discoideum. Vegetative wild-type (A and B) and corA-deficient cells (C and D) were washed and starved for 2 hr&#160;before being pulsed with (B and D) or without (A and C) 50 nM cAMP during 5 hr every 6.5 min in suspension. Cells were then washed, resuspended in BSS, and placed on a Petri dish at a density of 10 x 104 cells/cm2. Images were taken 20 hr after seeding of the cells. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53972/53972fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53972/53972fig4.jpg" /> 
Figure 4: corA-deficient Dictyostelium discoideum are able to produce all required factors for developmental induction but are unable to respond to them. Exponentially growing wild-type and corA-deficient cells were washed in PBM and seeded into multiwell plates at a density of (5, 10, 20, or 40) x 104 cells/cm2, incubated in conditioned medium obtained from wild-type (A) or corA-deficient starving cells (B). The images were taken 16 hr after seeding the cells. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53972/53972fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53972/53972fig5.jpg" /> 
Figure 5: Current model of Coronin A function in Dictyostelium discoideum. Coronin A is involved in the signal transduction of factor(s) secreted during the early starvation response leading to the induction of the cAMP cascade and the progression through the developmental cycle. Modified from Vinet et al., 201410. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53972/53972fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

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Universidad San Sebastian

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Biology

7.2K Views.  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.

Video: 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.

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

Genetics

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.

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

Biochemistry

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.

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

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.

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

Neuroscience

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.

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.

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.

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.

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.

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

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

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