Automatically Generated

Summary

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

Abstract

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Introduction

Macropinocytosis is an endocytic process responsible for internalizing a large amount of extracellular fluid and its content via the formation of dynamic and actin-driven plasma membrane protrusions called membrane ruffles¹. Many of these membrane ruffles form cups that close and fuse back onto the cell and separate from the plasma membrane as large, heterogeneous intracellular endosomes also known as macropinosomes¹. Although macropinocytosis is induced by growth factors such as macrophage colony-stimulating factor (M-CSF) and epidermal growth factor (EGF) in a wide range of cell types, an additional unique, calcium-dependent process known as constitutive macropinocytosis has been also observed in innate immune cells^{2,3,4,5,6,7,8}.

The ability of cells to internalize extracellular material via macropinocytosis has been shown to play an important role in a variety of physiological processes ranging from nutrient uptake to pathogen capture and antigen presentation^9,10,11. However, because this process is non-selective and inducible, it has also been implicated in a number of pathological conditions. Indeed, previous studies suggested that macropinocytosis plays an important role in Alzheimer's disease, Parkinson's disease, cancer, nephrolithiasis and atherosclerosis^{12,13,14,15,16}. Moreover, certain bacteria and viruses have shown to utilize macropinocytosis to gain entry into host cells and induce infection^17,18. Interestingly, stimulation of macropinocytosis can be also exploited for targeted delivery of therapeutic agents in various disease conditions^19,20.

Previous studies have explored macropinocytosis by quantifying internalized fluorescently-tagged fluid-phase markers in the absence and presence of pharmacological agents that inhibit macropinocytosis using flow cytometry and confocal imaging^21,22. Currently available pharmacological tools that inhibit macropinocytosis are limited to and comprise of 1) actin polymerization inhibitors (cytochalasin D and latrunculins), 2) PI3K blockers (LY-290042 and wortmannin) and 3) inhibitors of sodium hydrogen exchangers (NHE) (amiloride and EIPA)^{5,14,15,23,24,25}. However, because these inhibitors have endocytosis independent effects, it is difficult to selectively determine the contribution of macropinocytosis to solute uptake and disease pathogenesis especially in vivo²¹.

Scanning electron microscopy (SEM) is a type of electron microscope that produces ultra-high-resolution images of cells using a focused beam of electrons²⁶. In macropinocytosis research, SEM imaging is regarded as the gold standard technique to visualize topographical and morphological characteristics of the plasma membrane, quantify membrane ruffle formation, and investigate their progression towards macropinosome internalization. Furthermore, scanning electron microscopy combined with the quantification of solute uptake, in the presence and absence of macropinocytosis blockers, provides a reliable strategy to examine macropinocytotic solute internalization in vitro. This paper provides a detailed protocol on how to prepare cells for SEM, visualize the cell surface, quantify ruffle formation, and examine their progress towards cup closure and macropinosome internalization.

Protocol

NOTE: The following is a general protocol used to quantify membrane ruffle formation in RAW 264.7 macrophages using SEM microscopy. Optimization may be required for different cell types.

1. Cell line and cell culture

1. Grow RAW 264.7 macrophages in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (vol/vol) heat-inactivated Fetal Bovine Serum (FBS), 100 IU/mL of penicillin and 100 µg/mL streptomycin in a humidified incubator at 37 °C and 5% CO₂. Replace growth media every other day.
2. When cells become 80% confluent, wash the plate two times using sterile PBS.
3. Detach cells by adding 1,000 µL of 0.5% trypsin-EDTA and incubate the cell culture plate for 3-5 min in a humidified incubator at 37 °C.
4. Examine the plate under a light microscope to confirm cell dissociation. Add 10 mL of growth media containing 10% FBS to inactivate trypsin.
5. Collect the cell suspension in a 50 mL conical tube and centrifuge at 400 x g for 5 min at room temperature. Gently resuspend cells in the complete cell culture medium and determine the cell count and viability using Trypan Blue (0.4%) staining.
   NOTE: The recommended minimum cell viability for this experiment is >90%.
6. Place sterile glass coverslips in wells of a 24-well plate using autoclaved forceps. Seed cells onto coverslips at a density of 1 x 10⁶ cells/mL and incubate the plate overnight in a humidified incubator at 37 °C and 5% CO₂.
7. Change media of each well with fresh 500 µL complete media before treatment. Pretreat macrophages with vehicle (DMSO or other solvents used to dissolve EIPA) or the macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl)-Amiloride (EIPA, 25 µM²¹) for 30 min.
8. Treat cells with vehicle and stimulators of macropinocytosis to promote membrane ruffling: phorbol 12-myristate 13-acetate (PMA, 1 µM, 30 min²¹) and macrophage colony-stimulating factor (M-CSF, 100 ng/mL, 30 min³).
   NOTE: Alternative inhibitors [e.g., latrunculin A (1 µM, 30 min) or cytochalasin D (1 µM, 30 min)] and stimulators [e.g., epidermal growth factor (EGF, 100 ng/mL, 10 min) or platelet-derived growth factor (PDGF, 100 ng/mL, 15 min)] of macropinocytosis can be also used to characterize macropinocytosis in macrophages and other cell types^16,27,28,29. Cells may require overnight serum starvation prior to the treatment with physiological stimulators of macropinocytosis. It is important to note that the most effective concentrations and incubation times will have to be determined to stimulate macropinocytosis in other cell types.

