This work was supported by NIH grants R37 AI34867 and R01 GM064625 to N.W.A. We thank Dr. R. Tweten from the University of Oklahoma for the SLO expression plasmid.

1. Transcriptional Silencing of ASM
<ol>
 <li>Seed 1.5 x 105 HeLa cells in 2 ml of DMEM growth media (DMEM high glucose with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1% Penn-Strep) on 35 mm glass bottom dishes (MatTek) and incubate overnight at 37 &deg;C in a 5% CO2 incubator. </li>
 <li>On the following day, aspirate off the growth media and replace it with 2 ml DMEM reduced serum medium (DMEM with 4% FBS), at least 1 hr before adding the siRNA transfection mixture. </li>
 <li>Prepare 4...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol-dependent+cytolysins,+a+family+of+versatile+pore-forming+toxins.">Cholesterol-dependent cytolysins, a family of versatile pore-forming toxins.</a> Infect. Immun. 73, 6199-6209 (2005).</li><li>Zha, X., 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=Sphingomyelinase+treatment+induces+ATP-independent+endocytosis.">Sphingomyelinase treatment induces ATP-independent endocytosis.</a> J. Cell Biol. 140, 39-47 (1998).</li><li>Buckley, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossing+three+membranes.+Channel+formation+by+aerolysin.">Crossing three membranes. Channel formation by aerolysin.</a> FEBS Lett. 307, 30-33 (1992).</li><li>Parker, M. 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Earlier studies on mechanical injury and plasma membrane repair relied on diverse mechanisms of injuring cells, ranging from dropping glass beads, micropipetting, shearing, scratching or scraping. The readout of all these assays was qualitative rather than quantitative, and did not give precise information on the kinetics of the repair mechanism. The ability of cells to reseal their plasma membrane after these forms of injury was measured by the presence or absence of tracers added to the extracellular fluid in the cyt...

It has been known for several decades that plasma membrane repair after mechanical injury is a Ca2+-dependent process1,2. Later studies showed that Ca2+ influx through the wound triggers a vigorous process of exocytosis of intracellular vesicles at the site of injury, which is required for resealing3,4. The rate of loss of a fluorescent dye loaded into the cytoplasm of cells was used to assess the speed of repair, and concluded that resealing was completed within &lt;30 sec after injury5. Two models were initially proposed to explain the requirement for exocytosis in plasma membrane repair: 1) the "patch" model, which suggested that Ca2+ influx through the lesion triggers initially homotypic fusion of intracellular vesicles, forming a large "patch" that would then be applied to the plasma membrane to reseal the wound6 and 2) the tension reduction model, which proposed that membrane added by Ca2+-dependent exocytosis in the vicinity of the wound would reduce plasma membrane tension, facilitating resealing of the bilayer7. The role of Ca2+-triggered exocytosis in plasma membrane repair was further reinforced by studies showing that lysosomes contain a Ca2+ sensor molecule, synaptotagmin VII, which facilitates their exocytosis and plasma membrane repair in injured cells8,9,10,11.
However, additional evidence has indicated that exocytosis alone was not sufficient to promote plasma membrane repair. In addition to mechanical tears on the plasma membrane, a frequent form of cell injury is permeabilization by pore-forming toxins produced by bacteria12,13 or immune cells14,15. Unlike mechanical tears, pore-forming proteins insert themselves on the plasma membrane forming a stable, protein-lined pore that cannot be resealed simply by applying a membrane "patch" or by reducing membrane tension. Intriguingly, studies revealed that mammalian cells have an efficient mechanism to repair their plasma membrane after permeabilization with pore-forming proteins, and this process also requires the presence of extracellular Ca2+ 12. This finding raised the question of whether transmembrane pore removal from the cell surface was also a rapid process, as observed with mechanical wounds5. Surprisingly, our recent studies revealed that the resealing of cells permeabilized with pore-forming toxins has very similar properties to the repair of mechanical wounds: the process requires extracellular Ca2+, and is completed within ~30 sec. Investigating this process in more detail, we recently learned that in addition to Ca2+-regulated exocytosis of lysosomes, plasma membrane repair involves a rapid form of endocytosis, which is triggered by release of the lysosomal enzyme acid sphingomyelinase (ASM) and is essential not only for the removal of transmembrane pores, but also for the repair of mechanical wounds16.
To determine the kinetics of cell resealing after permeabilization with pore-forming proteins, in our laboratory we adapted a live imaging methodology that had been used previously to assess resealing of laser-injured isolated muscle fibers17. This assay relies on properties of the lipophilic dye FM1-43, which stably intercalates into the outer leaflet of lipid bilayers increasing in fluorescence intensity. When the plasma membrane bilayer is disrupted extracellular dye gains access to intracellular membranes, providing a sensitive assay to detect plasma membrane injury and repair18,16,19,20. To adapt this assay for the assessment of cell resealing after permeabilization with pore-forming proteins, we pre-incubated cells at 4 &deg;C with the bacterial toxin streptolysin O (SLO), which binds to membrane cholesterol21. Synchronous cell permeabilization can then be easily achieved by moving the cells from ice to warm medium in a heated microscope stage, which activates the oligomerization and change in conformation that leads to transmembrane pore formation. An advantage of this approach, over the previously published assays using laser wounding, is that a larger number of cells can be analyzed simultaneously in a microscopic field, providing better sampling of the cell population. Given the mechanistic similarities between the cell resealing process seen after mechanical injury and permeabilization with pore-forming toxins, the assay we describe here provides a very versatile and powerful method for dissecting factors involved in the fundamental process of plasma membrane repair. As an example, we show that it is possible to use this assay to identify Ca2+ dependent and independent steps of the repair process.

