Cell Biology Reveals the Size Shape and Location of Cell Components

Actual observation of cells awaited development of the first, crude microscopes in the early 1600s. A compound microscope, the most useful type of light microscope, has two lenses. The total magnifying power is the product of the magnification by each lens. As better lenses were invented, the magnifying power and the ability to distinguish closely spaced objects, the resolution, increased greatly. Modern compound microscopes magnify the view about a thousandfold, so that a bacterium 1 micrometer (1 ^m) long looks like it's a millimeter long. Objects about 0.2 ^m apart can be discerned in these instruments.

Microscopy is most powerful when particular components of the cell are stained or labeled specifically, enabling them to be easily seen and located within the cell. A simple example is staining with dyes that bind specifically to DNA to visualize the chromosomes. Specific proteins can be detected by harnessing the binding specificity of antibodies, the proteins whose normal task is to help defend animals against infection and foreign substances. In general, each type of antibody binds to one protein or large polysaccha-ride and no other (Chapter 3). Purified antibodies can be chemically linked to a fluorescent molecule, which permits their detection in a special fluorescence microscope (Chapter 5). If a cell or tissue is treated with a detergent that partially dissolves cell membranes, fluorescent antibodies can drift in and bind to the specific protein they recognize. When the sample is viewed in the microscope, the bound fluorescent antibodies identify the location of the target protein (see Figure 1-15).

Better still is pinpointing proteins in living cells with intact membranes. One way of doing this is to introduce an engineered gene that codes for a hybrid protein: part of the hybrid protein is the cellular protein of interest; the other part is a protein that fluoresces when struck by ultraviolet light. A common fluorescent protein used for this purpose is green fluorescent protein (GFP), a natural protein that makes some jellyfish colorful and fluorescent. GFP "tagging" could reveal, for instance, that a particular protein is first made on the endoplasmic reticulum and then is moved by the cell into the lysosomes. In this case, first the endoplasmic reticulum and later the lysosomes would glow in the dark.

▲ FIGURE 1-21 During the later stages of mitosis, microtubules (red) pull the replicated chromosomes (black) toward the ends of a dividing cell. This plant cell is stained with a DNA-binding dye (ethidium) to reveal chromosomes and with fluorescent-tagged antibodies specific for tubulin to reveal microtubules. At this stage in mitosis, the two copies of each replicated chromosome (called chromatids) have separated and are moving away from each other. [Courtesy of Andrew Bajer.]

Chromosomes are visible in the light microscope only during mitosis, when they become highly condensed. The extraordinary behavior of chromosomes during mitosis first was discovered using the improved compound microscopes of the late 1800s. About halfway through mitosis, the replicated chromosomes begin to move apart. Microtubules, one of the three types of cytoskeletal filaments, participate in this movement of chromosomes during mitosis. Fluorescent tagging of tubulin, the protein subunit that polymerizes to form microtubules, reveals structural details of cell division that otherwise could not be seen and allows observation of chromosome movement (Figure 1-21).

Electron microscopes use a focused beam of electrons instead of a beam of light. In transmission electron microscopy, specimens are cut into very thin sections and placed under a high vacuum, precluding examination of living cells. The resolution of transmission electron microscopes, about 0.1 nm, permits fine structural details to be distinguished, and their powerful magnification would make a 1-^m-long bacterial cell look like a soccer ball. Most of the organelles in eukary-otic cells and the double-layered structure of the plasma membrane were first observed with electron microscopes (Chapter 5). With new specialized electron microscopy techniques, three-dimensional models of organelles and large protein complexes can be constructed from multiple images. But to obtain a more detailed look at the individual macro-molecules within cells, we must turn to techniques within the purview of biochemistry.

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