Microscopy Techniques

In standard light (bright-field) microscropy, a beam of light from a source (usually placed below the specimen) is focused onto a specimen, passes through the specimen, is focused by a second series of lenses, and is then observed by eye or photographed. Samples are usually fixed to denature the proteins in the specimen, sectioned into thin slices (if needed), attached to a solid substrate (the slide), and stained using any of a series of chemicals that specifically react with cellular components such as DNA or protein. It is hoped that these treatments do not significantly alter the subcellular structures or their organization. Because of the small cell size of Saccharomyces, bright-field microscropy is very uninformative and researchers have developed other methods for visualizing subcellular structures.

FLUORESCENCE MICROSCROPY, IMMUNOFLUORESCENCE, AND GFP

Fluorescence microscropy allows the researcher to localize a specific protein to its subcellular site by providing a mechanism for a fluorescent dye to specifically bind to a particular protein or subcellular component. A fluorescent molecule is one that becomes activated by absorbing light of one wavelength (the excitation wavelength) and then returns to the resting state by emitting light at a longer wavelength (fluorescence wavelength) still in the visual range. The fluorescent molecule is visualized in the specimen using a fluorescence microscope that is designed to shine light of the excitation wavelength on the specimen (usually from above) and to allow one to observe the emitted light (again from above). The emitted light is passed through filters that block all except light of the fluorescent wavelength before it is viewed or photographed.

The most commonly used fluorescent dyes are rhodamine (which emits light in the red range) and fluorescein (which emits light in the green range). In immunofluorescence the dye is covalently conjugated to an antibody specific to the protein of interest. Cells are fixed and made permeable to the antibody. The sample is then treated with the fluorescent dye-conjugated antibody and the antibody then binds to its target antigen/protein. The position of the antibody is visualized using a fluorescence microscope. Microscopes can be fitted with several different sets of filter pairs thereby allowing one to observe, in a single cell, the location of two or even three antibodies each conjugated to a different dye and emitting light of a different fluorescent wavelength. In this way one can compare the localization of two or more different proteins within a single cell. For further information, the Handbook of Fluorescent Probes and Research Chemicals from Molecular Probes, a supplier of such reagents, is an excellent resource (http://www.probes.com).

A number of variations on the immunofluorescence theme have been developed for use in Saccharomyces and other cells. The fluorescent dye can be conjugated to a

Figure 2.2 Fluorescence in situ hybridization—FISH. Shown is the result of FISH analysis of centromere localization in the nuclei of diploid cells arrested in G1 of the cell cycle (using a temperature sensitive mutation of cdc4). Chromosomal DNA was stained with propidium iodide (grey) to show the outlines of the nucleus. Oligonucleotide probes were made from sequences tightly linked to CEN1, CEN4, and CEN16 and thus should hybridize to six chromosomal sites in these diploid cells. Each probe is tagged with an antigenic compound called digoxigenin and the location of the tagged oligonucleotide is visualized by immunofluorescence using dye-conjugated antibodies. The photograph shows that the centromeres are clustered and, using immunofluorescence to localize the position of the microtubule attachment to the nuclear envelope (data not shown), the authors demonstrate that the clustering is in the region closest to the spindle pole body. Taken from Guacci et at. (1997). Reproduced by permission of the American Society for Cell Biology molecule other than an antibody that specifically interacts with a particular cellular component. For example, phalloidin is a small cyclic peptide derived from the death cap fungus Amanita phalloides. It specifically binds to polymerized actin microfilaments. Rhodamine-conjugated phalloidin will enter permeablized cells and bind to the actin cytoskeleton thereby allowing this complex meshwork to be visualized by fluorescence microscropy (see Chapter 3, Figure 3.9). Fluorescence in situ hybridization, or FISH, uses nucleic acid hybridization to bind the fluorescent dye to specific DNA sequences in chromosomes. The fluorescent dye is conjugated to a DNA oligonucleotide probe, introduced into cells, and allowed to hybridize to the complementary site(s) on the chromosomes. Examination by fluorescence microscropy allows these positions to be visualized. FISH has been used to demonstrate the location of chromosomal telomeres in interphase Saccharomyces nuclei and to study chromosome separation during cell division (Figure 2.2). Finally, Walker (1998) lists several cytofluorescent dyes for yeast microscropy that interact with specific cellular molecules. One example is calcofluor white that binds to the chitin found at the site of bud scars. Another is DAPI (4,6-diamidino-2-phenylindole) that binds specifically to DNA and can be used to visualize the nucleus and even mitochondrial DNA (Figure 2.3).

