Name: assessment of dictyostelium discoideum response to acute mechanical stimulation
Uploaded: 2017-11-09T12:30:00
Description: Watch this Scientific Journal Video about Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation at JoVE.com

This work was supported by National Institutes of Health grant R35 GM118177 to PND.

1. Preparation of Solutions
<ol>
	<li>Prepare HL-5 media using the following: 10 g/L dextrose, 10 g/L proteose peptone, 5 g/L yeast extract, 0.965 g/L Na2HPO4&#183;7H2O, 0.485 g/L KH2PO4, and, unless otherwise indicated, 0.03 g/L streptomycin in deionized water. Autoclave the medium and store at room temperature. 
	NOTE: Due to differences between proteose peptone and yeast extract acquired from different suppliers, as well as between individual batches, pH of...

<ol>
	<li>Swaney, K. F., Huang, C. H., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+chemotaxis:+a+network+of+signaling+pathways+controls+motility,+directional+sensing,+and+polarity.">Eukaryotic chemotaxis: a network of signaling pathways controls motility, directional sensing, and polarity.</a> Annu Rev Biophys. 39, 265-289 (2010).</li><li>Sasaki, A. T., Chun, C., Takeda, K., 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=Localized+Ras+signaling+at+the+leading+edge+regulates+PI3K,+cell+polarity,+and+directional+cell+movement.">Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement.</a> J Cell Biol. 167 (3), 505-518 (2004).</li><li>Jeon, T. J., Lee, D. -. J., Merlot, S., Weeks, G., 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=Rap1+controls+cell+adhesion+and+cell+motility+through+the+regulation+of+myosin+II.">Rap1 controls cell adhesion and cell motility through the regulation of myosin II.</a> J Cell Biol. 176 (7), 1021-1033 (2007).</li><li>Parent, C. A., Blacklock, B. J., Froehlich, W. M., Murphy, D. B., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+Protein+Signaling+Events+Are+Activated+at+the+Leading+Edge+of+Chemotactic+Cells.">G Protein Signaling Events Are Activated at the Leading Edge of Chemotactic Cells.</a> Cell. 95 (1), 81-91 (1998).</li><li>Funamoto, S., Meili, R., Lee, S., Parry, L., 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=Spatial+and+Temporal+Regulation+of+3-Phosphoinositides+by+PI+3-Kinase+and+PTEN+Mediates+Chemotaxis.">Spatial and Temporal Regulation of 3-Phosphoinositides by PI 3-Kinase and PTEN Mediates Chemotaxis.</a> Cell. 109 (5), 611-623 (2002).</li><li>Iijima, M., Devreotes, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+Suppressor+PTEN+Mediates+Sensing+of+Chemoattractant+Gradients.">Tumor Suppressor PTEN Mediates Sensing of Chemoattractant Gradients.</a> Cell. 109 (5), 599-610 (2002).</li><li>Lim, C. J., Spiegelman, G. B., Weeks, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RasC+is+required+for+optimal+activation+of+adenylyl+cyclase+and+Akt/PKB+during+aggregation.">RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.</a> EMBO J. 20 (16), 4490-4499 (2001).</li><li>Kamimura, Y., 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=PIP3-Independent+Activation+of+TorC2+and+PKB+at+the+Cell's+Leading+Edge+Mediates+Chemotaxis.">PIP3-Independent Activation of TorC2 and PKB at the Cell's Leading Edge Mediates Chemotaxis.</a> Curr Biol. 18 (14), 1034-1043 (2008).</li><li>Kortholt, A., King, J. S., Keizer-Gunnink, I., Harwood, A. J., Van Haastert, P. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipase+C+Regulation+of+Phosphatidylinositol+3,4,5-trisphosphate-mediated+Chemotaxis.">Phospholipase C Regulation of Phosphatidylinositol 3,4,5-trisphosphate-mediated Chemotaxis.</a> Molecular Biology of the Cell. 18 (12), 4772-4779 (2007).</li><li>Huang, C. H., Tang, M., Shi, C., Iglesias, P. A., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+excitable+signal+integrator+couples+to+an+idling+cytoskeletal+oscillator+to+drive+cell+migration.">An excitable signal integrator couples to an idling cytoskeletal oscillator to drive cell migration.</a> Nat Cell Biol. 15 (11), 1307-1316 (2013).