Lab Module 7: Cell Adhesion Tissues are made of cells and materials secreted by cells that occupy the spaces between the individual cells. This material outside of cells is called the Extracellular Matrix (ECM) and is composed of a complex meshwork of macromolecules. Specialized anchoring junctions link the cells to the proteins of the ECM. The ECM can form several remarkable structures, including bone, tendons and the cornea of the eye. Different tissue types and organ systems have different matrix components, and different amounts of ECM. At one time, the ECM was thought to be an inactive filler material, but it is now recognized that the ECM plays an important role in many aspects of cell behavior, including differentiation, cell migration, and cell proliferation. For example, it has recently been demonstrated that stem cells grown on different matrix components differentiate into particular types of cells and express the genes appropriate for that cell type. Exactly how the ECM influences gene expression is not yet known, but is under intense investigation in many research labs. The ECM is composed primarily of (1) polysaccharide chains known as glycosaminoglycans (GAGs), which are often complexed with proteins to produce proteoglycans and (2) fibrous proteins such as collagens, fibronectin, laminin, elastin. Tissue culture cells attach to the ECM at specific sites called focal adhesions. Focal adhesions contain integral membrane proteins called integrins that bind to proteins in the ECM. The binding of integrins to the ECM is mediated by the extracellular domain of integrin heterodimers. The intracellular cytoplasmic tail of integrins binds proteins that link the integrins to the actin cytokeleton. Thus integrins form an important link from the extracellular material to the cytoskeleton. The actin cytoskeleton in tissue culture cells forms large bundles of actin, referred to as stress fibers, which terminate at the focal adhesions. In order to migrate, cells must continually form and then disassemble focal adhesions. Studies of the speed of cell motility have shown that cells that stick too tightly, or that fail to adhere to the substrate, move more slowly than cells that have an intermediate level of adhesion. 1
The ECM proteins laminin and fibronectin are especially important in mediating cell adhesion to fibrous ECM materials such as collagen fibrils, or to the basal lamina, a specialized layer of ECM that underlies many epithelial sheets. Laminin is a major component of the basal lamina; it is important for wound healing as well as for cell adhesion. These molecules also help cells to adhere to tissue culture petri dishes. In fact, fibronectin was discovered because it was noticed that tissue culturegrown fibroblasts have large amounts of this glycoprotein on their surfaces, while malignantly transformed fibroblasts have very little. It was also noted that transformed cells are less adherent than their normal counterparts, and there is now evidence which indicates that the reduced amounts of fibronectin on these cells is partly responsible for their reduced ability to adhere. When fibronectin is added to cultures of transformed cells, they adhere and spread out much better. The molecular details of the adhesion process have been studied in vivo and in vitro. Many in vitro studies use tissue culture cells grown on plastic or glass surfaces that have been coated with ECM materials. Depending on the types of integrins (many different combinations of integrins are possible) expressed by the cells, cells will attach to a greater or lesser extent. And once integrins bind to their appropriate ECM binding partner, adhesion-dependent signaling takes place, leading to survival and proliferation signals. These signals will then direct the machinery responsible for cell spreading, protrusion and motility. During this lab, you will examine cell adhesion in 2 types of tissue culture grown cells 3T3 mouse embryo fibroblasts and Drosophila S2 cells. Briefly, the experiment is as follows: you will receive dishes that have been coated with several different adhesion proteins, or a control solution. These dishes have a coverglass in the bottom so you can observe the cells without removing the coverslip from the dish, and you can return it to the incubator for further growth. You will then add cells to each dish, and observe the process of adhesion, spreading and motility. You will determine whether the adhesion proteins did or did not facilitate cell adhesion, by comparing (1) the number of cells adherent to each coated surface, compared to an uncoated control surface; and (2) the degree of spreading of the cells on the various surfaces, by measuring the area occupied by the cells. Materials Tissue culture medium without serum Dishes coated with various ECM components, or other adhesive molecules. Cultures of 2 different cell types, 3T3 fibroblasts and Drosophila S2 cells Procedure Cells will be plated on glass coverslips that are coated with different adhesive molecules. The cover glasses have been pre-treated for you (to save time it is not difficult) as follows: A clean glass coverslip (or the coverslip in the bottom of the dish) is incubated with the adhesive material, making sure that the entire surface is covered. The coverslip dishes are then incubated for sufficient time to coat the surface, the solution removed, and the coverslip rinsed and air dried. In the case of condition 6, the coverglass dish is baked at 60 C. Experiment #1: 3T3 cells Cover-glass 1: Distilled water (control) Cover-glass 2: Coated with 50 ug/ml fibronectin in PBS (isolated from bovine plasma) Cover-glass 3: Coated with 40 ug/ml collagen in PBS (Type IV, isolated from human placenta) Cover-glass 4: Coated with 1 mg/ml poly-l-lysine (>300K MW) in PBS (synthetic substrate) Cover-glass 5: Coated with 1 mg/ml poly-l-lysine (>300K MW) in PBS (synthetic substrate) and baked at 60 C. 2
