Читать книгу Cell Biology - Stephen R. Bolsover - Страница 29

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.

Figure 1.14. Super‐resolution microscopy. Fluorescence image of the surface of a eukaryotic nucleus by standard confocal microscopy and by STED.

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

Figure 1.15. (a) Fluorescence microscope image of two‐cell mouse embryo expressing GFP and RFP chimeras. Two images were acquired in succession using optical configurations suitable for GFP and RFP respectively. The two images were then combined in a computer. (b) Image (a) combined with a phase contrast transmitted light image; the clear envelope around the embryo is the zona pellucida.

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.