</li><li>Nishikawa, M., H&#246;rning, M., Ueda, M., Shibata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitable+Signal+Transduction+Induces+Both+Spontaneous+and+Directional+Cell+Asymmetries+in+the+Phosphatidylinositol+Lipid+Signaling+System+for+Eukaryotic+Chemotaxis.">Excitable Signal Transduction Induces Both Spontaneous and Directional Cell Asymmetries in the Phosphatidylinositol Lipid Signaling System for Eukaryotic Chemotaxis.</a> Biophys J. 106 (3), 723-734 (2014).</li><li>Gerisch, G., 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=Mobile+Actin+Clusters+and+Traveling+Waves+in+Cells+Recovering+from+Actin+Depolymerization.">Mobile Actin Clusters and Traveling Waves in Cells Recovering from Actin Depolymerization.</a> Biophys J. 87 (5), 3493-3503 (2004).</li><li>Gerisch, G., Ecke, M., Wischnewski, D., Schroth-Diez, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+modes+of+state+transitions+determine+pattern+in+the+Phosphatidylinositide-Actin+system.">Different modes of state transitions determine pattern in the Phosphatidylinositide-Actin system.</a> BMC Cell Biol. 12 (1), 42 (2011).</li><li>Bretschneider, T., 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=The+Three-Dimensional+Dynamics+of+Actin+Waves,+a+Model+of+Cytoskeletal+Self-Organization.">The Three-Dimensional Dynamics of Actin Waves, a Model of Cytoskeletal Self-Organization.</a> Biophys J. 96 (7), 2888-2900 (2009).</li><li>Weiner, O. D., Marganski, W. A., Wu, L. F., Altschuler, S. J., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Actin-Based+Wave+Generator+Organizes+Cell+Motility.">An Actin-Based Wave Generator Organizes Cell Motility.</a> PLOS Biol. 5 (9), e221 (2007).</li><li>Devreotes, P., Horwitz, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+Networks+that+Regulate+Cell+Migration.">Signaling Networks that Regulate Cell Migration.</a> Cold Spring Harbor Perspectives in Biology. 7 (8), (2015).</li><li>Artemenko, Y., Lampert, T. J., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+towards+a+paradigm:+common+mechanisms+of+chemotactic+signaling+in+Dictyostelium+and+mammalian+leukocytes.">Moving towards a paradigm: common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes.</a> Cell Mol Life Sci. 71 (19), 3711-3747 (2014).</li><li>Dalous, J., 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=Reversal+of+cell+polarity+and+actin-myosin+cytoskeleton+reorganization+under+mechanical+and+chemical+stimulation.">Reversal of cell polarity and actin-myosin cytoskeleton reorganization under mechanical and chemical stimulation.</a> Biophys J. 94 (3), 1063-1074 (2008).</li><li>Sato, M. J., 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=Switching+direction+in+electric-signal-induced+cell+migration+by+cyclic+guanosine+monophosphate+and+phosphatidylinositol+signaling.">Switching direction in electric-signal-induced cell migration by cyclic guanosine monophosphate and phosphatidylinositol signaling.</a> Proc Natl Acad Sci U S A. 106 (16), 6667-6672 (2009).</li><li>Zhao, M., Pu, J., Forrester, J. V., McCaig, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+lipids,+EGF+receptors,+and+intracellular+signals+colocalize+and+are+polarized+in+epithelial+cells+moving+directionally+in+a+physiological+electric+field.">Membrane lipids, EGF receptors, and intracellular signals colocalize and are polarized in epithelial cells moving directionally in a physiological electric field.</a> Faseb j. 16 (8), 857-859 (2002).</li><li>Decave, E., 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=Shear+flow-induced+motility+of+Dictyostelium+discoideum+cells+on+solid+substrate.">Shear flow-induced motility of Dictyostelium discoideum cells on solid substrate.</a> J Cell Sci. 116 (Pt 21), 4331-4343 (2003).</li><li>Artemenko, Y., Axiotakis, L., Borleis, J., Iglesias, P. A., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+mechanical+stimuli+act+on+common+signal+transduction+and+cytoskeletal+networks.">Chemical and mechanical stimuli act on common signal transduction and cytoskeletal networks.</a> Proc Natl Acad Sci U S A. 113 (47), E7500-E7509 (2016).