FISH

Figure 2.2 Fluorescence in situ hybridization—FISH. Shown is the result of FISH analysis of centromere localization in the nuclei of diploid cells arrested in G1 of the cell cycle (using a temperature sensitive mutation of cdc4). Chromosomal DNA was stained with propidium iodide (grey) to show the outlines of the nucleus. Oligonucleotide probes were made from sequences tightly linked to CEN1, CEN4, and CEN16 and thus should hybridize to six chromosomal sites in these diploid cells. Each probe is tagged with an antigenic compound called digoxigenin and the location of the tagged oligonucleotide is visualized by immunofluorescence using dye-conjugated antibodies. The photograph shows that the centromeres are clustered and, using immunofluorescence to localize the position of the microtubule attachment to the nuclear envelope (data not shown), the authors demonstrate that the clustering is in the region closest to the spindle pole body. Taken from Guacci et at. (1997). Reproduced by permission of the American Society for Cell Biology

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Figure 2.3 Visualization of mitochondria by different methods. Cells were grown to the midlog phase on a rich medium with galactose as the carbon source. Panel (a) shows the mitochondria stained by immunofluorescence. In panel (b) Alexa-phalloidin was used to detect polymerized actin. Panel (c) shows cells stained with the DNA binding dye DAPI. The large bright spot in the mother cell in panel (c) is the nucleus and the less brightly staining spots are the mitochondria, indicating that several copies of mtDNA can be found. Note that the mitochondria accumulate in the mother cell distal to the site of bud emergence and appear to lie along the length of the actin cables. Taken from Yang et al., 1999. Reproduced with permission from Elsevier Science

Figure 2.3 Visualization of mitochondria by different methods. Cells were grown to the midlog phase on a rich medium with galactose as the carbon source. Panel (a) shows the mitochondria stained by immunofluorescence. In panel (b) Alexa-phalloidin was used to detect polymerized actin. Panel (c) shows cells stained with the DNA binding dye DAPI. The large bright spot in the mother cell in panel (c) is the nucleus and the less brightly staining spots are the mitochondria, indicating that several copies of mtDNA can be found. Note that the mitochondria accumulate in the mother cell distal to the site of bud emergence and appear to lie along the length of the actin cables. Taken from Yang et al., 1999. Reproduced with permission from Elsevier Science

Perhaps the most powerful advance in fluorescence microscropy came with the development of GFP, by Martin Chalfie and coworkers (Chalfie et al., 1994). GFP is responsible for the bioluminescence exhibited by the jellyfish Aequorea victoria. What makes GFP such a valuable tool for the study of cell biology in the age of recombinant DNA technology? GFP fluorescence occurs in vivo in the living cell simply by shining light of the correct excitation wavelength on the cells. Since there is no fixation or staining necessary, it is believed that a more accurate view of the in vivo situation is obtained. The amino acid sequence and structure of GFP is solely responsible for its fluorescent activity, and no exogenously added cofactors or exogenously produced modifications are required. In fact, the GFP chromophore is synthesized autocatalytically by a series of intramolecular reactions involving the side-chains of several amino acid residues in the GFP sequence. Therefore, whether expressed in the native organism Aequorea victoria or heterologously in the cells of any other species, GFP undergoes this autocatalytic reaction to produce the chromophore and emits light at its characteristic fluorescent wavelength. GFP is nontoxic and thus can be expressed in all cell types. Most importantly, GFP fusions are usually functional and results obtained with these fusions are therefore biologically relevant.

To use GFP to study the localization of any protein, one constructs an in-frame fusion of the GFP ORF to that encoding the protein of interest. Plasmid vectors containing the full ORF of the GFP gene are available (Niedenthal et al., 1996). These contain multiple cloning sites positioned so as to allow the gene of interest to be inserted either upstream or downstream of the GFP. For use in Saccharomyces, a high-level constitutive promoter, such as the ADH1 promoter, is usually included. Using PCR-based methods, the sequence of the GFP ORF can be inserted at any position in any gene of interest. The GFP-fusion construction is then introduced into cells and transformants will produce a GFP-fusion protein whose subcellular localization can be visualized by fluorescence microscropy. In Saccharomyces time-lapse photography of cells expressing a GFP-fusion protein has been used to

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  • kaisa
    Is there any subcellular structures in saccharomyces cerevisiae?
    7 years ago
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    Is there any subcellular structure of Saccharomyces cerevisiae?
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