It was half a century ago, in 1962, when the Japanese marine biologist and organic chemist Osamu Shimomura isolated green fluorescent protein (GFP) from the jellyfish Aequore victoria. About 30 years later, with the advent of molecular genetic engineering, the GFP gene was cloned and introduced into E. coli bacteria, allowing for the production of the protein. This achievement paved the way for a new discipline which combines molecular biology with microscopy. Today there are countless methods in which scientists exploit fluorescent proteins in order to monitor different cellular and physiological processes. In the following video we will be introduced to several techniques that use fluorescent protein and non-protein markers.
This video was produced by Molecular Probes and depicts some of its products.
The video presented several methods for studying different biological processes using a common theme – the conjugation, by genetic engineering, of a fluorescent protein (reporter gene) with a protein of interest, introduction of the new gene into a cell and studying it by microscopy. The video does not explain in detail the scientific context of these experiments, so I will try to provide several possible explanations.
The biological activity of anti-cancer drugs: in this case the cytoskeleton was labeled green, the cell nucleus blue and the mitochondria (probably) red. These are probably cancer cells that are slowly dying after being treated with the drug. Notice the changes occurring in the cytoskeleton (green) and the mitochondria (red).
Quantum dots (Qdot®): fluorescent proteins have several disadvantages, including the fact that it usually takes a couple of days for them to accumulate to a detectable level. This delay might hinder the study of rapid or sensitive processes. In order to bypass this problem, there are different membrane-penetrating markers. One of those is known as Qdot®, which is a nanocrystal linked to a fluorescent marker and an antibody that recognizes our protein of interest. It is possible to use several different antibodies at a time, each associated with a different color marker. This way we can study several proteins in one sample. The video shows how Qdot® crystals penetrate into a cell.
Cell division (mitosis): cell division occurs in several stages, each characterized by the activity of different proteins. In the video, each fluorescently-labeled protein marks a different stage of cell division, so the scientists were able to examine the passage between the stages by monitoring the color of the cell.
Changes to the cytoskeleton: in order to examine intracellular processes that involve changes to the cytoskeleton (e.g. cell motility, morphological changes and cell division), scientists label the protein components of the cytoskeleton, called actin and microtubules, with different fluorescent markers (red and green, respectively). In the video we can see cell division. Another group of proteins that is often labeled are histones – DNA-binding proteins. During mitosis the DNA is coiled around histones so that it forms compact "packages". This packaging of DNA implies that the cell is preparing for division. When mitosis does not proceed as planned the cellular localization of the microtubules and histones would indicate this.
Acidity indicators for immune reaction: when phagocytes phagocytose ("swallow") a bacteria or a virus they transport it into an endosome – an organelle in which the acidity level gradually increases until the bacteria or virus become inactive (concomitantly with their breakdown by specialized enzymes). This process can be monitored using fluorescent acidity (pH) indicators (note the red dots in the cell).
Organelle movement within the cell: by labeling a protein that is found specifically in our organelle of interest, we can visualize the cellular localization of the organelle, and in the case of the mitochondrion we can also monitor its movement. By labeling different organelles in parallel to a protein of interest, we can determine the cellular localization of our protein.
Cell membrane structure: by labeling common membrane-associated proteins, one can monitor changes in the structure of the cell membrane.
Changes in intracellular calcium levels: calcium is an important mediator in many biological processes. Changes in calcium concentrations are indicative of various cellular activities. Some markers fluoresce (emit fluorescence) upon binding free calcium, while others change their color. These markers are not necessarily proteins. The video shows how such an indicator allows the scientists to estimate the calcium concentration inside the cell. Sometimes several such indicators would be used, each most efficient at a different calcium concentration, each fluorescing in a different color.
In summary, in this video we were introduced to several common fluorescent labeling techniques and usages in cellular biology. There are many more such markers and uses that were not discussed here, and what you saw was only the tip of the iceberg. These fluorescent markers, together with advanced microscopic techniques, allow us to investigate not only processes in cells or tissues, but also in whole, live animals.