Cell Biology. Stephen R. Bolsover
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Название: Cell Biology
Автор: Stephen R. Bolsover
Издательство: John Wiley & Sons Limited
Жанр: Биология
isbn: 9781119757788
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A series of advanced microscopy techniques start with confocal microscopy and then use clever optical approaches to dramatically improve the resolution so that objects considerably smaller than the wavelength of light are revealed. Collectively these techniques are known as super‐resolution microscopy. One such technique is Stimulated Emission Depletion Microscopy (STED), in which the excitation spot is surrounded by a doughnut of light of a different wavelength that actually de‐excites the dye. Figure 1.14 shows one use of STED. Over 100 proteins are associated with the nuclear pores that perforate the nuclear envelope (Figure 1.2). Göttfert and coworkers used an antibody that recognizes one of these proteins, called gp210, labeled with a dye that emits red light, and a second antibody, labeled with a green emitter, against the transport machinery inside the pore. Figure 1.14 shows how an uninterpretable fuzz of red and green fluorescence in the standard confocal image is resolved into beautiful images by STED, revealing how eight gp210 molecules surround the pore. Data like these contribute to our present understanding of the eightfold symmetry of the structure that braces the pore to keep it open (Figure 12.10 on page 211). Super‐resolution microscopes can resolve objects down to tens of nanometers in size and can visualize biological structures previously thought to be unresolvable using light.
Fluorescent Proteins
Instead of introducing fluorescent dyes into fixed or living cells, we can cause cells to make them. This technology began with the description in 1962 of Green Fluorescent Protein ( GFP), a protein from the jellyfish Aequorea victoria that glows green when excited with blue light. Since the original description of GFP, a family of fluorescent proteins of different colors has become available, some through artificially mutating the original GFP, some found in other organisms. Using recombinant DNA technology (Chapter 8), the gene for a fluorescent protein can be introduced into a living cell, which then makes the protein.
Source: Göttfert et al. (2013). Coaligned Dual‐Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution. Biophysical Journal, 105(1), L01 ‐L03. doi:10.1016/j.bpj.2013.05.029
Source: Images by Lia Paim and Adelaide Allais, University of Montreal.
This basic technique is useful in itself for labeling a population of cells. However a battery of more and more sophisticated techniques has been developed from this starting point. Most use the approach of fusing the gene for a fluorescent protein with the gene for another protein of interest so that the cell makes a chimera – a single protein comprising the protein of interest plus the fluorescent protein. For example, Figure 1.15 shows a fluorescence image of a mouse embryo at the two‐cell stage engineered to express a GFP chimera that concentrates at the plasma membrane together with a chimera of red fluorescent protein (RFP, from coral) and histone H2B (page 39) that concentrates in the nuclei.
In this example the two parts of the chimeras worked independently, so that the GFP and RFP simply showed where their respective partners were located. However, clever protein design has created more complex chimeras of the calcium‐binding protein calmodulin (page 115) with GFP mutants so that the fluorescence changes according to the concentration of calcium. Calcium concentrations change dramatically as cells respond to stimuli (Chapter 10) and these fluorescent calmodulin chimeras can be used to report these changes. Even more clever, if the calcium‐measuring chimera is fused to a third protein with a known specific location in the cell, then the protein can be used to report the calcium concentration in that specific location.
Source: The Nobel Foundation. Photo: U. Montan.
Many proteins are colored, but in most cases the color is generated by a prosthetic group (for example the heme group in hemoglobin (page 118) and in chlorophyll). However, in 1979 Osamu Shimomura, working at Princeton University in the USA, showed that the colored moiety in a GFP made by the jellyfish Aequorea aequorea was a reaction product of the amino acids themselves. This opened up the possibility of using the cell's own machinery to make genetically encoded labeling proteins that could be targeted to precise tissues and even specific sites within the cell. However, the suspicion was that one or more specialized enzymes in the jellyfish cells would be needed to carry out the conversion of the amino acids to the fluorophore, so that simply introducing the gfp gene would do nothing. In 1994 Martin Chalfie, working at Columbia University in New York, showed that this was not the case: the gfp gene product, on its own, converted itself into fluorescent GFP. The next leap in technology was to engineer GFP and GFP chimeras to be more than markers, and instead to be reporters of cell behavior. From 1992 onward, working at the University of California at San Diego, Roger Tsien and his lab engineered an ever‐increasing family of fluorescent proteins that are now used universally by cell biologists and drug companies to study almost all aspects of cell behavior, creating both beautiful science and beautiful images, such as Figure 1.15. Shimomura, Chalfie, and Tsien were awarded the Nobel Prize in Chemistry in 2008.
Answer to thought question: Only transmission electron microscopy reveals all the structures present in a particular volume of the cell at sufficient resolution to determine whether it is malformed. Super‐resolution microscopy has the resolution to reveal individual molecules on or within the Golgi, but only those individual molecules that the scientist chose to study are revealed, not the overall structure. Malformation of the endoplasmic reticulum and Golgi apparatus is thought to underlie one type of inherited spastic СКАЧАТЬ