ABN, 15, April 2010
New probe technology illuminates the activation of light-sensing cells
Ultimately, Charles Darwin’s “endless forms most beautiful and most wonderful” can be boiled down to a scant 20 or so amino acids, the basic building blocks of life. From this parsimonious palette, nature paints the proteins that make up the wild diversity of life on earth, from the simplest bacteria to the most complicated structure in the known universe — the human brain. Now, in work published today online by Nature, researchers from The Rockefeller University reveal a new technique for tagging
Seeing (infra)red. Scientists designed genetically encoded probes to examine the workings of the visual pigment rhodopsin (pictured above) with infrared spectroscopy. The probes revealed that light causes changes in the protein much faster than previously believed proteins with non-natural amino acids to scrutinize details about how they function.
The experiments in Nature yield new findings about rhodopsin, the light sensitive cell receptor that is crucial to dim-light vision, showing that light causes changes in the structure of the protein much faster than previously believed — on the order of tens of microseconds rather than milliseconds. Thomas P. Sakmar, head of the Laboratory of Molecular Biology and Biochemistry, and postdoctoral associate Shixin Ye, worked with colleagues in Germany, England, Spain and Switzerland, to combine a variety of genetic engineering techniques to introduce an amino acid, azidoF, a relative of phenylalanine, into several points on rhodopsin. The three-nitrogen-atom azido is an especially good probe for three reasons: In contrast to other tags, azido does not exist naturally in mammals, which makes it easier to “see,” or distinguish from other molecules in the cell; it is small enough to not interfere with a protein’s normal functioning; and it has chemical properties that make it a good handle on which to hang other molecules, like fluorescent probes.
In fact, the method could in principle be applied to place a fluorescent probe at any point in any protein in a mammalian cell. “The long-term goal is to label receptors in live cells and do single molecule fluorescent studies,” says Sakmar, who is Richard M. and Isabel P. Furlaud Professor. Such experiments could illuminate the minute functional differences that differentiate proteins the world over.
Similar approaches have been successfully used in bacteria, but last year, the researchers first showed that their method could be applied to mammalian cells with such specificity and efficiency, the scientists say. Extensive genetic screening allowed the team to target the azido probes efficiently. They then confirmed the presence of azido with fourier transform infrared (FTIR) difference spectroscopy, which measures stretching frequencies of the atoms in the amino acids that make up a protein.
Because azido has a unique vibration frequency that is sensitive to its surroundings, the team was able to use the spectroscopic data to confirm structural changes rhodopsin undergoes in light versus dark. “What you want is a probe that doesn’t perturb the protein and one that can tell you something about its structure and function,” Sakmar says. “That’s what we have here.”
The scientists were able to see previously unobserved changes in the structure of rhodopsin, which is a model for the ubiquitous G protein coupled receptors (GPCRs), heptahelical, transmembrane receptors found in eukaryotic cells. There are more than 700 GPCRs in the human genome alone that constitute different signaling systems, activated by light-sensitive molecules, odors, neurotransmitters, hormones and pheromones. The scientists looked at regions of the GPCR, in this case rhodopsin, which are broadly shared or conserved among related receptors.
“We have found that the activation process that begins moving the helices apart — the earliest stage of signal transduction — is faster than predicted, maybe an order of magnitude faster,” Sakmar says. He hopes to use the technique to identify the mechanical components of the switch machinery that activate the receptors, he says, which are involved in a wide range of diseases and are the targets of many pharmaceuticals.
Cell division orchestrated by multiple oscillating proteins, new research finds
Cell division is a crucial but dangerous business. It unfolds in a cycle of many steps, including DNA replication, spindle formation, mitosis and others, and they must happen in the right order to prevent abnormal cell death and cancer formation. New research from Rockefeller University examines the activity of two proteins at the heart of the cell-cycle control system and finds that the cycle has not just one, but several independent processes that help to maintain order. The work suggests that autonomous oscillating proteins
Cellular synchrony. Scientists blocked yeast cells from dividing to observe the behavior of key proteins that control cell-cycle events. Above, Cdc14 (green) oscillates, separating from the nucleolus (red), and sometimes overlapping with it (yellow).
may coordinate the events of the cell cycle through a phenomena called “phase-locking,” similar to how our circadian rhythm syncs to the light-dark cycle of our environment.