</li><li>Artemenko, Y., Swaney, K. F., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+development+and+chemotaxis+in+Dictyostelium+discoideum+mutants.">Assessment of development and chemotaxis in Dictyostelium discoideum mutants.</a> Methods Mol Biol. 769, 287-309 (2011).</li><li>Gaudet, P., Pilcher, K. E., Fey, P., Chisholm, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Dictyostelium+discoideum+with+plasmid+DNA.">Transformation of Dictyostelium discoideum with plasmid DNA.</a> Nat. Protocols. 2 (6), 1317-1324 (2007).</li><li>Veltman, D. M., Lemieux, M. G., Knecht, D. A., Insall, 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=PIP3-dependent+macropinocytosis+is+incompatible+with+chemotaxis.">PIP3-dependent macropinocytosis is incompatible with chemotaxis.</a> J Cell Biol. 204 (4), 497-505 (2014).</li><li>Cai, H., Huang, C. H., Devreotes, P. N., Iijima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+chemotaxis+in+Dictyostelium.">Analysis of chemotaxis in Dictyostelium.</a> Methods Mol Biol. 757, 451-468 (2012).</li><li>Schindelin, J., 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Meth. 9 (7), 676-682 (2012).</li><li>Brenner, M., Thoms, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caffeine+blocks+activation+of+cyclic+AMP+synthesis+in+Dictyostelium+discoideum.">Caffeine blocks activation of cyclic AMP synthesis in Dictyostelium discoideum.</a> Dev Biol. 101 (1), 136-146 (1984).</li><li>Bretschneider, T., 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=Dynamic+Actin+Patterns+and+Arp2/3+Assembly+at+the+Substrate-Attached+Surface+of+Motile+Cells.">Dynamic Actin Patterns and Arp2/3 Assembly at the Substrate-Attached Surface of Motile Cells.</a> Curr Biol. 14 (1), 1-10 (2004).</li><li>Swaney, K. F., Borleis, J., Iglesias, P. A., Devreotes, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+protein+Callipygian+defines+the+back+of+migrating+cells.">Novel protein Callipygian defines the back of migrating cells.</a> Proc Natl Acad Sci U S A. 112 (29), E3845-E3854 (2015).</li><li>Meili, R., Ellsworth, 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+novel+Akt/PKB-related+kinase+is+essential+for+morphogenesis+in+Dictyostelium.">A novel Akt/PKB-related kinase is essential for morphogenesis in Dictyostelium.</a> Curr Biol. 10 (12), 708-717 (2000).</li><li>Westendorf, 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=Actin+cytoskeleton+of+chemotactic+amoebae+operates+close+to+the+onset+of+oscillations.">Actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations.</a> Proc Natl Acad Sci U S A. 110 (10), 3853-3858 (2013).</li></ol>

The methods described here offer a convenient way to assess both population and individual cell behavior in response to shear flow. Importantly, while previous studies analyzed localization of leading and lagging edge markers during migration, the current approach allows investigation of immediate effects by applying the shear flow acutely. Using this method we demonstrated that, in fact, the initial response of cells to shear flow does not require cell migration. Instead, the rapid and transient response to the initial ...

Migration of eukaryotic cells is biased by diverse chemical and physical cues in the environment, including gradients of soluble or substrate-bound chemoattractants, variable stiffness of the substrates, electric fields, or shear flow. Although there have been many advances in our understanding of the molecular mechanisms driving chemotaxis, less is known about other types of directed migration and how these diverse signals are integrated at the cellular level to produce a unified migratory response.
Directed migration toward an increasing concentration of a chemoattractant involves three behavioral components: motility, directional sensing, and polarity1. Motility refers to random movement of cells achieved by pseudopod protrusion. Directional sensing is the ability of a cell to detect the source of a chemoattractant, which can occur even in immobilized cells. Polarity refers to the more stable asymmetrical distribution of intracellular components between the leading and lagging edge of a cell, which leads to increased persistence in movement.