1) The cells will be removed from the flask that they are growing in by treating with the enzyme Trypsin. The cells will be spun in a low speed centrifuge, and the pellet resuspended in medium. Observe the procedure. 2) Taking turns using the sterile hood, seed 0.5 ml of the cell suspension per dish. Add 1 ml medium dish and mix gently. Note the time that the cells were added to the dishes. 3) Examine the cells at low magnification and take an image of a few fields of view. 4) Put the cells at 37 C. Let the cells incubate for ~45 minutes. 5) While the cells are incubating, take a small aliquot of the cells that were plated, and perform a cell count. Record the number of cells/ml. A procedure is provided. 6) After incubation, take the dishes (one at a time) to the microscope and observe. Take representative images at low mag. Return each dish to 37C after observation. Check them again at 1 hour and 2 hours. When you observe the cells, take images of several fields of view at low (10X) or intermediate (40X) magnification. It is probably not necessary to use 100X. record the time of your observations. Experiment #2: Drosophila S2 cells Cover-glass 1: Distilled water (control) Cover-glass 2: Coated with Concanavalin A (Sigma). There are three different concentrations of ConA that we will test; two groups will get each concentration (0.1, 0.5 and 1.0 mg/ml). The S2 cells are weakly adherent to tissue culture plastic, so they can be removed from the flask by pipetting. Go to the tissue culture hood, and place 0.5 ml of S2 cells in each dish. Add an additional 1 ml of medium. Swirl gently. Note the time that the cells were added to the dish. Observe the cells and take a few fields of view. These cells are small, so 40X lens might be a good choice. Incubate these cells at room temperature. S2 cells spread well on ConA, and should spread within 30 min. Take observations every ~ 10 minutes for an hour. Alternate your observations of the S2 cells with the 3T3 cells. Experiment 3: The S2 cells express fluorescent tubulin. When they flatten, you should be able to record the distirubtion of microtubules in these cells. You can also examine S2 cells that you plate on ConA and then treat with nocodazole or with cytochalasin. What happens under these conditions? Does disruption of the cytoskeleton alter the kinetics of adhesion? Data analysis: Note how fast the cells attach on the different matrices. Do you see differences in speed of attachment? Order the substrates according to the speed of cell attachment. Do you observe differences in extent of attachment between 3T3 and S2 cells? From the images that you collect, determine the area occupied by the cells. To do this, trace the outline of several cells in each field of view; plot the area as a function of time after plating. Make a figure showing representative cells at the various time points on the different substrates. Follow up: when you have observed the 3T3 cells for ~2 hours, and the S2 cells for at least 1 hour, you can store the dishes in an incubator until Wednesday. On Wednesday: 1. Acquire images of the cells on each substrate. What is the cell morphology, cell density, and extent of attachment? Order the substrates according to their quality for 3T3 and S2 cell growth. 3
2. Determine the number of dead cells per substrate as follows: add 50 ul of 0.5% Trypan blue to the dish. Swirl. Dead cells take up the dye and appear dark blue. Living cells exclude the dye and appear clear. On the microscope, take low magnification images; use the 10X objective lens and collect images of 10 fields of view for each treatment. Estimate the % of dead cells from these images. (alternatively, you could trypsinize the cells in the dish, add the trypan blue to the resuspended cells, and then count the number of live and dead cells using a Hemocytometer. This is more accurate, but because we have only one tissue culture hood, it is not practical). Discussion and Data Analysis (Wednesday): The quiz on adhesion will be on 3/31. After you have finished collecting the remainder of the data for this lab, we will have a group discussion of the data. Each group should be prepared to share with the class the following: Data on the adhesion of S2 cells to concanavalin A (speed, degree of attachment; area of cells, %dead). From the class data, what is the best concentrations of ConA to utilize? Data on 3T3 cells. Did the choice of substrate make a difference in cell morphology? The extent of spreading? The number of cells that survive (% dead)? Do determine the answer to these questions, you will need to show the data collected on mon and wed. For example, you can show the group images of cell spreading, quantification of cell area, etc. 4
Counting the Cells 1. Prepare a suspension of cells. 2. Gently mix your cells--it s very important that your cells be evenly dispersed before removing a sample to count. 3. With a coverslip in place, use a pasteur pipette to transfer a small amount of cell suspension to one chamber of a hemocytometer by carefully touching the edge of the coverslip with the pipette tip and allowing capillary action to fill the counting chamber. DO NOT overfill or underfill. 4. Notice that there are nine counting squares, and that each of the four corner counting squares is divided into 16 smaller squares (to help you keep track of the cells while counting). Do not mistake the smaller squares for counting squares. Starting with the upper left chamber, count all cells that you see. Count the cells in the three other corner squares. If you have not counted at least 100 cells, then also count the cells in the middle square. 5. Each square of the hemocytometer represents a total volume of 0.1 mm 3 (10-4 cm 3 ) under the coverslip. Since one cubic cm is approximately 1 ml, you can determine the cell concentration and the total number of cells using the following calculations: Cells / ml = total # of cells x 10 4 # of squares counted For example, if 200 cells were counted in 5 squares, the concentration would be: 200 x 10 4 = 8 x 10 5 / ml 5 To determine the total # of cells in the culture: Total cells = cells / ml x original volume of cell suspension 5