Cell Membrane Repair Assay Using a Two-photon Laser Microscope

Summary

Cell membrane wounding via two-photon laser is a widely used method for assessing membrane resealing ability and can be applied to multiple cell types. Here, we describe a protocol for in vitro live-imaging of membrane resealing in dysferlinopathy patient cells following two-photon laser ablation.

Abstract

Numerous pathophysiological insults can cause damage to cell membranes and, when coupled with innate defects in cell membrane repair or integrity, can result in disease. Understanding the underlying molecular mechanisms surrounding cell membrane repair is, therefore, an important objective to the development of novel therapeutic strategies for diseases associated with dysfunctional cell membrane dynamics. Many in vitro and in vivo studies aimed at understanding cell membrane resealing in various disease contexts utilize two-photon laser ablation as a standard for determining functional outcomes following experimental treatments. In this assay, cell membranes are subjected to wounding with a two-photon laser, which causes the cell membrane to rupture and fluorescent dye to infiltrate the cell. The intensity of fluorescence within the cell can then be monitored to quantify the cell's ability to reseal itself. There are several alternative methods for assessing cell membrane response to injury, as well as great variation in the two-photon laser wounding approach itself, therefore, a single, unified model of cell wounding would beneficially serve to decrease the variation between these methodologies. In this article, we outline a simple two-photon laser wounding protocol for assessing cell membrane repair in vitro in both healthy and dysferlinopathy patient fibroblast cells transfected with or without a full-length dysferlin plasmid.

Introduction

The cell membrane of eukaryotic cells consists of a double-layer of protein-studded phospholipids which defines the intra/extracellular environment of the cell and is essential for maintaining cellular homeostasis and cell survival. Cell membrane injuries arising from mechanical or chemical insults are commonplace in various mammalian cell types, including skeletal and cardiac muscle, stomach, and lung cells^1,2,3,4. In addition to injuries consequent to daily physiological function, cell membranes can also be damaged by environmental insults, bacterial toxins, and ischemic reperfusion⁵. Failure to reseal ruptures in the cell membrane leads to an unregulated influx of extracellular Ca²⁺- along with other potentially toxic extracellular components - into the cell, triggering downstream signal cascades which can quickly result in cell death^1,4,5,6.

To date, there have been several proposed models for membrane repair in cells. Different repair mechanisms may be activated depending on the size and nature of the membrane rupture. For example, it is suggested that cell membranes may utilize lateral flow or protein clogging to repair small disruptions (<1 nm). The lateral fusion model proposes that membrane ruptures are rapidly mended through lateral recruitment of dysferlin-containing membrane⁷, while the protein clogging model suggests that small perforations are mended through protein aggregations (mostly annexins)⁸. Conversely, larger membrane lesions trigger Ca²⁺-dependent, dysferlin-mediated vesicle fusion and formation of a repair patch. In the repair patch model, a rapid Ca²⁺ influx into the cell triggers the recruitment of multiple proteins (dysferlin, annexins, mitsugumin-53, and EDH proteins) which form a complex or "patch", along with a fusion of intracellular vesicles or lysosomes at the site of membrane damage^4,9,10,11,12. It is important to note that these models are not necessarily mutually exclusive and may work in concert to facilitate membrane repair. Failure to properly reseal cell membrane damage is associated with multiple disease states, including muscular dystrophy (dysferlinopathy - including Miyoshi myopathy, limb-girdle muscular dystrophy type IIB, and distal myopathy with anterior tibial onset)¹³, cardiomyopathy¹⁴, and Chediak-Higashi syndrome (CHS)¹⁵.

Given that proper cell membrane integrity and resealing plays such an important role in health and disease, a deeper understanding of the underlying molecular mechanisms of cell membrane repair would be beneficial in the search for novel therapeutic strategies. It is, therefore, necessary to possess appropriate experimental techniques to monitor the kinetics and evaluate the ability of cell membrane repair. Several in vitro methods for modeling membrane repair have been designed. One strategy involves mechanical injury, which can be facilitated through cell scraping with a pipette/surgical blade/razor or by rolling glass beads over the cells¹⁶. However, this type of mechanical injury generates larger lesions, and creates a high degree of variation in cell injury, both within and between cultures.