Cellular response to a chemoattractant depends on the activity of four conceptually defined regulatory networks: receptor/ G protein, signal transduction, actin cytoskeleton, and polarity1. Chemoattractant binding to the G protein-coupled receptor transmits the signal via heterotrimeric G proteins &#945; and &#946;&#947; to the downstream signal transduction network, which amplifies the directional signal. Multiple pathways within the signal transduction network act in parallel and feed into the actin cytoskeleton network to bias actin polymerization, and consequent pseudopod protrusion, in the direction of the gradient. Among important regulators of chemotaxis are Ras GTPase, TorC2, phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog (PTEN), and guanylyl cyclase. Feedback mechanisms within the signal transduction network and between the signal transduction and actin cytoskeleton networks further amplify the response. Finally, the poorly defined polarity network receives input from the actin cytoskeleton, and further biases the signal transduction network to promote persistent migration in the direction of the gradient.
Much of our mechanistic understanding of chemotaxis was made possible because of the development of fluorescently-tagged biosensors for various components of the regulatory networks. Many chemotaxis regulators have an asymmetrical distribution either of the regulatory molecule itself or its activity. For example, biosensors that recognize activated versions of small GTPases Ras and Rap1 &#8211; Ras-binding domains of Raf1 (referred to as RBD here) and RalGDS, respectively &#8211; localize to the leading edge of a chemotaxing cell2,3. Similarly, PI3K and its product phosphatidylinositol (3,4,5)-trisphosphate (PIP3), recognized by a pleckstrin homology (PH) domain, also show localization at the front of a cell4,5. In contrast, a 3-phosphatase PTEN, which converts PIP3 back to phosphatidylinositol (4,5)-bisphosphate, localizes to the lagging edge of the cell6. Importantly, these biosensors change their localization in response to global stimulation with a chemoattractant. Leading edge markers, which are cytosolic or on the tips of protrusions in a resting cell, relocalize to the cortex, whereas lagging edge markers, which have cortical localization and are absent from the tips of protrusions in a resting cell, become cytosolic after stimulation. Analysis of biosensor distribution in response to global stimulation with a chemoattractant minimizes the contribution of motility and polarity, which often confound the observations. Global or uniform stimulation of a cell suspension with a chemoattractant is also used as a tool to assess population-wide changes in protein activation, often detected by protein phosphorylation7,8,9. This biochemical assay is primarily used to obtain temporal information, whereas microscopy is used to gather both temporal and spatial information about the behavior of various components of the regulatory networks.
The signal transduction network incorporates features of an excitable system10,11. Responses to supra-threshold chemotactic stimuli are &#34;all-or-none&#34; and display refractory periods. Responses are also triggered stochastically and can show oscillatory behavior. Signal transduction events are localized to regions of the cortex that propagate as waves12,13,14,15. Front, or back, markers are recruited to, or dissociate from, the active zones of the propagating waves. Due to the refractory region trailing the active zone, the oppositely directed waves annihilate as they meet. The propagating signal transduction waves underlie the cellular protrusions that mediate cell migration10.
Much of the aforementioned information on chemotaxis came from the studies on the social amoeba Dictyostelium discoideum, although similar regulatory mechanisms are also applicable to neutrophils and other mammalian cell types16. Dictyostelium is a well-established model organism that has a robust chemotactic response during starvation, when thousands of single cells migrate toward an aggregation center, eventually forming a multicellular fruiting body containing spores. Chemotaxis is also essential during the single-cell growth stage of this organism for locating bacterial food sources. Importantly, migration of single Dictyostelium cells is remarkably similar to the migration of mammalian neutrophils or metastatic cancer cells, all of which undergo very rapid amoeboid-type migration. In fact, both the overall topology of the regulatory networks, as well as many of the individual signal transduction pathways involved in chemotaxis are conserved between Dictyostelium and mammalian leukocytes17. Furthermore, other cells, such as fibroblasts, use receptor tyrosine kinases (RTK) instead of GPCRs; however, RTKs may feed into similar networks.
In contrast to chemotaxis, thorough understanding of the signaling mechanisms that drive various other modes of directed migration is lacking. Similarly to cells migrating in a chemoattractant gradient several studies have reported activation and/or localization of typical leading edge markers, including actin polymerization, PIP3 and/or extracellular signal-regulated kinase (ERK) 1/2, at the front of cells undergoing directed migration in response to shear flow or changes in electric fields18,19,20,21. However, in these studies continuous exposure to the stimulus also resulted in cell migration, leaving open the question whether, for example, the leading edge markers localize specifically in response to a stimulus, or if they simply localize at the leading edge because of increased number of pseudopods at the front of a migrating cell.