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		 </tr>
		 <tr>
			<td>HeLa 229 cell line</td>
			<td>ATCC</td>
			<td>CCL2-1</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose </td>
			<td>Invitrogen</td>
			<td>11965</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM High Glucose No Calcium</td>
			<td>Invitrogen</td>
			<td>20168</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FM1-43</td>
			<td>Invitrogen</td>
			<td>T3163</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Optimem Reduced Serum</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Lipofectamine RNAiMax</td>
			<td>Invitrogen</td>
			<td>13778</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Control medium GC content RNAi oligo</td>
			<td>Invitrogen</td>
			<td>12935300</td>
			<td></td>
		 </tr>
		 <tr>
			<td>SMPD1 RNAi oligo</td>
			<td>Invitrogen</td>
			<td>HSS143988</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal Bovine Serum Heat-inactivated</td>
			<td>Gemini-Bioproducts</td>
			<td>100-106</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Sphingomyelinase from Bacillus cereus</td>
			<td>Sigma</td>
			<td>S7651</td>
			<td></td>
		 </tr>
		 <tr>
			<td>35 mm-glass bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-0-14</td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Material</td>
		 </tr>
		 <tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Camera</td>
			<td>Hamamatsu Photonics</td>
			<td>C9100-50</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Spinning disk confocal microscope</td>
			<td>PerkinElmer</td>
			<td>UltraViewVoX</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software analysis software</td>
			<td>PerkinElmer</td>
			<td>Volocity Suite</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Environmental chamber</td>
			<td>Pathology Devices</td>
			<td>LiveCell System Chamber</td>
		 </tr>
		</tbody>
 </table>

live imaging assay for assessing the roles of ca2+ and sphingomyelinase in the repair of pore-forming toxin wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Low concentrations of bacterial sphingomyelinase (SM) rescue plasma membrane repair in cells depleted in lysosomal acid sphingomyelinase.
Using the FM1-43 imaging assay, we previously showed that cells deficient for the lysosomal enzyme ASM and exposed to SLO wounding in the presence of Ca2+ have a plasma membrane defect is rescued by extracellular addition of the recombinant human enzyme19. Since the human lysosomal ASM has a low pH optimum, we investigated whether resealin...

Watch this Scientific Journal Video about Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds at JoVE.com

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

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11.6K Views.  University of Maryland. Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.