Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 79
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In a DIC image, cells appear inpseudorelief. Because only a narrow in-focus region is imaged, aDIC image is an optical slice through the object. [Courtesy ofin thick specimens (e.g., an intact Caenorhabditis elegansroundworm) can be observed in a series of such optical sections, and the three-dimensional structure of the object can bereconstructed by combining the individual DIC images.light up when illuminated by the exciting wavelength, a technique called immunfluorescence microscopy (Figure 5-45).Staining a specimen with two or three dyes that fluoresce atdifferent wavelengths allows multiple proteins to be localizedwithin a cell (see Figure 5-33).N.
Watson and J. Evans.]Fluorescence Microscopy Can Localizeand Quantify Specific Moleculesin Fixed and Live CellsLaminapropiaLateralmembraneBrushborderPerhaps the most versatile and powerful technique for localizing proteins within a cell by light microscopy is fluorescentstaining of cells and observation by fluorescence microscopy.A chemical is said to be fluorescent if it absorbs light at onewavelength (the excitation wavelength) and emits light (fluoresces) at a specific and longer wavelength. Most fluorescentdyes, or flurochromes, emit visible light, but some (such asCy5 and Cy7) emit infrared light.
In modern fluorescence microscopes, only fluorescent light emitted by the sample isused to form an image; light of the exciting wavelength induces the fluorescence but is then not allowed to pass the filters placed between the objective lens and the eye or camera(see Figure 5-42a, c).20 mImmunological Detection of Specific Proteins in Fixed CellsThe common chemical dyes just mentioned stain nucleicacids or broad classes of proteins. However, investigatorsoften want to detect the presence and location of specificproteins.
A widely used method for this purpose employsspecific antibodies covalently linked to flurochromes. Commonly used flurochromes include rhodamine and Texas red,which emit red light; Cy3, which emits orange light; and fluorescein, which emits green light. These flurochromes can bechemically coupled to purified antibodies specific for almostany desired macromolecule. When a flurochrome–antibodycomplex is added to a permeabilized cell or tissue section, thecomplex will bind to the corresponding antigens, which then▲ EXPERIMENTAL FIGURE 5-45 One or more specificproteins can be localized in fixed tissue sections byimmunofluorescence microscopy.
A section of the rat intestinalwall was stained with Evans blue, which generates a nonspecificred fluorescence, and with a yellow green–fluorescing antibodyspecific for GLUT2, a glucose transport protein. As evident fromthis fluorescence micrograph, GLUT2 is present in the basal andlateral sides of the intestinal cells but is absent from the brushborder, composed of closely packed microvilli on the apicalsurface facing the intestinal lumen.
Capillaries run through thelamina propria, a loose connective tissue beneath the epitheliallayer. [See B. Thorens et al., 1990, Am. J. Physio. 259:C279; courtesy ofB. Thorens.]188CHAPTER 5 • Biomembranes and Cell Architectureused to visualize the expression and distribution of specificproteins that mediate cell–cell adhesion (see Figure 6-8).In some cases, a purified protein chemically linked to afluorescent dye can be microinjected into cells and followedby fluorescence microscopy. For example, findings fromcareful biochemical studies have established that purifiedactin “tagged” with a flurochrome is indistinguishable infunction from its normal counterpart.
When the tagged protein is microinjected into a cultured cell, the endogenous cellular and injected tagged actin monomers copolymerize intonormal long actin fibers. This technique can also be used tostudy individual microtubules within a cell.Determination of Intracellular Ca2 and H Levels withIon-Sensitive Fluoresent Dyes Flurochromes whose fluo-▲ EXPERIMENTAL FIGURE 5-46 Expression of fluorescentproteins in early and late mouse embryos is detected byemitted blue and yellow light. The genes encoding bluefluorescent protein (ECFP) and yellow fluorescent protein (EYFP)were introduced into mouse embryonic stem cells, which thenwere grown into early-stage embryos (top) and late-stageembryos (bottom).
These bright-field (left) and fluorescence (right)micrographs reveal that all but four of the early-stage embryosdisplay a blue or yellow fluorescence, indicating expression ofthe introduced ECFP and EYFP genes. Of the two late-stageembryos shown, one expressed the ECFP gene (left) and oneexpressed the EYFP gene (right). [From A.-K.