“Our research suggests that the modern eukaryotic cell-cycle may start from multiple oscillatory modules,” says Ying Lu, a former graduate fellow in Frederick R. Cross’s Laboratory of Yeast Molecular Genetics, who led the research. “That modularity may provide a functional robustness to cell division.”
At the center of the cell-cycle control system is a protein called cyclin-dependent-kinase (Cdk); Cdk’s independent oscillating activity can establish the pace and order of cell cycle events. The researchers, led by Lu, reasoned that if Cdk oscillation was the only cycle-setting pacemaker in the cell, blocking it would cause the cell cycle to stall. In experiments published Thursday in Cell, they tested the hypothesis by watching what happens to another important protein in the cell cycle known as Cdc14, which normally moves away from the nucleolus, activates and begins antagonizing Cdk as the cell exits mitosis. Using quantitative time-lapse microscopy, the researchers were able to capture the transient Cdc14 movement and activation process. They then blocked Cdk oscillation and overt cell-cycle progression, and surprisingly found that the periodic Cdc14 activation/inactivation continued just as it would in a normally dividing cell. They also discovered a negative feedback pathway underlying this Cdc14 oscillator, a finding which indicates that the cell cycle may be composed of multiple autonomous pacemakers.
The existence of these pacemakers raises another question, says Lu, who is now a postdoc in Marc Kirschner’s lab at Harvard University. How do oscillators with different intrinsic frequencies coordinate with each other to form a coherent cell cycle progression? The experiments suggest that, although Cdc14 activity oscillated at constant Cdk levels, its frequency was controlled by several different Cdk activities, which indicates that autonomous cell-cycle oscillators may coordinate each other through a phenomena called phase-locking. Such a system, which is analogous to day-night cycles entraining our circadian clocks, would help explain the evolution of the cell cycle, and to ensure its accuracy and reliability.
“We think multiple oscillators, as they exist independently in the cell cycle, could achieve coherence through interactions affecting their frequencies,” Lu says.
Scientists identify potential new target for schizophrenia drugs
Rockefeller University scientists have identified a protein that boosts the signaling power of a receptor involved in relaying messages between brain cells, a finding that suggests a new target for the development of treatments for schizophrenia and Parkinson’s disease. The protein, called Norbin, directly interacts with a receptor for the neurotransmitter glutamate, which is critical to the process by which individual brain cells send messages to one another and plays a key role in learning and memory.
Receptors such as the one linked to Norbin, known as mGluR5, are proteins that are embedded in the plasma membrane of a cell and respond to small molecules called ligands. As mGluR5 binds to its ligand it responds by changing its shape, which ultimately leads to the initiation of an intracellular signal. Several classes of receptors exist, the most common of which is the G-protein coupled receptor (GPCR).
“Proteins that modulate GPCR functions are becoming as important as the receptors themselves because they represent novel therapeutic targets,” says Marc Flajolet, a senior research associate in Rockefeller’s Laboratory of Molecular and Cellular Neuroscience headed by Paul Greengard. “About half of the drugs commercially available today target this group.”
Glutamate, one of the most important neurotransmitters, acts through several GPCRs. These receptors are involved in normal processes such as development, learning and memory, as well as in various diseases.
Flajolet and first author Hong Wang searched for proteins that physically interact with mGluR5 and found several, including Norbin. The researchers paired Norbin and mGluR5 in cultured neurons and found that Norbin not only stimulated mGluR5 activity but also increased the amount of mGluR5 on the surface of the neurons.
Fajolet and Wang then looked at Norbin’s effects on mGluR5 in the adult mouse brain, and found Norbin in the hippocampus, amygdala, septum and nucleus accumbens, similar to where mGluR5 is typically found.
The researchers went on to create conditional knockout mice, which are genetically engineered to shut off Norbin production after birth. These mice showed deficits in synaptic plasticity and behavior similar to those found in a rodent model of schizophrenia. For example, the Norbin knockouts were startled by a loud noise as much as normal mice, but were unable to suppress a startle response with subsequent exposure to noise.
“Our work further demonstrates the importance of GPCR regulatory proteins,” says Flajolet. “We are now investigating the mechanism by which Norbin affects mGluR5 and are searching for regulatory pathways that could modify Norbin function and, indirectly, mGluR5 activity.”