Another method of generating membrane wounds is laser ablation by two-photon microscopy. In contrast to traditional laser confocal microscopy that employs single photon excitation, the two-photon laser simultaneously uses two long-wavelength, low-energy photons to facilitate the excitation of a high-energy electron¹⁷. This non-linear process results in excitation exclusively within the focal plane and not along the entire light path¹⁷ (Figure 1). This reduced excitation volume helps to minimize photodamage when imaging live cells^18,19. Researchers are therefore able to generate precise lesions in the cell membrane and monitor membrane resealing in real time by using fluorescent dyes and observing changes in the fluorescence intensity as the cell membrane ruptures and then reseals.

This approach has been used repeatedly to study membrane wounding in vitro and in vivo and in multiple cell types^20,21. For example, membrane repair defects in fibroblasts and myotubes derived from dysferlinopathy patients were assessed using this technique^22,23. Also, single muscle fibers isolated from mice were used to monitor repair patch formation^13,24,25. The movement of fluorescently tagged proteins can also be observed during membrane repair in single muscle fibers⁹. Furthermore, the process of sarcolemmal repair following two-photon laser wounding in zebrafish embryos can be observed in real-time in vivo²⁶.

In this article, we outline a methodology for assessing cell membrane repair dynamics in fibroblasts using two-photon laser wounding, although this methodology can be applied to various cell types for the purpose of quantifying plasma membrane resealing ability in vitro. In this method, cells are incubated with FM4-64, a lipophilic, cell-impermeable dye which quickly fluoresces as it binds to negatively charged phospholipids within the cytoplasm upon entering the cell through the membrane lesion (Figure 2&3). Quantification of dye fluorescence adjacent to the membrane lesion allows for monitoring the time that it takes for the cell membrane to reseal itself. To exemplify the utility of this method, we use dysferlinopathy patient fibroblasts transfected with GFP-conjugated full-length dysferlin (DYSF) plasmids to assess the rescue of cell membrane repair.

Protocol

Human fibroblast cells used in this study were used with approval from the Human Ethics Committee of the Faculty of Medicine and Dentistry at the University of Alberta.

1. Transfection of cells with full-length DYSF plasmid

1. Culture fibroblast cells in a T225 flask containing 40 mL of growth media - 36 mL Dulbecco's Modified Eagle Medium (DMEM), 4 mL of fetal bovine serum (FBS), and 20 µL of penicillin/streptomycin (P/S) - in a CO₂ incubator at 37°C.
   NOTE: Cells should be cultured to approximately 70-80% confluency and should not be allowed to become completely confluent.
2. To prepare cells for seeding, rinse the cells with 40 mL of phosphate buffered saline (PBS) twice, then add 5 mL of 0.05% trypsin to detach the cells. Return the cells to the 37 °C CO₂ incubator and incubate the cells for 5 min.
   1. When the cells are fully detached, add 40 mL of growth media to stop the trypsin reaction.
   2. Count the cells using a hemocytometer or another cell counting device.
3. Seed 2 mL of cells at 1x10⁵ cells/mL into 35 mm collagen-coated glass-bottom dishes. Incubate the cells overnight in a CO₂ incubator at 37 °C.
   NOTE: Cells should be approximately 50-60% confluent the next day.
4. Remove the growth media and replace it with 2 mL of serum-deprived media (growth media without FBS).
5. Transfect the cells with full-length dysferlin plasmid using lipotransfection.
   1. Prepare 150 µL of serum-deprived media in a 1.5 mL tube (one tube for each dish to be transfected).
   2. Add 3 µL of transfection reagent to each 1.5 mL tube containing serum-deprived media. Incubate it at least 5 min at room temperature.
   3. In a separate 1.5 mL tube, add 175 µL of serum-deprived media and 3.5 µg of plasmid DNA.
      NOTE: Multiply the above amounts for each additional dish to be transfected.
   4. Combine 150 µL of media containing plasmid DNA into a tube with 150 µL of serum-deprived media and transfection reagent. Incubate it for 20 min at room temperature.
   5. Evenly distribute 300 µL of media with plasmid and transfection reagent across the dish. Incubate the dishes for 24 h in a CO₂ incubator at 37°C.
6. Replace the media with fresh growth media containing serum and incubate it for an additional 24 h in a CO₂ incubator at 37 °C.