2. SEM fixation

1. After the treatment, aspirate the media from wells and wash coverslips with ice-cold PBS twice.
2. Fix cells (4% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate) for 30 min at room temperature, followed by overnight incubation in the fixative at 4 °C.
3. Gently wash and incubate coverslips with 500 µL of following reagents without disturbing the cell monolayer.
   1. Wash once in 0.1 M sodium cacodylate and incubate for 15 min.
   2. Wash twice with dH₂O with 10 min incubation in each wash.
   3. Wash twice with 25% ethanol with 10 min incubation in each wash.
   4. Wash twice with 50% ethanol with 10 min incubation in each wash.
   5. Wash twice with 75% ethanol with 10 min incubation in each wash.
   6. Wash twice with 80% ethanol with 10 min incubation in each wash.
   7. Wash twice with 95% ethanol with 10 min incubation in each wash.
   8. Wash twice with 100% ethanol. Perform 10 min of incubation in each wash.
      NOTE: Changing/replacing of reagents should be done fast to prevent air drying of cells. Coverslips can be left in 100% ethanol for several days at 4 °C. For long-term storage, add additional 100% ethanol and cover wells with parafilm to prevent air drying of the sample.

3. Critical point drying

1. Place coverslips in a Critical Point Dryer and cover with 100% ethanol. Press the Power button and open the CO₂ tank.
2. Press the Cool button for approximately 30 s until the temperature decreases to 0 °C. Press the Fill button until a bubble appears in the chamber window.
3. Press the Purge button until the smell of ethanol from the purge exhaust disappears. Press the Cool button again until the temperature decreases to 0 °C.
4. Repress the Fill and Purge buttons to turn them off and close the CO₂ tank. Repress the Cool button to turn it off and press the Heat button.
5. Set the temperature at 42 °C and pressure at 1,200 psi. Once the pressure and temperature stabilize, press the Bleed button to allow the pressure to decrease slowly.
6. Once the chamber pressure reaches 150 psi, press the Vent button and wait until the pressure decreases to 0 psi. Turn off the critical point dryer and remove the coverslips.
7. Mount coverslips on SEM aluminum specimen mounts using Carbon Adhesive tabs and subject to sputter coating using gold/palladium in a sputter coater.
8. Turn on the Power button and wait until the vacuum reaches 30 mTorr. Flush the chamber to remove humidity and air by turning off the gas switch and turning the Fine gas valve counterclockwise.
9. Once the vacuum increases to 200 mTorr turn off the gas switch and wait until the vacuum reaches 30 mTorr. Repeat this step 3 times.
10. Push the Timer button and adjust the Voltage knob until the gauge reads 10 mA. Remove coated coverslips from the chamber.
    NOTE: Follow manufacturer's instruction to perform critical point drying and sputter coating of the sample.

4. Imaging and quantification

1. Insert sample coverslips into the chamber of a scanning electron microscope. Close the door and press the Evac button.
2. Open the SEM operation software. Set the accelerating voltage to 15 kv and the working distance to 10 mm.
3. Press the Coordinates button and move around the controller until the cells appear in the center of the observation screen. Set the magnification to 3,500x and image the sample by clicking the Photo button.
   NOTE: Use a higher level of magnification (8,500x to 16,000x) to show the plasma membrane at greater details.
4. Take images from at least 10 random locations on the coverslips, making sure that each microscopic field contains multiple cells. Save images as .tif files.
5. From the images, locate membrane ruffles, defined as protruding blanket-like folds of the plasma membrane, with a size greater than 500 nm (Figure 1). Count the number of ruffles per cell.
   NOTE: C-shaped ruffles were identified as curved membrane protrusions that did not fuse on their lateral ends [(Figure 1 (M-CSF treatment) and Figure 2B]. Fused circular membrane ruffles, prior to their closure, were identified as macropinocytotic cups (Figure 2C).
6. Normalize the number of membrane ruffles to the total number of cells in the microscopic field evaluated. Repeat this analysis for the samples from each group.

