Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.
The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5.
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-β-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration.
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.
1. Transfection of HUVEC with GFP-actin
2. Setting up the live-cell imaging chamber and stage heater
3. Acquisition of data with live-cell imaging microscope
4. Data analysis
5. Representative Results:
With our transfection protocol, we typically see expression of GFP-actin in at least 50% of the HUVEC transfected, and often can find areas on the coverslip where >90% of the HUVEC in the region express GFP-actin. An example of a live cell imaging experiment with subconfluent HUVEC expressing GFP-actin is shown in Movie 1. For this particular experiment, an image was acquired once per minute. As can be seen in the movie, GFP-actin in the HUVEC can be observed throughout the cytoplasm, as well as in filamentous structures and in local lamellipodia protruding along the cell edge. Also apparent in the movie is that GFP-actin expression is not uniform between cells. Cells chosen for study typically have enough GFP-actin present to visualize various structures containing filamentous actin. Cells expressing very high levels of GFP-actin can be problematic for study because in these cells it is typically difficult to distinguish F-actin structures from the high amount of G-actin present.
Movie 2 shows an example of the behavior of confluent HUVEC expressing GFP-actin. Like the subconflent HUVEC, active lamellipodia formation and turnover along the perimeters of cells was apparent. However, these lamellipodia often gave rise to membrane ruffles, indicating less efficient protrusion7. This is probably due to the presence of adjacent cells blocking the nearby substratum. Actin cortical fibers and stress fibers are also visible in Movies 1 and 2. Although the cells we observed remained stationary, the stress fibers are similar to transverse arc fibers in migrating cells, forming near the cell edge and moving laterally toward the cell center where they disassemble8, 9. An additional dynamic feature observed in these cells was the formation of actin ring structures that expanded concentrically, previously named actin clouds10.
An example of how the distance, persistence, and velocity of cell protrusions are quantified from these time-lapse image sets using kymograph analysis is shown in Figure 1. In Figure 1A, a single pixel line is drawn roughly perpendicular to the cell edge for generation of a kymograph (Figure 1B). In this kymograph, the region defined by the line is placed vertically and the images from throughout the time-lapse are stacked horizontally. Looking from left to right in the kymograph, protrusions are represented as upward movements in the edge of the cell. In Figure 1C, a line was superimposed on the edge of one of these protrusions, and the pixel data associated with that line were collected and are shown in the Results window at the bottom of the panel. This analysis allows quantification of protrusion dynamics, and can also be used to estimate the protrusion frequency (number of protrusions/time) in this region of the cell.
An example of the analysis of stress fiber movement is shown in Figure 2. Most stress fibers we observed formed near the cell periphery and moved toward the cell center, where they eventually disassembled. This can also be quantified by kymograph analysis. A line is drawn perpendicular to the cell edge and to the stress fibers (Figure 2A) and a kymograph is generated (Figure 2B). The stress fibers appear as continuous lines in the cytoplasmic area in the kymograph, often moving down and to the right (toward the cell center). Sometimes the fibers are difficult to see in the original kymograph, and in these cases we use the unsharp mask filter to sharpen the image (Figure 2C). Lines are drawn on the stress fibers and the pixel data are collected using the measure function (Figure 2D). An alternative way to collect this data is to draw a line from the start to finish of the identified stress fiber to get the average slope for the duration that the fiber was observed (Figure 2E). This analysis allows quantification of stress fiber lateral movement and can also be used to quantify the number of stress fibers observed in this region of the cell.