Hadjantonakis et al.,rescence depends on the concentration of Ca2 or H haveproved useful in measuring the concentration of these ionswithin live cells. As discussed in later chapters, intracellularCa2 and H concentrations have pronounced effects onmany cellular processes. For instance, many hormones orother stimuli cause a rise in cytosolic Ca2 from the restinglevel of about 107 M to 106 M, which induces various cellular responses including the contraction of muscle.The fluorescent dye fura-2, which is sensitive to Ca2,contains five carboxylate groups that form ester linkageswith ethanol. The resulting fura-2 ester is lipophilic and can2002, BMC Biotechnol.
2:11.]Expression of Fluorescent Proteins in Live Cells A naturally fluorescent protein found in the jellyfish Aequorea victoria can be exploited to visualize live cells and specificproteins within them. This 238-residue protein, called greenfluorescent protein (GFP), contains a serine, tyrosine, andglycine sequence whose side chains have spontaneouslycyclized to form a green-fluorescing chromophore. With theuse of recombinant DNA techniques discussed in Chapter 9,the GFP gene can be introduced into living cultured cells orinto specific cells of an entire animal. Cells containing the introduced gene will express GFP and thus emit a green fluorescence when irradiated; this GFP fluorescence can be usedto localize the cells within a tissue. Figure 5-46 illustrates theresults of this approach, in which a variant of GFP that emitsblue fluorescence was used.In a particularly useful application of GFP, a cellular protein of interest is “tagged” with GFP to localize it.
In this technique, the gene for GFP is fused to the gene for a particularcellular protein, producing a recombinant DNA encoding onelong chimeric protein that contains the entirety of both proteins. Cells in which this recombinant DNA has been introduced will synthesize the chimeric protein whose greenfluorescence reveals the subcellular location of the protein ofinterest. This GFP-tagging technique, for example, has been▲ EXPERIMENTAL FIGURE 5-47 Fura-2, a Ca2+-sensitiveflurochrome, can be used to monitor the relative cytosolicCa2+ concentrations in different regions of live cells. (Left) In amoving leukocyte, a Ca2+ gradient is established.
The highestlevels (green) are at the rear of the cell, where corticalcontractions take place, and the lowest levels (blue) are at thecell front, where actin undergoes polymerization. (Right) When apipette filled with chemotactic molecules placed to the side ofthe cell induces the cell to turn, the Ca2+ concentrationmomentarily increases throughout the cytoplasm and a newgradient is established. The gradient is oriented such that theregion of lowest Ca2+ (blue) lies in the direction that the cell willturn, whereas a region of high Ca2+ (yellow) always forms at thesite that will become the rear of the cell. [From R.
A. Brundage etal., 1991, Science 254:703; courtesy of F. Fay.]5.6 • Visualizing Cell Architecturediffuse from the medium across the plasma membrane intocells. Within the cytosol, esterases hydrolyze fura-2 ester,yielding fura-2, whose free carboxylate groups render themolecule nonlipophilic, and so it cannot cross cellular membranes and remains in the cytosol. Inside cells, each fura-2molecule can bind a single Ca2 ion but no other cellularcation. This binding, which is proportional to the cytosolicCa2 concentration over a certain range, increases the fluorescence of fura-2 at one particular wavelength. At a secondwavelength, the fluorescence of fura-2 is the same whether ornot Ca2 is bound and provides a measure of the totalamount of fura-2 in a region of the cell.
By examining cellscontinuously in the fluorescence microscope and measuringrapid changes in the ratio of fura-2 fluorescence at these twowavelengths, one can quantify rapid changes in the fractionof fura-2 that has a bound Ca2 ion and thus in the concentration of cytosolic Ca2 (Figure 5-47).Similarly to fura-2, fluorescent dyes (e.g., SNARF-1) thatare sensitive to the H concentration can be used to monitor the cytosolic pH of living cells.Confocal Scanning and DeconvolutionMicroscopy Provide Sharp Imagesof Three-Dimensional ObjectsConventional fluorescence microscopy has two major limitations.
First, the physical process of cutting a section destroys material, and so in consecutive (serial) sectioning a(a) Conventional fluorescence microscopy189small part of a cell’s structure is lost. Second, the fluorescentlight emitted by a sample comes from molecules above andbelow the plane of focus; thus the observer sees a blurredimage caused by the superposition of fluorescent imagesfrom molecules at many depths in the cell.
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