We developed assays that allow us to observe the response of cells to acute mechanical perturbation delivered as shear flow both at the population level and as individual cells22. Similar to global stimulation with a chemoattractant, acute stimulation with shear flow allows the study of a cellular response to a mechanical stimulus without the confounding contribution from motility or polarity. Combining these biochemical and microscopic assays with genetic or pharmacological perturbations allows us to learn about how mechanical stimuli are perceived and transmitted. Moreover, this approach also provides a novel method for tapping into the system downstream of the chemoattractant receptor in the absence of a chemoattractant, thereby isolating the signal transduction and actin cytoskeleton networks from the receptor/G protein network.
Using the techniques described below we recently demonstrated that acute shear stress leads to activation of multiple components of the chemotactic signal transduction and actin cytoskeleton networks22. By applying the acute mechanical stimulus at varying intervals, we demonstrated that, similarly to chemoattractants, response to mechanical stimuli also exhibits features of an excitable system, including the all-or-none behavior of the response under saturating conditions and the presence of a refractory period. Finally, by combining mechanical and chemical stimulation we showed that the two stimuli share signal transduction and actin cytoskeleton networks, which likely allow for integration of multiple stimuli to bias cell migration.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher</td>
			<td>D16-1</td>
			<td>Use to prepare HL-5 media and SM plates (steps 1.1, 1.2)</td>
		</tr>
		<tr>
			<td>Proteose peptone</td>
			<td>Fisher</td>
			<td>DF0120-17-6</td>
			<td>Use to prepare HL-5 media (step 1.1)</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Fisher</td>
			<td>50-550-445</td>
			<td>Use to prepare HL-5 media and SM plates (steps 1.1, 1.2)</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;7H2O</td>
			<td>Fisher</td>
			<td>S373-500</td>
			<td>Use to prepare HL-5 media, and 10X Phosphate Buffer (steps 1.1, 1.3)</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher</td>
			<td>P386-500</td>
			<td>Use to prepare HL-5 media, SM plates, and 10X Phosphate Buffer (steps 1.1-1.3)</td>
		</tr>
		<tr>
			<td>Streptomycin (dihydrostreptomycin sulfate)</td>
			<td>Fisher</td>
			<td>ICN10040525</td>
			<td>Use to prepare HL-5 media (step 1.1)</td>
		</tr>
		<tr>
			<td>Peptone (Bacto)</td>
			<td>Fisher</td>
			<td>DF0118-17-0</td>
			<td>Use to prepare SM plates (step 1.2)</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Fisher</td>
			<td>P288-100</td>
			<td>Use to prepare SM plates (step 1.2)</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher</td>
			<td>BP1423-500</td>
			<td>Use to prepare SM plates (step 1.2)</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher</td>
			<td>M65-500</td>
			<td>Use to prepare DB Buffer (step 1.4)</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td>Use to prepare DB Buffer (step 1.4)</td>
		</tr>
		<tr>
			<td>Caffeine</td>
			<td>Fisher</td>
			<td>O1728-500</td>
			<td>Use to prepare caffeine (step 1.5)</td>
		</tr>
		<tr>
			<td>cAMP (EMD Millipore Calbiochem Adenosine 3 ft.,5 ft.-cyclic Monophosphate, Sodium Salt)</td>
			<td>Fisher</td>
			<td>11-680-1100MG</td>
			<td>Use to prepare cAMP (step 1.6)</td>
		</tr>
		<tr>
			<td>Folic acid</td>
			<td>Sigma</td>
			<td>F7876</td>
			<td>Use to prepare folic acid (step 1.7)</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher</td>
			<td>BP152-500</td>
			<td>Use to prepare 3X sample buffer (step 1.8)</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Fisher</td>
			<td>BP166-500</td>
			<td>Use to prepare 3X sample buffer (step 1.8)</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>G33-500</td>
			<td>Use to prepare 3X sample buffer (step 1.8)</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Bio-Rad</td>
