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Figure 2.4 Green fluorescent protein fusions for visualization of living cells. The photo shows a time-course of Migl repressor exit from the nucleus. These cells are expressing a fully functional Miglp GFP fusion protein. Following growth on glucose, the cells are harvested and placed in medium lacking glucose. The first panel shows the cells 30 seconds after glucose removal. Miglp-GFP is seen localized to a discrete subcellular site that, by DAPI staining, is shown to be the nucleus (data not shown). Very little fluorescence is observed in the cytoplasm. The same cells are photographed every 30 seconds for 1.5 minutes during which time the fluorescence can be seen to leave the nucleus and accumulate in the cytoplasm. The large poorly fluorescent region seen in the cytoplasm is the vacuole (based on Nomarski optics). Taken from De Vit et al. (1997). Reproduced by permission of John Wiley & Sons Publishers

Figure 2.4 Green fluorescent protein fusions for visualization of living cells. The photo shows a time-course of Migl repressor exit from the nucleus. These cells are expressing a fully functional Miglp GFP fusion protein. Following growth on glucose, the cells are harvested and placed in medium lacking glucose. The first panel shows the cells 30 seconds after glucose removal. Miglp-GFP is seen localized to a discrete subcellular site that, by DAPI staining, is shown to be the nucleus (data not shown). Very little fluorescence is observed in the cytoplasm. The same cells are photographed every 30 seconds for 1.5 minutes during which time the fluorescence can be seen to leave the nucleus and accumulate in the cytoplasm. The large poorly fluorescent region seen in the cytoplasm is the vacuole (based on Nomarski optics). Taken from De Vit et al. (1997). Reproduced by permission of John Wiley & Sons Publishers demonstrate changes in subcellular location of the fusion protein resulting from changes in growth conditions, such as carbon source or temperature. An example of this type of analysis is shown in Figure 2.4, which demonstrates the time-course of Miglp GFP-fusion protein exit from the nucleus after cells are shifted from a glucose-containing medium to a medium lacking glucose. To control for the possibility that fusion to GFP inactivates some or all of the functions of the protein under study, it is essential that the GFP-fusion construction be tested for its ability to complement the mutant phenotypes of a null allele of the gene of interest.

The introduction of a limited number of amino acid changes in the wild-type GFP gene has allowed for improvements in the fluorescence characteristics of GFP, such as increased emission of light or spectral resolution, and has even produced mutant products that absorb and emit light at slightly different wavelength ranges (for example, blue fluorescent protein or BFP). The coexpression of GFP- and BFP-fusion genes allows the researcher to compare the subcellular localization of these two proteins in vivo. Several different variants are now available.

CONFOCAL SCANNING MICROSCROPY

Most of the high-resolution subcellular localization studies done using fluorescence microscropy would not have been possible without the development of an improved imaging technique called confocal scanning microscropy. Fluorescence microscropy is generally done using whole cells because the embedding media used for sectioning often is fluorescent and obscures the fluorescence derived from the sample. Since eukaryotic cells, even Saccharomyces, have a thickness, the fluorescence one observes is coming not only from the molecules in the plane of focus but also from molecules above and below. The greater the thickness of the sample the greater the problem one will have in resolving specific structures.

The confocal microscope uses a laser light source to produce the excitation light beam that can be focused into a narrow focal plane allowing only a thin optical section of the sample to be illuminated. The laser beam set at a specific excitation wavelength rapidly scans the sample. The position and intensity of the emitted light is recorded and the information stored for computer analysis. The results of these scans are combined to generate a composite digital image of the fluorescence from a sample. Because only information from a narrow focal plane is used this method produces a high-resolution map of the subcellular position of the fluorescent molecule.

Other forms of image analysis can also be carried out. The amount of fluorescence can be quantified by computer analysis of the digital image and used as a measure of gene expression. Depending on the laser source and the filter sets available for the particular microscope, one can create images of the fluorescence produced by different antibody-dye conjugates. These images, when superimposed by the computer software system, can very accurately demonstrate whether the two antibodies are colocalized in the cell. Confocal imaging is also used to create a three-dimensional image by a method referred to as optical sectioning. The microscope stage is moved vertically in small steps thereby moving the focal plane through the cell. At each step an image is generated and these are then combined to create a single three-dimensional image that can be rotated in space on the computer screen.

NOMARSKI INTERFERENCE MICROSCOPY

Nomarski interference microscopy (sometimes called DIC) can be used to visualize live unstained cells or tissue samples. It makes use of the differences in thickness and refractive index of different parts of the cell and gives a three-dimension-like image. Light moves more slowly through material with a higher refractive index. Nomarski imaging requires a microscope equipped with special polarizing lenses and prism. A

Figure 2.5 Nomarski optics view of S. cerevisiae. Nomarski optics can be used to visualize live or fixed cells. The vacuole is by far the most predominant organelle of S. cerevisiae and is very clearly observable by this method. The multiple punctate structures in the cytoplasm surrounding the vacuole are vesicles of various types. Taken from Lang et al. (2000). Reproduced by permission of the American Society for Microbiology

Figure 2.5 Nomarski optics view of S. cerevisiae. Nomarski optics can be used to visualize live or fixed cells. The vacuole is by far the most predominant organelle of S. cerevisiae and is very clearly observable by this method. The multiple punctate structures in the cytoplasm surrounding the vacuole are vesicles of various types. Taken from Lang et al. (2000). Reproduced by permission of the American Society for Microbiology plane-polarized light beam is split into two and allowed to pass through the sample at nearly adjacent sites after which the two beams are rejoined. If the two sites differ in refractive index, then the beams of light will be out of phase when they exit the sample and when joined will interfere with each other thereby reducing the intensity of the light beam. If there is no difference between the two sites, then the intensity of light will be high. If the difference is substantial, then the beams will completely interfere and a dark region will result. When observed by Nomarski optics, the most prominent organelle in Saccharomyces is the vacuole (Figure 2.5).

ELECTRON MICROSCROPY

The electron microscope uses an electron beam instead of a light beam to visualize the cell and its components. The transmission electron microscope passes the electron beam through the sample and to do so the sample must be sliced into very thin sections using special tools and an embedding material. In order to see cellular structures the sample must be stained with an electron dense agent such as osmium. The electron beam is focused with magnetic lenses and this is projected onto a viewing screen or photographed.

The major problem with EM analysis is sample preparation. EM is not often used for Saccharomyces but it has proved to be very useful for some studies, particularly on the cytoskeleton. The development of antibody localization techniques for EM work has encouraged more researchers to attempt this difficult method of analysis. The method is referred to as immuno-gold localization because it uses antibodies that are bound to gold particles via Staphylococcus aureus Protein A. The surface of the gold particle (about 4nm in diameter) is covered with Protein A, which also binds very tightly to the constant region of IgG antibodies. The EM sectioned sample is treated with the antibody-gold complex under conditions that allow binding to the specific antigen. The gold particles appear as black dots in the transmission electron microscope and their subcellular location can be determined in osmium stained samples. Figure 2.6 shows an example of the immuno-gold labeling method to localize Ste2p, the a-factor receptor to endocytic vesicles forming at the plasma membrane. Double label methods are now available for the colocalization of proteins at the EM level.

The surface of a Saccharomyces cell can be studied using scanning electron microscropy (SEM). Cells are fixed, allowed to adhere to a solid support, and coated with a heavy metal film, such as platinum. The coating process is carried out in a vacuum chamber and the vaporized metal is allowed to deposit on the sample while the sample is rotated for even coating. The electron beam scans the sample and excited secondary electrons are released and visualized on a monitor. This method gives a three-dimensional appearance to the sample.

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