Representative Results

Here, we describe the results from the presented technique. Representative SEM images shown in Figure 1 demonstrate membrane ruffle formation in RAW 264.7 macrophages following treatment with PMA and M-CSF. Images were first captured at a magnification of 3,500x for quantification purposes and then at higher levels of magnification (8,500x to 16,000x) to show the plasma membrane at greater details. Pretreatment of macrophages with the macropinocytosis inhibitor EIPA attenuated membrane ruffle formation (Figure 1). Macropinocytosis-associated plasma membrane activities can be divided into five consecutive steps: 1) initiation of membrane ruffling, 2) circularization, 3) cup formation, 4) cup closure, and 5) internalization of extracellular fluid and its associated solutes via macropinosome formation. Figure 2 shows representative images of these morphologically distinct plasma membrane activities following M-CSF stimulation. Large sheet-like ruffles (Figure 2A), circularized C-shaped ruffles (Figure 2B), and macropinocytic cups (Figure 2C) were captured at a magnification of 6,000x to 7,000x. Scanning electron microscopy can be used to provide high-resolution images of the cell surface but cannot be utilized to visualize internalized macropinosomes. Quantification of membrane ruffles following PMA and M-CSF treatment is shown in Figure 3. Macropinosome formation can be confirmed by alternative imaging techniques, including confocal microscopy, as shown in Figure 4. Finally, SEM quantification of membrane ruffling should be complemented by flow cytometry quantification of a fluorescent fluid-phase marker (e.g., FITC- or Texas red-dextran) to confirm stimulation of macropinocytosis (Figure 5).

Figure 1: Scanning electron microscopy images of RAW 264.7 macrophages. RAW 264.7 macrophages were pre-treated with vehicle (DMSO control) or EIPA (25 µM) for 30 min and incubated with PMA (1 µM, 30 min) or M-CSF (100 ng/mL, 30 min) to stimulate membrane ruffling. Images on the right show higher magnification of the cell surface. Red arrows: membrane ruffles. Green arrow: c-shaped ruffle. Please click here to view a larger version of this figure.

Figure 2: Morphological stages of macropinocytosis captured by scanning electron microscopy. RAW 264.7 macrophages were treated with M-CSF (100 ng/mL) for 30 min and cells were processed for SEM imaging. (A) sheet-like membrane protrusion, (B) C-shaped membrane ruffle, and (C) macropinocytic cup. Please click here to view a larger version of this figure.

Figure 3: Quantification of membrane ruffle formation. RAW 264.7 macrophages were pre-treated with vehicle (DMSO control) or EIPA (25 µM) for 30 min and incubated with PMA (1 µM, 30 min) or M-CSF (100 ng/mL, 30 min) to stimulate membrane ruffling. Bar graph shows the number of membrane ruffles normalized to total cell number (n = 3). Data represent the mean ± SEM. One-way ANOVA; *p < 0.05 vs. vehicle, ^#p < 0.05 vs. PMA or M-CSF treatment. Please click here to view a larger version of this figure.

Figure 4: Macropinosome formation. Cells were pre-treated with PMA for 30 min and incubated with Texas red-dextran (25 µg/mL, red) and FM4-64 (5 µg/mL, green) for 10 min. Imaging were performed using a confocal microscope. Blue arrows: membrane ruffling, yellow arrows: macropinosome containing Texas red-dextran, green arrows: macropinosomes. Please click here to view a larger version of this figure.

Figure 5: Quantification of macropinocytosis. RAW 264.7 macrophages were pre-treated with vehicle (DMSO control) or EIPA (25 µM) for 30 min and incubated with PMA (1 µM) and FITC-dextran (100 µg/mL; 70,000 MW) for 2 h. Bar graph shows the mean fluorescence intensity (MFI) fold change normalized to vehicle treatment (n = 3, performed in triplicates). Data represent the mean ± SEM. One-way ANOVA; *p < 0.05 vs. vehicle, ^#p < 0.05 vs. PMA. Please click here to view a larger version of this figure.