Figure 1. Kymograph analysis of the cell edge to determine the protrusion distance, persistence, and velocity of local lamellipodia. A. A single pixel line is drawn roughly perpendicular to the edge of the cell. This region is extracted from each image of the time-lapse set to generate a montage of the region over time. B. In the resulting kymograph, the x-axis represents time, moving from left to right, and the y-axis shows distance. Movement of the cell edge over time can be evaluated in this kymograph, and lamellipodia are identified regions where the cell edge, moving rightward, goes upward. C. A line is drawn on the cell edge where a protrusion by a lamellipodium has been identified. The dimensions of the bounding rectangle for the line drawn are then acquired (shown in superimposed window). The width is used to calculate the protrusion time or persistence. The height is used to calculate the protrusion distance. The protrusion velocity is calculated by dividing the height by the width. In this example, the upward distance was 96 pixels x 0.16125 μm/pixel = 15.5 μm, and the time was 11 pixels x 1 min./pixel = 11 min. The velocity was calculated as 15.5 μm/11 min. = 1.4 μm/min.
Figure 2. Kymograph analysis of the movement of actin stress fibers. A. A single pixel line is drawn perpendicular to the edge of the cell to generate a kymograph. B. As shown in Fig. 1, in the resulting kymograph, the x-axis represents time and the y-axis distance. Stress fibers are observed as continuous lines in the cell, often going downward and to the right (arrows) C. To better visualize the stress fibers, the unsharp mask filter can be used. In this example, a radius of 3 pixels and mask weight of 0.60 was used. D. Single pixel lines are then drawn over the identified stress fiber, and data collected. In this panel three lines were drawn and then “delete” was pressed to make a permanent annotation (white line) of their locations after each measurement. E. Alternatively, if the average distance, time, and velocity of the stress fiber is desired, a line can be drawn from the start and finish points (yellow line), and the data for that can be acquired (highlighted in the Results window). For this example, the measurements were made on this stress fiber for 37 frames x 1 frame/min. = 37 min. The distance traveled over this time period was 75 frames x 0.16125 μm/pixel = 12.1 μm. The resulting lateral velocity of the stress fiber was 12.1 μm/37min = +0.33 μm/min. A positive value for velocity is assigned for fibers moving toward the cell center, and negative for fibers moving toward the periphery.
Movie 1. Time-lapse images of live HUVEC expressing GFP-actin. The interval between images is 1 min. Local lamellipodia protruded along the entire perimeter of cells. In addition, transverse arc stress fibers moved laterally toward the center of cells. When thrombin (1 U/ml) was added to the bath, the cells contracted slightly and the outward protrusions of lamellipodia paused for about 10 min. After the cells contracted, lamellipodia formation and turnover resumed. Click here to watch the movie.
Movie 2. Confluent HUVEC expressing GFP-actin, before and after treatment with thrombin. Elapsed time is shown in the bottom right corner as minutes:seconds. The interval between images is 30 s. Thrombin (1 U/ml) was added after the 45 min. baseline period. Annotated events include local lamellipodia formation and turnover (arrowheads near cell edges, 0:59 – 37:29), actin clouds (arrows, 1:59 – 21:29), and a gap at a tricellular junction that widens after thrombin is added (arrow, 62:30 – 70:00). Transverse arc stress fibers are also apparent in several of the cells. The cells displayed active formation and turnover of local lamellipodia along their perimeters, with many giving rise to membrane ruffles. Thrombin caused a pause in lamellipodia formation and turnover, and the brief appearance of some small gaps between cells. Click here to watch the movie.
The imaging of GFP-actin in live endothelial cells enables a detailed analysis of the dynamics of the actin cytoskeleton in response to inflammatory stimuli. This method may also be useful to build upon previous findings showing remodeling of the cytoskeleton in response to physical forces like shear stress11. In addition, this method allows a detailed assessment of the contribution of actin cytoskeletal dynamics to various endothelial cell activities, including migration, mitosis, formation of intercellular junctions and junctional maturation, and maintenance of barrier function.
In the data shown, the behavior of the endothelial actin cytoskeleton can be observed before and after treatment with thrombin. Local lamellipodia all along the edges of endothelial cells were observed forming and regressing over time in both nonconfluent and confluent cell monolayers. Treatment with thrombin briefly interrupted lamellipodia formation and turnover. Thrombin also caused the cells to contract slightly, in agreement with previous reports that thrombin causes actin stress fiber formation and increased centripetal tension development in endothelial cells12-14. However, from live cell imaging studies such as this, the origin of the stress fibers can now be determined. In HUVEC, most of the stress fibers originate at the cell periphery and resemble transverse arc fibers in migrating cells8, 9. Another strength of this method over using fixed cells is that the number of stress fibers can be quantified in individual cells before and after thrombin treatment, eliminating selection bias between experimental groups.
