Exercise #3 -
Effect of Cytochalasin D on Microfilament
Organization.
Author: Lee
Weber, Biology Dept, University of Nevada, Reno
Minor modifications by Grant Mastick
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to BIO 395 Schedule]
I. Overview
The purpose of this laboratory is to examine the effect of the antimitotic drug cytochalasin D on the organization of actin in mouse fibroblasts. Cytochalasin D is an alkaloid drug produced by the mold Helminthosporium sp. It is a potent inhibitor of actin-dependant cellular processes such as cell motility and cell division. In this exercise you will treat cells with increasing concentrations of cytochalasin D. You will determine the effect of each treatment on actin organization by staining with fluorescein conjugated phalloidin and examining the distribution of F-actin in cells using fluorescence microscopy. By careful inspection of your results, you should be able to describe the effects of cytochalasin D on the organization of the different actin containing structures in a fibroblastic cell.
II. Background
A. Actin, Microfilaments and Stress Fibers:
Actin is a major protein component of all eukaryotic cells. Most multicellular organisms produce several different forms of actin in muscle and non-muscle cells. All forms of actin are highly conserved at the amino acid sequence level, both within a species and between species. Actin exists in two general forms. G-actin is the monomeric subunit form and has a molecular weight of approximately 42,000 da. Under appropriate conditions, G actin can polymerize into filamentous F-actin, which is the form of actin making up the 10 micron thin filament structures found in both muscle and non-muscle cells. F-actin thin filaments are organized into higher order fibrous structures by interaction with a variety of actin-binding proteins. Cytoplasmic F-actin in fibroblastic cells is organized as a crosslinked network of thin filaments found primarily in the vicinity of the plasma membrane (cortical actin fibers). Cortical actin is composed of dynamic actin filaments that are constantly polymerizing and depolymerizing, crosslinking and breaking up, all under control of specific actin binding proteins. Cortical actin has been shown to be involved in cellular processes such as pinocytosis, endocytosis, cytokinesis (cell division), membrane ruffling, microvilli and lamellipodia formation and cell movement. When fibroblasts move along a surface to which they can adhere tightly, a second type of F-actin-containing filament forms. Structures know as stress fibers attach to the inner surface of the plasma membrane at sites called focal adhesion plaques. These plaques are sites at which the plasma membrane comes in contact with the substratum through specific transmembrane receptor proteins. Stress fibers are composed of bundles of parallel F-actin fibers held in register by specific actin bundling proteins. Stress fibers tend to be oriented parallel to the direction of cell movement and contain actin fibers of opposite polarity. These extend between adhesion plaques in the anterior and posterior region of the cell. Non-muscle myosin is also associated with stress fibers. It is believed that shortening of stress fibers is brought about by myosin ATPase activity as the anterior and posterior actin fibers slide against each other in opposite directions. Thus, stress fibers are thought to be involved in contraction of the posterior region of the cell as it moves. The F-actin filaments in stress fibers tend to be much more stable than those found in cortical actin. Individual stress fibers persist in living cells for time to the order of 10-15 min before disassembly.
Much of our understanding of the function of actin filaments in cell physiology comes from studies using drugs that have specific effects on actin polymerization. Phalloidin, an alkaloid compound produced by the mushroom Amanita phalloides, binds to F-actin and prevents depolymerization. Fibroblasts treated with phalloidin stop moving and dividing. The cells remain spread out and lamellipodia and microvilli remain in place. F-actin containing structures such as stress fibers and cortical microfilaments are stabilized by this drug. Cytochalasin D, an alkaloid produced by Helminthosporium and other molds, also blocks actin dependant processes in cells. However, cells treated with cytochalasin round up and the lamellipodia and microvilli disappear. The total F-actin content of treated cells diminishes as stress fibers and cortical thin filaments are no longer visible in the microscope. Thus, cytochalasin D is believed to bind to G actin and prevent polymerization of actin monomers. Existing F-actin fibers then depolymerize as the effective concentration of free G-actin becomes limiting. In some types of cells, binding of cytochalasin D to G-actin also results in proteolytic degradation of monomeric actin.
B. Staining Cells with Fluorescent Probes:
The ability to stain cells with labeled molecular probes can readily demonstrate both the presence and subcellular localization of specific macromolecules. Molecular probes can be antibodies, ligands that bind specifically with target macromolecules, or nucleic acids. Nucleic acid probes can be used to identify cells containing a defined gene sequence or mRNA species by the method of in situ hybridization. Antibodies can be used to localize almost any protein or polysaccharide that is immunogenic. Drug or hormone ligands can be used to identify their target molecules in cells. In order to localize the binding of a probe to its target, the probe molecule must be marked with some type of label to allow detection under the microscope. This is typically accomplished by labeling the probe with a tag that is either radioactive, fluorescent, or contains an enzyme activity. Radioactive probes can be detected by autoradiography. This involves coating the microscopic preparation with a photographic emulsion. Radioactive emissions from the isotopic tag reduce silver grains in the immediate vicinity of the target molecule which become visible when the preparation is treated with photographic developer. Enzymatically tagged probes that can be detected colorimetrically upon reaction with a substrate that produces a colored product. Recently, luminescent substrates have been developed that emit visible light upon reaction with the enzymatic tag. Alkaline phosphatase and peroxidase are two enzymes that are often conjugated with molecular probes as enzymatic tags. Fluorescent probes labeled by conjugation with fluorescent dyes, such as fluorescein (FITC), Texas red, or rhodamine, are very commonly used for subcellular localization studies. Each of these compounds has a characteristic absorption and fluorescence emission spectrum. Fluorescence microscopes equipped with the appropriate filters can simultaneously detect and document as many as three different targets in a cell labeled by probes tagged with different dyes.
