Tag: bioassays

For cells, too, 2-D is not the same as 3-D

Cell culture experiments to screen for compounds that can inhibit cell migration – and potentially metastasis of cancer cells – are typically carried out in a 2-D environment, but researchers at Johns Hopkins University and the University of Washington suggest that results from such experiments may be, at best, misleading. The team has shown that the way in which cells move in a 3-D environment, such as the human body, is different both qualitatively and quantitatively from the way they move in a 2-D environment such as a culture dish. When cells are grown in 2-D, they develop broad fan-shaped protrusions called lamella along their leading edge which help them to move forward. Macromolecular assemblies known as focal adhesions which can last for up to several minutes are also formed. These focal adhesions mediate cell signalling, force transduction and adhesion. In 3-D, the cells take on a more spindle-like appearance, with two pointed protrusions at opposite ends and focal adhesions – if they form at all – are so small and short-lived that they cannot be resolved by microscopy. The authors suggest that the shape and movement of cells in 2-D culture experiments are artifacts of the environment and could produce misleading results in studies to test the effects of drugs on cell motility. This may explain why positive results from cell culture experiments do not always translate into efficacy in animal models.

Even in cell culture systems designed to more closely mimic a 3-D environment, the cells may be only partially embedded in a matrix and produce misleading results. Using live-cell microscopy, the team showed that, when cells are fully embedded in a 3-D matrix, focal adhesion proteins do not form aggregates, but are distributed throughout the cytoplasm. The focal adhesion proteins still modulate cell motility, but not in the same way as in a 2-D environment. Because loss of adhesion and increased motility are hallmarks of cancer cells, it is important to understand cell motility under physiological conditions and to use culture techniques that most closely mimic this.

The study is published in Nature Cell Biology.

Role for Green Fluorescent Protein in Electron Transfer

Green fluorescent protein (GFP), originally isolated from the jellyfish, Aequorea victoria, fluoresces green when exposed to blue light. Because the protein is easily detected, its gene has become widely used as a reporter gene in biological experiments. The gene is fused with the gene for the protein of interest and, when this gene is switched on, GFP is also produced by the cell and production of the target protein can be monitored by measuring the green fluorescence.

Aequorea victoriaAlthough the protein has proved so useful to biologists, the natural function of GFP is not well understood. Writing in the journal Nature Chemical Biology, scientists from the Shemiakin-Ovchinnikov Institute have now shown that, when exposed to light, GFP can act as an electron donor. It was already known that under low (<1%) oxygen conditions, GFP undergoes a photoconversion into a red fluorescent state, a phenomenon the authors call ‘redding’. Although the GFP red state is stable for many hours in the absence of oxygen, its structure and mechanism of formation were not understood. The authors found that redding could also be brought about by treatment with electron acceptors under both aerobic and anaerobic conditions. In the presence of electron acceptors such as nicotinamide adenine dinucleotide (NAD) or cytochrome c, the green glow reddened – the same effect as seen under low-oxygen conditions –suggesting that transfer of electrons could be changing the structure of the chromophore. The authors were also able to demonstrate redding in living cells, although there was a high variability of redding rate between individual cells within a number of different cell lines. Rather than just passive light absorbers/emitters, the study points to a new role for GFPs in light-induced electron transfer – a role that should be kept in mind when designing experiments using these proteins.