Discussion

The present SEM imaging protocol provides a tool to visualize and quantify membrane ruffle formation, circular protrusions, and macropinocytic cups on the cell surface in vitro. Although the current protocol focuses on macrophages, studies have shown that membrane ruffle formation also occurs on various other cell types including dendritic cells, fibroblasts, neurons, and cancer cells^{11,12,14,21,30}. Given this, optimization of cell culture conditions and the use of different stimulators or treatment conditions may be required for the visualization of membrane ruffles in other cell types.

Although some leeway exists for sample preparation, the dehydration and critical point drying steps must be followed exactly to preserve the cell surface architecture. In addition, the presence of water or other solvents such as ethanol would disturb the vacuum, which is essential for SEM imaging and therefore, it must be removed in a controlled manner to keep the specimen morphology intact¹⁴.

Electron microscopes use a beam of accelerated electrons as a source of illumination. The first electron microscopes were developed when the wavelength became the limiting factor in light microscopes³¹. Electrons have much shorter wavelengths thus electron microscopes have a higher resolving power than light microscopes and can be used to provide high-resolution images and investigate the ultrastructure of a wide range of biological specimens. A major limitation of this method revolves around the fact that SEM provides only a snapshot of the plasma membrane morphology, as opposed to the fluorescent live cell imaging, which would allow visualization of membrane ruffles, their transition to macropinocytic cups, and formation of macropinosomes. Live cell imaging, transmission electron microscopy (TEM), and fluorescent dextran uptake assays can be used to investigate the internalization of fluorescent solutes via macropinocytosis, macropinosome translocation and maturation, and intracellular signaling mechanisms regulating macropinocytosis. Because SEM requires fixation onto coverslips, only the dorsal side and the peripheral region of the cell can be visualized, which is another limitation to this technique.

We would like to add that sometimes it is difficult to distinguish between macropinocytosis-independent formation of membrane protrusions and macropinocytic membrane ruffling. We standardized our quantification by identifying membrane ruffles with at least 0.5 µm minimum distance between any two points at the edge of the protrusion. The number of membrane ruffles per cell is highly variable and depends on numerous factors including the stimulant, its concentration, incubation time and cell type³². With PMA and M-CSF, we did see cells with multiple membrane ruffles but chose representative images that better represent our quantified data (1-2 ruffles per cell). SEM remains the gold standard technique for assessing topographical and morphological characteristics of the plasma membrane in vitro. The combination of this method along with live cell imaging and flow cytometry analysis of solute internalization provides a reliable strategy for analyzing macropinocytosis in various cell types.

Materials

0.5% Trypsin-EDTA	Gibco	15400-054
2% Glutaraldehyde	Electron Microscopy Sciences	16320
4% Paraformaldehyde	Santa Cruz Biotechnology	281692
5-(N-ethyl-N-isopropyl)-Amiloride	Sigma Life Science	A3085
Accuri C6 Flow Cytometer
Carbon Adhesive Tabs	Electron Microscopy Sciences	77825-09
Dimethyl Sulfoxide	Corning	25-950-CQC
Dulbecco's Modified Eagle Medium	Cytiva Life Sciences	SH30022.01
Falcon 24-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate	Falcon	353047
Fetal Bovine Serum	Gemini Bio	900-108
FitC-dextran	Thermo Fisher Scientific	D1823
FM 4-64	Thermo Fisher Scientific	T13320
HERAcell 150i CO2 incubator	Thermo Fisher Scientific	51026282
Hummer Model 6.2 Sputter Coater	Anatech USA	58565
JSM-IT500HR scanning electron microscope
Microscope Cover Glass	Thermo Fisher Scientific	12-545-82
Pen Strep	Gibco	15140-122
phorbol 12-myristate 13-acetate	Millipore Sigma	524400
RAW 264.7 macrophage	ATCC	ATCC TIB-71
Recombinant Human M-CSF	Peprotech	300-25
Samdri-790 Critical Point Dryer	Tousimis Research Corporation	8778B
SEM Aluminum Specimen Mounts	Electron Microscopy Sciences	75220
Sodium Cacodylate	Electron Microscopy Sciences	12300
Texas red-dextra	Thermo Fisher Scientific	D1864
Trypan Blue Solution	Thermo Fisher Scientific	15250061
Zeiss LSM 780 confocal microscope