How cells customize compounds
24 July 2006 (Volume 1 Issue 7)
New laser-based imaging technology resolves a protein trafficking debate
Figure 1: An electron micrograph of a section through the Golgi apparatus of a higher plant. The cis side is the entrance, and the trans side, the exit.
enlarge imageA research team from RIKEN has developed and used high-speed, sensitive, live-imaging technology to help resolve a debate over how cells process the biochemical compounds they make, transport and secrete.
The work is significant because the preparation and delivery of such compounds is fundamental to the operation and communication of all cells and organisms. In addition, the new imaging technology the group developed can be applied to many other areas of interest such as tracking drug delivery and observing the invasion of cells by viruses.
Biologically active compounds in cells are typically constructed around a protein core that is assembled in a cluster of membranes called the endoplasmic reticulum. From there, the compounds pass into a second set of membranes known as the Golgi apparatus where the protein core is modified and customized for action by the addition of chemical groups, such as sugars and phosphates.
In higher organisms the Golgi apparatus resembles a stack of flattened bladders called cisternae (Fig. 1). Proteins from the endoplasmic reticulum enter at one end of the stack, the cis end, and move through the Golgi apparatus during processing. The finished compounds emerge from the other end, the trans end.
Figure 2: A yeast cisterna (white arrow) changes colour over time as the proteins attached to the membranes change during processing.
enlarge imageUntil now, there has been a debate as to how this maturation process took place. One typical model suggests that each cisterna is a permanent structure and that proteins move from one cisterna to another during processing. An alternative is that new cisternae form at the Golgi entrance and move with their entire contents up the stack as they mature to break down and release the processed compounds at the exit.
As reported in Nature1, researchers from RIKEN’s Discovery Research Institute in Wako were able to resolve this debate with a series of experiments using yeast. They chose yeast because unlike higher organisms the individual cisternae are not stacked together but independent of each other, scattered freely in the cell, and can be tracked easily. The researchers took pairs of membrane components—one associated with the early cis part of the processing and the other associated with a later stage—and labeled each with a different color fluorescent protein, one red and the other green.
With their new, live-imaging technology—a high-speed, confocal microscope linked to the latest, ultra-sensitive cameras—the team can observe weakly fluorescent signals within living cells. So the researchers could detect and track their fluorescently labeled compounds, and use them to determine whether cisternae were either at the cis or trans stage or somewhere in between. In yeast, they found cisternae typically showed primarily red or green fluorescence confirming that cis and trans forms scatter independently through the cell.
The researchers then followed the progress of individual cisternae. They reasoned that if maturation occurred all within one cisterna, then the components associated with the membranes, hence the colors, should change over time. And that is exactly what they detected (Figs. 2 and 3). What’s more, the color changes were always unidirectional—from red to green, or cis to trans-related membrane proteins, never the reverse. Cis-stage membrane proteins were not evident at the trans-stage.
Figure 3: Typical graphs of the change in the relative fluorescence of red and green-tagged components (colored accordingly) in a cisterna over time.
enlarge imageBut if that were so, the researchers argued, somehow the cis-stage membrane proteins would have to be moved out of the maturing cisternae after their job was done, and back to where the new cisternae were forming. The standard explanation for this involves transportation in small membrane-bound bags called COPI vesicles.
In search of an answer, the research team examined a mutation which results in the formation of defective coats for COPI vesicles and prevents them from functioning properly. In yeast cells carrying this mutation the team detected that the speed of transition from cis to trans was more than three times slower, indicating that COPI vesicles are an important part of the maturation process. But as the mutation did not stop the process completely, other additional mechanisms are likely to be involved.
“We now want to look at the Golgi apparatus in higher organisms to see if our findings are also true for them,” says project director Akihiko Nakano. “In future, we would like to use our technology to examine the dynamic events of how proteins are trafficked and sorted within cells.”
Many more questions about Golgi apparatus processing remain to be answered, he commented. Why is it that different protein cargoes are processed at different rates, for instance, and how is this achieved? And, what mechanism ensures that the maturation process only runs one way?
The team hopes that further development of their technology in terms of speed and sensitivity will open further fields in cell biology for exploration.
- Matsuura-Tokita, K., Takeuchi, M., Ichihara, A., Mikuriya, K. & Nakano, A. Live imaging of yeast Golgi cisternal maturation. Nature 441, 1007–1010. |article|
About the Researcher
Akihiko Nakano
Akihiko Nakano was born in 1952 in Hokkaido and received a Doctor of Science degree from the University of Tokyo in 1980. From 1980 to 1986, he worked as a research associate at the National Institute of Health in Tokyo. From 1984 to 1986, he served as a post doctoral researcher at the University of California, Berkeley, and then returned to the institute in Tokyo as a senior researcher. In 1998, he was appointed as a lecturer at the University of Tokyo, and became an associate professor in 1991. Since 1997, he has been serving as the Chief Scientist and Director of the Molecular Membrane Biology Laboratory at RIKEN's Discovery Research Institute in Wako. He is also a professor at the University of Tokyo Graduate School of Science.
