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, whic....

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 d....

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