2. Preparation of cells for two-photon wounding assay

1. Remove the media by pipetting and rinse the cells once with 1 mL of Tyrode's solution, then add 1 mL of fresh Tyrode's solution containing 1 µL of 2.5 mM FM 4-64 dye.
   NOTE: The final concentration of FM 4-64 dye is 2.5 µM.
2. If assessment of membrane repair in a calcium-depleted environment is desired, rinse cells with 1 mL of PBS and then add 1 mL of PBS containing 1 mM EGTA.
   NOTE: As PBS will cause the cells to detach over time, the wounding assay should be completed as quickly as possible.

3. Two-photon laser wounding assay

1. Using an inverted confocal microscope and associated program software, create a 0.2 µm x 2 µm target and place it at the edge of the cell membrane so that the target line overlaps and lies perpendicular to the direction of the cell membrane (Figure 2) .
2. Use a 543-nm HeNe laser to excite FM dye signal and set the detection range for 600-760 nm. Use a 488-nm Argon laser to excite GFP signal (Figure 4) and set the detection range for 500-550 nm.
   1. Create a 5 min time series of sequential image scans, imaging cells every 5 s.
   2. To generate a membrane lesion, bleach cells using a two-photon laser set to 820-nm, using 15% laser power with 10 iterations and bleach cells 25 s after the beginning of the time series.
3. To quantify FM 4-64 dye fluorescence, use the software to draw a 6 µm x 6 µm region of interest (ROI) and place it adjacent to the wounding location and within the cell (Figure 3).
   NOTE: Avoid sampling areas of pixel oversaturation by using the "Range Indicator" function in the software - in this application, red pixels represent oversaturated pixels. Export raw fluorescence values into an electronic spreadsheet for statistical analysis.
4. Calculate relative fluorescence values for each time point by subtracting the background value (mean fluorescence value of timepoints prior to membrane wounding) and dividing the net increase by the fluorescence value at t = 0 (Figure 5, 6).

Results

Healthy human fibroblasts, non-treated dysferlinopathy patient fibroblasts, and patient fibroblasts transfected with a plasmid containing the full-length dysferlin sequence were subjected to two-photon laser wounding to assess membrane resealing ability in real time. Healthy human fibroblast cells displayed low levels of FM 4-64 fluorescence activation following laser wounding and non-treated patient fibroblasts exhibited a high degree of relative fluorescence intensity following injury (...

Discussion

Two-photon laser wounding of the cell membrane is a precise and versatile technique for assessing the dynamics of membrane resealing in vitro. In this article, we described a protocol for determining cell membrane resealing ability in dysferlinopathy patient cells using the two-photon laser wounding assay. Our results show that dysferlinopathy patient cells are defective in cell membrane resealing, which is consistent with findings by other researchers and which further highlights the striking similarities in ce...

Materials

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium (DMEM)	Thermo Fisher	11320033
Fetal Bovine Serum	Sigma-Aldrich	F1051
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher	15140122
Trypsin-EDTA (0.05%), phenol red	Thermo Fisher	25300062
Dulbecco’s Phosphate Buffered Saline	Sigma-Aldrich	D8537
35mm collagen-coated glass-bottom dishes	MatTek	P53GCOL-1.5-14-C
Serum-deprived media	Thermo Fisher	31985070
Transfection reagent	Thermo Fisher	15338100
FM 4-64 Dye	Invitrogen	T13320
Tyrode’s Salts Solution	Sigma-Aldrich	T2397
Confocal laser scanning microscope	Carl Zeiss	NA
Chameleon Two-photon laser	Coherent	NA