With this protocol we evaluate dynamic motion of the cell edge and actin stress fibers. To understand actin monomer dynamics in endothelial cells, more advanced techniques such as fluorescence recovery after photobleaching (FRAP) or fluorescence speckle microscopy (FSM) can be applied15, 16. In addition, because microvascular endothelial cells may represent a better model of microvascular barrier function, optimization of transfection protocols to effectively express GFP-actin in microvascular endothelial cells represents a logical future direction.
In summary, imaging of live endothelial cells expressing GFP-actin represents a powerful tool to determine how the endothelial cell actin cytoskeleton responds to various types of stimuli. Studies using tightly confluent endothelial monolayers will help determine the roles of dynamic structures such as actin-rich lamellipodia and transverse arc stress fibers in endothelial barrier function. In addition, live cell imaging of endothelial cells expressing GFP-actin or other fusion proteins that allow visualization of other subcellular structures will provide detailed spatiotemporal information needed to understand the signaling and structural mechanisms that determine barrier integrity.
The authors have nothing to disclose.
The GFP-β-actin plasmid was generously provided by Dr. Wayne Orr, LSUHSC-S Department of Pathology, and was amplified in the laboratory of Dr. Becky Worthylake, LSUHSC-NO Department of Pharmacology. This work was supported by grants from the National Institutes of Health (P20 RR-018766) and the American Heart Association (05835386N).
1. Ringer 5x Stock | |||
Chemical | Company | Catalog Number | Amount |
Sodium Chloride | EMD | SX0420-3 | 35 g |
Potassium Chloride | J.T. Baker | 3040 | 1.75 g |
Calcium Chloride | Sigma | C-3881 | 1.47 g |
Magnesium Sulfate | Sigma | M-9397 | 1.44 g |
Sterile Filtered Water | N/A | N/A | Bring to 1 L |
Sterile filter into autoclaved bottles and stores at 4°C | |||
2. MOPS buffer | |||
Chemical | Company | Catalog Number | Amount |
MOPS | Sigma | M3183 | 125.6 g |
Sterile Filtered Water | N/A | N/A | Bring to 1 L |
Sterile filter into autoclaved bottles and stores at 4°C | |||
3. Albumin Physiological Salt Solution (APSS) | |||
Chemical | Company | Catalog Number | Amount |
Ringer stock (5x) | N/A | N/A | 200 mL |
Mops Buffer | N/A | N/A | 5 mL |
Sodium Phosphate | Sigma | S-9638 | 0.168 g |
Sodium Pyruvate | Sigma | P5280 | 0.22 g |
EDTA sodium salt | Sigma | ED2SS | 0.0074 g |
Glucose | Sigma | G7528 | 0.901 g |
Albumin, Bovine | USB | 10856 | 10 g |
Sterile Filtered Water | N/A | N/A | Bring to 1 L |
Adjust pH to 7.4 at 37°C, then sterile filter into autoclaved bottles and store at 4°C. | |||
4. 0.9% Saline | |||
Chemical | Company | Catalog Number | Amount |
Sodium Chloride | EMD | SX0420-3 | 9 g |
Sterile Filtered Water | N/A | N/A | Bring to 1 L |
Sterile filter into autoclaved bottles and stores at 4°C | |||
5. 1.5 % Gelatin Solution | |||
Gelatin from porcine skin | Sigma | G2500 | 15 g |
0.9% Saline | N/A | N/A | Bring to 1 L |
Warm the solution to 37°C to dissolve gelatin sufficiently. While still warm, sterile filter into autoclaved bottles and store at 4°C |