 |
| Fluorescent Staining of Cytoskeletal Elements |
Fluorescent antibody staining is a precise technique for marking elements of the cytoskeleton so they become visible under a microscope. The technique has revealed much about the location and functions of the cytoskeleton within a cell. It also offers some of the most visually appealing of the microscopic images available to biologists.
Cells require an internal system of fibers in order to maintain and change their shape. Perhaps the most straightforward function of the cytoskeleton is to provide cell support, a scaffolding that gives each cell a distinctive three-dimensional shape.
Without such support every cell would be shaped like a fried egg. The fibers found in the cytoskeleton serve other functions as well: for example, as rails along which substances shuttle from one part of the cell to another.
    |
Equally important is the role of the cytoskeleton in cell movement and change in cell shape. The various cytoskeletal fibers can be rearranged, dissolved, and reconstructed at new locations when need be. The cytoskeleton is also a key component of some highly specialized cell structures, such as cilia and flagella.
Fluorescent antibody staining techniques allow scientists to observe exactly how various cytoskeletal fiber types are oriented within the cell. This technique has greatly increased knowledge of how the cytoskeleton does its jobs. It is also among the most aesthetically beautiful of all the procedures used by cell biologists.
The Cytoskeleton
The cytoskeleton is composed of three different kinds of fibers, each with different, specialized functions. Microfilaments are the smallest (6 nanometers in diameter).
They are made up of a protein similar to the actin protein in muscle. It is thus not surprising that microfilaments participate in cell movement and shape changes. They usually are found in bundles just inside the surface of the cell, where they are best situated to help the cell change shape.
Intermediate filaments are somewhat larger. Unlike microfilaments, they seem to serve as passive scaffolding elements within the cell. They typically form an interlaced meshwork in the cytoplasm. The proteins that make up these fibers are subtly different in different kinds of cells; the significance of such variation is not clear.
The largest fibers are called microtubules (25 nanometers in diameter). They are hollow spirals of protein building blocks. The microtubules have important, specialized roles in particular regions of cells and at certain times in a cell’s life.
For example, they help move the chromosomes around during division of one cell into two. They are important components of both cilia and flagella—whiplike structures on the surfaces of some cells that serve as oars to help them swim about or to move substances along their surfaces.
Technique
The properties of the cytoskeleton depend upon its precise three-dimensional architecture within the intact cell. Thus, it is important that the researcher use methods designed to analyze the intact cytoskeleton inside whole cells.
The most popular method is to fasten a fluorescent dye onto an antibody. The most commonly used dyes are rhodamine (for red fluorescence) and fluorescein (for greenish-yellow fluorescence). When such a fluorescent antibody is then added to cells, it can be located using a special fluorescence microscope.
Although this procedure works reasonably well, it can be improved by amplifying the signal—that is, by devising a method to add more than one molecule of fluorescent dye to each anticytoskeleton antibodymolecule, which will increase the amount of fluorescent light emitted.
The brighter light is much easier to see. This can be done using the socalled secondary-antibody procedure. A “secondary antibody” must be prepared using the first antibody (against the cytoskeletal protein) as an antigen. This time, the secondary antibody is made fluorescent.
The cytoskeletal proteins are then tagged in two steps: First, the primary antibody is added to cells, then the fluorescent secondary antibody is added. Because of the nature of the secondary antibody, numerous molecules of it can adhere to each molecule of primary antibody.
In this way, many fluorescent molecules can be attached to each molecule of primary antibody. The result is that the researcher can now see strikingly beautiful images in the fluorescence microscope: brilliantly colored glowing strands of the cytoskeleton, arranged in various patterns depending on what is happening inside the cell.
What Biologists Have Learned
Fluorescent antibody techniques have helped scientists learn that micro filaments and microtubules are dynamic fibers,made up of many kinds of protein (perhaps as many as several hundred), which grow and shrink as necessary. (Intermediate fibers are more stable.)
In addition to proteins, whose primary role is to construct the filament itself, there are proteins associated with each fiber type whose role is to make the decisions about when, where, and how fast to assemble or disassemble the filaments. They can be assembled or disassembled like a set of building blocks.
The building blocks can be moved from one part of a cell to another quite rapidly if required. Within a few seconds, the distribution of fibers can change dramatically within a living cell. This phenomenon occurs, for example, if a moving cell encounters an obstacle and changes direction.
Under certain conditions, microtubule proteins can be added to one end of a microtubule at the same time that they are removed from the opposite end. The result is that the microtubule appears to “move” toward the growing end. This process has been likened to the movement of the tread on a Caterpillar-type tractor.
The first cell function to be definitely attributed to the cell’s cytoskeletonwas cell division. This process is an elaborate, highly choreographed minuet in which the microtubules move two sets of chromosomes (the structures that carry the genetic information) apart from one another and the micro filaments squeeze the cell in two between the chromosome sets.
The result is two cells where there was only one, and each has a complete set of genes. How are the activities of the two kinds of fibers coordinated in both time and space, so that this elegant process occurs properly? It is now understood that changing concentrations of calcium atoms inside cells help to coordinate the actions of the several fiber types.
Fluorescent antibody staining has revealed that the chromosome-moving microtubules change in length during movement and that they generate a force sufficient to drag the chromosomes through the cell.
