Probing the Complex World of the Cell

A living cell is a world in itself, its insides consisting of highly organized networks of structures.

The nucleus is the hereditary headquarters, the source of instructions to run the cellís activities encoded in DNA. From near the nucleus, labyrinths of tubules extend, along which the cells build proteins using genetic blueprints. Lipids and sugars are added to the proteins to fashion secretions.

Throughout the cell, a vast protein scaffolding, or cytoskeleton, supports the varied cellular contents, reaching toward the periphery and providing a distinct overall shape. Finally, the cellís outer membrane is like an oily ocean with proteins afloat. Not merely a covering, the cell membrane, particularly its proteins, is a sensitive monitor of incoming information. In response, the cell membrane alerts "second messenger" molecules within the cell in a complex biochemical dance called signal transduction. The cell then responds to the outside information, perhaps by secreting, dividing or moving.

Without the use of stains and dyes, the exquisitely complex cell would appear to be little more than a collection of transparent bags within bags. Fluorescent dyes and fluorescence microscopy provide the contrast necessary for researchers to view the workings of the cell at the molecular level, without killing the cell and thereby altering its appearance.

Fluorescence microscopy utilizes fluorescent dyes that bind to specific biochemicals in a cellñsuch as a protein girder of the cytoskeleton, or a key enzyme in a signal transduction pathway. A fluorescent dye absorbs light of one wavelength and emits light of a longer wavelength, which imparts the dyeís characteristic color.

Two commonly used fluorescent dyes are rhodamine and fluorescein. Rhodamine is excited with yellow-green light and emits deep red; fluorescein is excited with blue wavelengths and emits a yellow-green color. A fluorescence microscope is a variation of a light microscope that has two filters: one to direct the exciting wavelengths to the sample, the other to collect and transmit back the emitted light.

Molecular Probes Inc. of Eugene, Ore., has developed and marketed fluorescent dyes for 25 years. Today, its products help cell biologists observe the molecular interactions of life at the cellular level. "Our BioProbes are our biggest family of new molecular biology products. They are unique, state-of-the-art probes," says David Phelps, director of technology at the company. Molecular Probes offers standard dyes as well as new ones, such as paclitaxel (Taxol) to highlight tubulin, the cytoskeletal building block responsible for cell division.

A cell first senses an outside signal when a molecule binds a protein receptor that is part of the cell membrane. By identifying chemicals that bind to specific receptor types and hooking them to fluorescent dyes, cell biologists can then visualize receptor densities on certain cell types. This can explain physiology.

A classic example of highlighting receptors uses the snake venom bungarotoxin. It binds selectively to receptors on the muscle end of a neuromuscular junction that receive the neurotransmitter acetylcholine. Fluorescence microscopy has shown that these acetylcholine receptors cluster densely, at 20,000 or more per michrometer, at the site of the junction, but are relatively rare elsewhere in the muscle cellís membrane. Molecular Probes offers several different fluorescent dyes that can be linked to bungarotoxin.

Molecular Probes also offers a series of membrane dyes that insert into the outer lipid layer of a cell membrane and then become fluorescent. The dye styrylpyridinium, for example, is used on nerve cells. It becomes incorporated into a sending-cellís membrane as the spent versicles that ferry neurotransmitters between cells are recycled back into the membrane. Therefore, the more the sending cell fluoresces, the more active it has been. Time-lapse video-fluorescence microscopy can trace the dyeís fate further in the cell.

Some membrane proteins form channels that admit ions (charged atoms) into cells. A toxin from the dingflagellate ptychodiscus brevis, which causes "red tides," binds selectively to channels that admit sodium ions, but only when these channels are open. Molecular Probes offers a sampler kit that includes five different fluorescent tags to the redtide brevetoxin.

The company also has probes for the subsequent steps in signal transduction. Probes detect the second messenger molecule cyclic ADP ribose, which receives messages from a variety of surface receptors, and three types of protein kinases, which are enzymes within the cell that control responses to the received message.

Some incoming messages eventually make their way to the cellís nucleus, activating genes that produce specific proteins in response. Molecular Probes offers a new product, NanoOrange, which binds proteins in solution and can be used on broken apart cells to detect proteins. "This is the first dye for protein solution determination. It is extremely easy to use, with little signal variability," says Phelps.

The probe is added to the sample and heated at 95 ∞C for 10 m, after which fluorescence can be measured for up to six hours. The dye is excited at a wavelength of 485 nm and emits at 590 nm. In a solution lacking protein, it does not fluoresce. Existing methods for protein determination present problems. They either precipitate the protein, work for only ten minutes, are difficult to use, or do not work when certain reagents are present.

Existing fluorescence-based techniques can be applied to emerging areas of cell biology, such as apoptosis, or programmed cell death. Apoptosis is in contrast to necrosis, in which an injured cell swells and bursts, causing inflammation.

In the genetically orchestrated appoptosis dance of death, the cell rounds up, and its membrane undulates, forming bulges called blebs. The nuclear membrane breaks, admitting enzymes that clip chromosomes into equal-sized DNA pieces. The structures that carry out the cellís activities, called organelles, collapse in on themselves. Then the cell breaks apart, with pieces of membrane enclosing toxins, preventing inflammation. Finally, nearby cells consume the remains. So fast does apoptosis occur that researchers have had difficulty sorting out the precise sequence of events.

Cell death is a normal part of life. In the embryo apoptosis carves fingers and toes from weblike precursors. The brain and the immune system form by cells dying according to a plan. Each embryo initially has too many cellsñonly the needed ones survive apoptosis. Later in life, a peeling sunburn is apoptosis, the bodyís way of ridding itself of skin cells so damaged that the alternative could become cancerous. Apoptosis is beginning to receive attention from the medical research community because the process gone awry may lie behind cancer and autoimmune disorders.

Two approaches utilizing fluorescence can detect the chopping up of DNA in apoptosis. This process is called DNA laddering, because the uniformly sized DNA pieces align on an electrophoresis gel in a pattern resembling a ladder. Molecular Probesí SYBR Green 1 Nucleic Acid Gel Stain displays the ladder with 10-to -100-fold greater sensitivity than standard staining procedures. A variety of products can also label the ends of the DNA fragments, revealing the process at different stages. "We are working on a kit that will look at the characteristics of apoptotic DNA to form laddering patterns, and we are making probes for the enzymes diagnostic of apoptosis," says Phelps.

Nucleic acid stains can reveal a nucleusí breakdown into several blobs of color. The companyís SYTO 13 used with propidium iodide shows normal-sized green nuclei in living cells; large, red nuclei in cells undergoing necrosis; and small, green nuclear pieces in cells in the death throes of apoptosis. Another approach targets the collapsing cell membrane. SYTOX GreenFluorescent Dead Cell Stain only enters cells when the integrity of the cell membranes is shattered, as happens when blebs form. Then, nuclei of dead cells glow a bright green when excited by a 488-nm argon laser.

In life and in death, fluorescent labels are providing exciting and informative glimpses of cells in action.

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