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

Summary

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.

Abstract

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⁺.

Introduction

It has been known for several decades that plasma membrane repair after mechanical injury is a Ca²⁺-dependent process^1,2. 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 resealing^3,4. 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 <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 cells^8,9,10,11.

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 bacteria^12,13 or immune cells^14,15. 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 Ca^{2+ 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 repair^18,16,19,20. To adapt this assay for the assessment of cell resealing after permeabilization with pore-forming proteins, we pre-incubated cells at 4 °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.

Protocol

1. Transcriptional Silencing of ASM

1. 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 °C in a 5% CO₂ incubator.
2. 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.
3. Prepare 4 tubes for the siRNA oligo transfection mixture. In tubes A and C, add 250 μl Optimem reduced serum and 4 μl Lipofectamine RNAiMax, per 35 mm dish to be transfected. In tube B, add 250 μl of Optimem reduced serum and 8 μl (160 pmoles) of control medium GC content oligo (Control siRNA), per 35 mm dish to be transfected. In tube D, add 250 μl of Optimem reduced serum and 8 μl (160 pmoles) of SMPD1 oligo (siASM), per 35 mm dish to be transfected. Incubate the tubes at room temperature for 5 min.
4. Combine tubes A and B to form the transfection complex for Control siRNA, and tubes C and D for the ASM siRNA . Incubate the reactions at room temperature for 20 min.
5. Add the Control siRNA and ASM siRNA transfection reaction mixtures slowly, dropwise, into the respective 35-mm glass bottom dishes and incubate at 37 °C/5% CO₂. At 24 hr post-transfection, gently aspirate off media and replace with fresh DMEM growth media. For efficient ASM knockdown, the dishes should be further incubated at 37 °C/5% CO₂ for about 31 hr (total 55 hr) before live imaging.

2. Preparation of Reagents for Live Microscopy

1. Prepare a solution of recombinant pore-forming toxin Streptolysin O (SLO) at a concentration of 100 ng/μl in cold 1x PBS without Ca²⁺ and Mg²⁺. The SLO used in these assays is a constitutively active cysteine mutant that does not require reducing agents for activity, kindly provided by Dr. Rod Tweten, University of Oklahoma. The toxin was expressed in E. coli BL21 DE3 and purified under native conditions, as described previously¹⁶.
2. Prepare the FM1-43 stock solution: Resuspend FM1-43 lyophilized powder in DMSO to create a 25 mM solution.
3. Prepare Solution A: Dilute the FM1-43 stock solution in cold DMEM with Ca²⁺ to a final concentration of 4 μM (1.5 ml are needed per MatTek dish) and store on ice until needed.
4. Prepare Solution B: Dilute the FM1-43 stock solution in cold DMEM without Ca²⁺ and 10 mM EGTA (Ca²⁺-free DMEM) to a final concentration of 4 μM (1.5 ml needed per MatTek dish) and store on ice until needed.
5. Pre-warm (37 °C) 1 ml aliquots of Solution A and Solution B in Eppendorf tubes.
6. Keep on ice solutions of DMEM with Ca²⁺, and Ca²⁺-free DMEM.

3. Live Cell Imaging of FM1-43 Influx into SLO Treated and Untreated Cells

1. Fill a medium size shallow metal container with crushed ice, invert it into a large glass container with ice, and add additional ice leaving only the bottom surface of the metal container exposed. Place a wet paper towel on the exposed metal bottom to allow for even thermal transfer.
2. Take one Control siRNA-treated MatTek dish (Sample 1) and place it on the cold wet paper towel. Gently aspirate off all media and wash 3x with cold Ca²⁺-free DMEM. After the last wash, aspirate off all media in the dish.
3. To image cell-associated FM1-43 in cells with no SLO wounding (Sample 1) in the presence of Ca²⁺, add 180 μl of cold Solution B to the center glass coverslip of the 35-mm glass bottom dish and incubate for 5 min on ice (Table 1).
4. Place MatTek dish on the stage of a confocal microscope heated to 37 °C and equipped with an environmental chamber (which maintains temperature, humidity and CO₂ levels). Find the field of cells that will be imaged looking through a 40X NA 1.3 objective (Nikon). If available, turn on PerfectFocus or a similar device to correct for thermal drift.
5. Add 1 ml of prewarmed Solution A gently to the side of the 35-mm dish and replace cover of environmental chamber (Table 1).
6. Acquire images for 4 min at 1 frame every 3 sec using appropriate imaging software (Volocity in the UltraView Vox Perkin Elmer spinning disk confocal microscopy system). Excitation of FM1-43 is accomplished with a 488 nm laser line using a dichroic filter with a 488 nm band pass, and emission discrimination is achieved through use of a 527(W55) emission filter. Laser power levels and camera sensitivity are set to achieve image exposures of 100 msec.
7. Repeat steps 3.2 to 3.6 to detect FM1-43 in Control siRNA-treated cells with no SLO wounding (Sample 2) in the absence of Ca²⁺, except that at step 3.5 Solution B should be added (Table 1).
8. To measure FM1-43 influx into Control siRNA-treated cells wounded with SLO either in the presence (Sample 3) or absence of Ca²⁺ (Sample 4), add 180 μl of cold Solution B containing SLO to the center glass coverslip of the 35-mm glass bottom dish and incubate for 5 min on ice as in step 3.3. Repeat steps 3.2 through 3.6; adjust adding either Solution A or Solution B at step 3.5 (Table 1).
9. To determine the role of ASM in plasma membrane repair, repeat steps 3.2-3.6 using ASM siRNA-treated cells for all conditions (Samples 5-8) (Table 1).
10. To determine the role of extracellular recombinant sphingomyelinase (SM) on the kinetics of plasma membrane repair (reflected by the inhibition in FM1-43 influx) (Samples 9-10), SM is added (0-50 μU) to either Solution A or Solution B in step 3.5 and then added to cells during live cell imaging in a total volume of 1 ml (Table 1).

4. Quantification of FM1-43 Influx Into Cells

1. After movies have been acquired, measure the intracellular fluorescence intensity of FM1-43 using image analysis software (Volocity Suite, PerkinElmer). Draw a region of interest in the cytosolic region of each cell in the field and apply the software tools to determine the mean FM1-43 fluorescence intensity throughout all frames of the video.
2. To adjust for variations in the initial FM1-43 staining, the data for each video is expressed as fold increase in fluorescence intensity over time, and plotted for each different condition. The fold increase in fluorescence of each individual timepoint per cell is calculated as F_T/F₀, where F_T is the mean fluorescence at the specific timepoint, and F₀ is the mean fluorescence at t = 0 (Figures 1-2).

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent
HeLa 229 cell line	ATCC	CCL2-1
DMEM High Glucose	Invitrogen	11965
DMEM High Glucose No Calcium	Invitrogen	20168
FM1-43	Invitrogen	T3163
Optimem Reduced Serum	Invitrogen	31985
Lipofectamine RNAiMax	Invitrogen	13778
Control medium GC content RNAi oligo	Invitrogen	12935300
SMPD1 RNAi oligo	Invitrogen	HSS143988
Fetal Bovine Serum Heat-inactivated	Gemini-Bioproducts	100-106
Sphingomyelinase from Bacillus cereus	Sigma	S7651
35 mm-glass bottom dishes	MatTek	P35G-0-14
Material
Inverted microscope	Nikon	Eclipse Ti
Camera	Hamamatsu Photonics	C9100-50
Spinning disk confocal microscope	PerkinElmer	UltraViewVoX
Software analysis software	PerkinElmer	Volocity Suite
Environmental chamber	Pathology Devices	LiveCell System Chamber