The most common method used to visualize the subcellular localization and organization of specific proteins is known as indirect immunofluorescence. In this procedure, cells are fixed with a treatment that immobilizes proteins. Typically, agents that can crosslink proteins such as formaldehyde or glutaraldehyde are used for fixation followed by permeabilization of the cell membrane with a mild detergent. An organic precipitating reagent such as methanol or acetone is sometimes used in the case of abundant structural protein components such as actin, tubulin, or intermediate filaments. Organic solvents also extract the cell membrane so a permeabilization step is unnecessary. The cell preparations are then treated with an antibody preparation that is specific for the target molecule. After rinsing away unbound antibody, the preparation is then treated with a fluorescent dye-tagged second antibody specific for the first class of immunoglobin. For example, if the first unlabeled antibody was produced in a rabbit, the second labeled antibody might be goat antiserum raised against rabbit immunoglobin. This two step process increases the sensitivity of the detection process since several labeled molecules can bind to each molecule of first antibody bound to antigen. After rinsing the preparation to remove unbound labeled antibody, the preparation is viewed under a fluorescence microscope.
The drug phalloidin is a specific ligand for F-actin. That is, F-actin is the only molecule in the cell to which phalloidin will bind. Phalloidin also stabilizes F-actin against depolymerization. Thus, phalloidin conjugated with a reactive form of fluorescein, fluorescein isothiocyanate (FITC), is a convenient molecular probe for F-actin. Phalloidin also binds very rapidly with F-actin and takes less time than the indirect immunofluorescence procedure. In this lab exercise you will use FITC-phalloidin to visualize F-actin in mouse fibroblasts and determine the effect of the drug cytochalasin on F-actin organization.
III. Procedure:
A. Cytochalasin D Treatment:
1. Prior to the laboratory period, your instructor will have treated chamber slide cultures with 0, 0.1, 1, or 10 :M cytochalasin D. The cells should be exposed to the drug for 1-2 hr. (record the actual time of exposure)
2. Obtain a chamber slide, and note which chambers contain cells, and which have been treated with the different concentrations of drug. Examine each group of treated cells using the inverted microscope and note any differences in cell shape that may result from cytochalasin D treatment.
B. Fixation and FITC-Phalloidin to Localize F-actin:
1. WEAR GLOVES WHEN HANDLING THE SLIDES DURING THE REST OF THE PROCEDURE.
2. Carefully remove the chambers, and quickly rinse slides twice in PBS for a few seconds.
3. Fix in 3.7% formaldehyde in PBS for 20 min at room temperature.
4. Rinse slides twice in PBS for a few seconds.
5. Permeabilize cells for 45 min with 0.1% saponin in PBS.
6. Dilute FITC-phalloidin stock (33 ug/ml) fifty-fold in PBS.
7. Place your slide into a plastic box containing a moistened paper towel. Pipette 2-100 ul drops of the diluted phalloidin stock onto each patch of cells. Place a coverslip on each drop of phalloidin solution and incubate for 40 min in the dark. Seal the container to minimize evaporation. (Reminder: phalloidin is another actin-binding toxin, so avoid contact with skin.)
8. Remove the coverslip, and rinse off the phalloidin solution once in PBS. Next, you will stain your cells with DAPI to reveal the nuclei. (DAPI is a blue fluorescent dye that strongly binds DNA, intercalating into the DNA much like Hoechst dye. Thus, DAPI is a potential carcinogen, so avoid skin contact.) Cover the slide with PBS + DAPI solution, and let sit for 1 minute. Rinse your slide 3 X 2 min in PBS.
9. (Get help from instructor.) Place two drops of slide mounting solution onto the microscope slide and carefully lay down a large coverslip. (Prolong, Molecular Probes Inc. This solution contains a potential carcinogen, so again avoid skin contact)
10. View each cytochalasin D treated cell preparation under the fluorescence microscope in Dr. Mastick’s laboratory. Initially view each group of treated cells using the same illumination gain setting that allows visualization of the untreated cells. This should provide information about the relative amount of F-actin present after treatment with each concentration of cytochalasin D. Capture an image of each preparation for your laboratory report.
11. Adjust the amount of illumination such that cells and cell structures are clearly visible for each treatment. In order to view the cells treated with the higher concentrations of cytochalasin D, greater amounts of incident light must be used.
Save the images from the fluorescence microscope as high resolution TIFF or JPG files. Label all relevant subcellular structures.
Visually analyze the micrographs of your phalloidin stained preparations. What is the effect of cytochalasin on the stress fibers? On cortical actin filaments? Describe the effect of each concentration of cytochalasin D on F-actin content and organization.
Compare your results and those of your classmates to the literature and discuss the known effects of cytochalasin D on cellular structure and function.
REFERENCES
Wolf, S.L., Molecular and Cellular Biology, Wadsworth Publishing, Belmont, CA, 1993. pp. 452-460, 482-487.
Harlow, E. and Lane, D., Antibodies, A laboratory manual, Cold Spring Harbor Laboratory, 1988. pp. 361-411.