			<td>1610610</td>
			<td>Use to prepare 3X sample buffer (step 1.8)</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Bio-Rad</td>
			<td>1610404</td>
			<td>Use to prepare 3X sample buffer (step 1.8)</td>
		</tr>
		<tr>
			<td>NaF</td>
			<td>Fisher</td>
			<td>S299-100</td>
			<td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td>
		</tr>
		<tr>
			<td>Na3VO4</td>
			<td>Fisher</td>
			<td>50-994-911</td>
			<td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td>
		</tr>
		<tr>
			<td>Sodium pyrophosphate decahydrate</td>
			<td>Fisher</td>
			<td>S390-500</td>
			<td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td>
		</tr>
		<tr>
			<td>Complete EDTA-free protease inhibitor cocktail (Roche)</td>
			<td>Sigma</td>
			<td>11873580001</td>
			<td>Use to prepare sample buffer with protease and phosphatase inhibitors (step 1.8)</td>
		</tr>
		<tr>
			<td>Latrunculin A</td>
			<td>Enzo Life Sciences</td>
			<td>BML-T119-0100</td>
			<td>Use to prepare Latrunculin A (step 1.9)</td>
		</tr>
		<tr>
			<td>4-15% Tris-HCl polyacrylamide gel</td>
			<td>Bio-Rad</td>
			<td>3450029</td>
			<td>Use for immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>Polyvinylidene fluoride membrane</td>
			<td>Bio-Rad</td>
			<td>1620177</td>
			<td>Use for immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>Phospho-PKC (pan) (zeta Thr410) (190D10) Rabbit antibody</td>
			<td>Cell Signaling</td>
			<td>2060</td>
			<td>Primary antibody for use in immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP antibody</td>
			<td>Cell Signaling</td>
			<td>4370</td>
			<td>Primary antibody for use in immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>Rabbit IgG HRP Linked Whole Ab (from Donkey)</td>
			<td>GE Healthcare</td>
			<td>NA934-100UL</td>
			<td>Secondary antibody for use in immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>Enhanced chemiluminescence substrate (Clarity Western ECL Substrate)</td>
			<td>Bio-Rad</td>
			<td>1705060</td>
			<td>Use for immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>Stripping buffer (Restore Plus Western blot stripping buffer)</td>
			<td>Pierce</td>
			<td>PI46430</td>
			<td>Use for immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue</td>
			<td>Fisher</td>
			<td>PI20278</td>
			<td>Use for immunoblotting (step 3.3)</td>
		</tr>
		<tr>
			<td>K. aerogenes strain (non-pathogenic)</td>
			<td>Dicty Stock Center (dictybase.org)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials and Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker (model G-33, or a comparable alternative)</td>
			<td>New Brunswick Scientific</td>
			<td></td>
			<td>Use to grow K. aerogenes (step 2.1.1), and to basalate and stimulate D. discoideum cells (step 3). The radius of gyration for this shaker is 9.5 mm.</td>
		</tr>
		<tr>
			<td>10 cm Petri dish</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td>Use to prepare SM plates (step 1.2)</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Fisher</td>
			<td>02-671-51B</td>
			<td>Use to count D. discoideum cells (step 2.1.2)</td>
		</tr>
		<tr>
			<td>35 mm dish (Corning Falcon Easy-Grip Tissue Culture Dish)</td>
			<td>Fisher</td>
			<td>08-772A</td>
			<td>Use to plate D. discoideumcells for mechanical stimulation followed by cell lysis (step 3.1.1)</td>
		</tr>
		<tr>
			<td>Polystyrene cup (5 mL)</td>
			<td>VWR</td>
			<td>13915-985</td>
			<td>Use to stimulate D. discoideum cell suspension with a chemoattractant (step 3.2.2)</td>
		</tr>
		<tr>
			<td>Fluidic unit with pump (ibidi Pump System)</td>
			<td>Ibidi</td>
			<td>10902</td>
			<td>Use to deliver mechanical stimulation to D. discoideum cells in a channel (steps 4.1, 4.2, and 5)</td>
		</tr>
		<tr>
			<td>Slide with a channel (&#956;-Slide III 3in1 ibiTreat: #1.5 polymer coverslip, tissue culture treated, sterilized)</td>
			<td>Ibidi</td>
			<td>80316</td>
			<td>Use to plate D. discoideum cells for mechanical stimulation delivered by a fluidic device (steps 4.1, 4.2, and 5)</td>
