Monday, December 08, 2003

Biology Lab Primer - Of glowing green-eyed flies, calcium, and brains

So I said I'd talk about how my research on fruit flies has a practical application. The eye of a fruit fly is very different from mammalian eyes. They are basically arrays of tiny motion detectors, designed to sense movement of a predator rather than to discern the intricacies of color and form like human eyes. The electrical responses of fruit flies are 100 times faster than those of human eyes. That's what makes them so darn hard to swat with a fly swatter.

While insect vision is interesting in itself (especially if you want to be able to swat them effectively), what I'm really interested in is how nerve cells or neurons function in all animals. The photoreceptors in the fly eye are actually modified neurons. They receive an input (light) and transmit an output (histamine) at their synapses. Flies are ideal model systems to use to study neurons because they have simple nervous systems, they are easy and inexpensive to maintain, there are many mutant flies that can be tested, and you can do experiments on them that you couldn't do on humans. Yes, ripping the eyes off a human being and jabbing electrodes into the freshly dissociated eyes IS unethical and illegal in case you were wondering.

The fly photoreceptor cell signals in a way that is similar to certain types of neurons in the mammalian brain. This pathway contains a receptor, the protein rhodopsin (also found in mammalian eyes) in this case, on the surface of the cell that responds to an external stimulus, light. Once light activates rhodopsin, rhodopsin then turns on a whole cascade of other signaling proteins. The proteins in the signaling cascade had different functions. Some turn on other proteins, some turn others off. Some breakdown certain biological compounds, while other synthesize new compounds. Some proteins undergo conformational changes that allow them to transport other molecules into or out of the cell. These proteins are called ion channels. The end result of light stimulating rhodopsin results in ion channels opening and allowing high concentrations of calcium from outside to rush into the cell. High amounts of calcium are eventually toxic to cells so it is then pumped out of the cell at the end of the signaling event.

But high calcium inside of cells is needed for other signaling events that help make vision and other neuronal processes more efficient and dynamic. Adaptation, which occurs when you enter a darkened room, is a calcium-dependent process. Memory is also calcium dependent. And more and more research is showing that its not just the amount of calcium that's important for neuronal signaling, but also the location of the calcium influx. Localized calcium influxes (also called calcium microdomains) have been shown to occur in the inputs and outputs of neurons. There are billions of neurons in the human brain, and each neuron can have thousands of inputs and outputs. Now combine that with localized calcium signals and the dynamic range and computational power of the brain is enormous. Memories are probably stored by the coding of various inputs and outputs along with the localization of calcium signals.

Proteins in the inputs and outputs of neurons are organized into little signaling complexes so that they can respond to local increases in calcium. In fly photoreceptors, there are scaffolding proteins that bind together ion channels and calcium-dependent signaling proteins. If you disrupt the complex by mutations in the scaffolding protein, the ion channels still function and calcium flows into the cell, but the calcium-dependent proteins don't function properly. We think this is because they need to be close to the source of calcium, the ion channels, in order to do their job right.

So my thesis project involves testing this idea by measuring the calcium microdomains in normal flies and in flies where the protein signaling complexes have been disrupted. I've made flies that have calcium "detectors" fused to different proteins in the signaling complex. The calcium "detector" is a protein that fluoresces in the presence of calcium. I can image these "detectors" inside a fly photoreceptor cell with a microscope and a camera. The brighter the cells, the more calcium is near the "detector."

Falling asleep yet? That's your biology lab lesson for today. Here's a picture of me taking images of fly photoreceptor cells in the dark.

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