by Laura Lane
Liposuction and the like can leave people thinking that all our extraneous fat serves little or no function (except to remind us of our gluttonous excesses). This conclusion might be true. However, recent findings are showing that the maligned substance plays a much more significant role in cell physiology than previously thought. Mounting evidence shows that lipids, in their various forms and shapes, act as signaling molecules, thus improving our understanding of the vast cellular communication networks. With yet another player to keep track of, we’re all the more fortunate to have access to a number of tools and techniques. From improved efficiency to advanced technology, the market can put you on the right track toward deciphering the maze of cell signaling.
The new insight into lipids stems partly from changing conceptions of gene expression. “Traditionally, focus has been given to the characterization of individual genes/proteins or individual interactions during cellular events,” according to a study published in PLoS Computational Biology1. “However, many phenotypes and behaviors … arise from characteristics of cellular networks, which represent connections between molecules in cells.”
This realization has driven the creation of not only genomics and proteomics, but also their counterparts, transcriptomics, metabolomics, lipodomics, glycomics, and a growing list of “omics.” They all seek to define the functions and interactions of the cell’s molecules. Such studies are crucial to the ultimate goal of improving medical treatments. For example, research focused on lipids has revealed that metabolic syndrome, a cluster of diseases affecting about 65 million Americans, stems from abnormalities in certain lipids that are involved in the cell signaling pathway of insulin 2.
Be they lipids, small peptides, hormones, neurotransmitters, or cytokines, signaling molecules bind to receptors located in the membrane or cytoplasm. The binding touches off a string of events that aims to translate the signal into a change in gene expression. First, the receptor responds by instigating the production of second messengers, which are molecules that transduce the signal into the cell. Kinases take it from there by catalyzing a cascade of phosphorylations. This involves facilitating the transfer of ATP’s terminal phosphate molecule to the serine, threonine, or tyrosine residue of specific proteins. The protein largely responds according to which combination of amino acid residues are phosphorylated.
Uncovering more details on cell signaling requires assessing the presence, measuring the amounts, and even visually tracking the various components. For these efforts, you can choose from more traditional radioactive probes and colorimetric dyes. Or, you may be ready for (and in need of) the advantages of fluorescent and luminescent dyes. While tackling cell signaling will never be easy, you’ll find that kits can significantly simplify the task. These include microplates that are pre-aliquoted with reagents, and beads that are prepared with the appropriate antibodies or other molecules.
Cell signaling assays typically measure the cell’s response to certain small molecules, from known signaling molecules to novel drug candidates. The substantial proliferation of second messengers makes them a good target to track, as their amounts correlate with the level of activation of the signal receptor. You can choose from a multitude of assays for studying all kinds of second messengers, such as cyclic AMP, calcium ions, cyclic GMP, diacylglycerol and inositol triphosphate, nitric oxide, and carbon monoxide. The products include those packaged for high throughput screening, homogeneous cell lysates, and live cell assessments. ELISA-type assays are certainly abundant. You’ll find beads displaying antibodies against most second messengers.
Or, you might be interested in measuring levels of ATP to examine kinases, whose activity depletes ATP levels. Firefly luciferase could be the way to go. In the presence of ATP, along with luciferin and oxygen, luciferase produces oxyluciferin and light. By providing plentiful luciferin and luciferase, the light emitted will be directly proportional to the amount of ATP present.
To study kinases, you may consider looking at the protein that is phosphorylated by a specific protein. Companies have a large selection of anti-phosphoproteins that are labeled to allow tracking. With more than 500 protein kinases estimated to exist, you may want to take advantage of fluorescent dyes. The multiple colors allow you to simultaneously screen for several different phosphoproteins. To increase sensitivity, biotinylated antibodies with strepavidin dyes can help with detection.
The vast selection is more than any one can consume at once; you can try one or all. Pile your plate high. Come back for seconds. You’ll go the distance with cell signaling—all without gaining an extra pound.
References:
1 Y Qi and H Ge, “Modularity and Dynamics of Cellular Networks," PLoS Comput Biol, 2(12):e174, December 29, 2006.
2 NB Ruderman and AK Saha, "Metabolic Syndrome: Adenosine Monophosphate-activated Protein Kinase and Malonyl Coenzyme A," Obesity, 14:25S-33S, 2006.