		</tr>
		<tr>
			<td>50 mL reservoirs (Ibidi Reservoir Holder for Fluidic Unit)</td>
			<td>Ibidi</td>
			<td>10978</td>
			<td>Use to setup the fluidic device for delivering mechanical stimulation to D. discoideum cells in a channel (steps 4.1, 4.2, and 5)</td>
		</tr>
		<tr>
			<td>Lines for the fluidic unit (Perfusion Set YELLOW-and-GREEN)</td>
			<td>Ibidi</td>
			<td>10964</td>
			<td>Use to setup the fluidic device for delivering mechanical stimulation to D. discoideum cells in a channel (steps 4.1, 4.2, and 5). Note: the two lines can also be cut from the 1.6 mm tubing (catalog number 10842).</td>
		</tr>
		<tr>
			<td>Line for the drain (1.6 mm ID tubing)</td>
			<td>Ibidi</td>
			<td>10842</td>
			<td>Use to setup the fluidic device for delivering mechanical stimulation to D. discoideum cells in a channel (steps 4.1, 4.2, and 5).</td>
		</tr>
		<tr>
			<td>Zeiss Observer.Z1 inverted microscope equipped with a 40X/1.3 oil objective and a 20X/0.3 air objective (or a comparable alternative)</td>
			<td>Zeiss</td>
			<td></td>
			<td>Used to image D. discoideum cells with phase-contrast or fluorescence illumination (steps 4 and 5)</td>
		</tr>
		<tr>
			<td>UltraView spinning disk confocal microscope equipped with a 40&#215;/1.25&#8211;0.75 oil objective (or a comparable alternative)</td>
			<td>Perkin-Elmer</td>
			<td>DM 16000</td>
			<td>An alternative used to image D. discoideum cells (step 4.1.4)</td>
		</tr>
		<tr>
			<td>FemtoJet Microinjector (Eppendorf; model 5247, or a comparable alternative) with Eppendorf InjectMan</td>
			<td>Eppendorf</td>
			<td>5252000021D and 5192000027</td>
			<td>Use to deliver mechanical stimulation to D. discoideum cells by a micropipette (step 4.3). Note: catalog number are for the newest version of the equipment (4i).</td>
		</tr>
		<tr>
			<td>Micropipette (Femtotip; 1 &#956;m outside diameter, 0.5 &#956;m inside diameter )</td>
			<td>Eppendorf</td>
			<td>930000035</td>
			<td>Use to deliver mechanical stimulation to D. discoideum cells by a micropipette (step 4.3)</td>
		</tr>
		<tr>
			<td>Microloader tips (Eppendorf Femtotips Microloader Tips for Femtojet Microinjector)</td>
			<td>Eppendorf</td>
			<td>5242956.003</td>
			<td>Use to fill the micropipette (femtotip) with buffer (step 4.3.1)</td>
		</tr>
		<tr>
			<td>1-well chamber (Thermo Scientific Nunc Lab-Tek Chambered Coverglass)</td>
			<td>Fisher</td>
			<td>12-565-472</td>
			<td>Use to plate D. discoideum cells for mechanical stimulation delivered by a micropipette (step 4.3.2)</td>
		</tr>
		<tr>
			<td>8-well chamber (Thermo Scientific Nunc Lab-Tek II Chambered Coverglass)</td>
			<td>Fisher</td>
			<td>12-565-338</td>
			<td>Use to plate D. discoideum cells for mechanical stimulation delivered by bulk buffer addition (step 4.4.1)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analysis Software (Fiji)</td>
			<td>NIH</td>
			<td>https://fiji.sc/</td>
			<td>Use to quantify response to mechanical stimulation (step 4.5)</td>
		</tr>
		<tr>
			<td>Migration analysis software (Tracking Tool PRO)</td>
			<td>Gradientech</td>
			<td>http://gradientech.se/tracking-tool-pro/</td>
			<td>Use to quantify cell speed and persistence (step 5.4)</td>
		</tr>
	</tbody>
</table>

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.

Temporal response of chemotaxis regulators to acute mechanical stimulation
To assess the response of D. discoideum cells to mechanical stimulation, adherent aggregation-competent cells were exposed to a brief pulse of shear flow. Aggregation-competent D. discoideum cells secrete cAMP, which can be sensed by neighboring cells. To overcome the contribution of cell-cell signaling, cells were treated with caffeine, wh...

Watch this Scientific Journal Video about Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation at JoVE.com

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.

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 ...

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