Fluorescence Technique Reveals Structural Protein Movements In Vivo

The choreography of structural proteins that occurs during the budding of a yeast daughter cell has just become clearer, thanks to a groundbreaking technique that uses orientationally constrained green fluorescent protein (GFP). The technique was designed by Alina Vrabioiu, research fellow in the laboratory of Timothy Mitchison, the Hasib Sabbagh professor of systems biology at HMS. In the Sept. 28 Nature, lead author Vrabioiu demonstrates that structural proteins called septins appear to have an ordered longitudinal organization that precisely rotates 90 degrees during the process of cytokinesis. Mitchison and Vrabioiu expect that their technique could be used to visualize the movements of a variety of structural proteins in vivo.

Image courtesy of Alina Vrabioiu

Rank and file. Polarized fluorescence microscopy showed that in living yeast, septin filaments line up along the bud neck (left) and rotate 90 degrees (right) at the onset of cytokinesis to form two ring structures around the circumference of the neck.

Septin proteins are essential for cytokinesis in most eukaryotes except plants. They were recently found to form filaments in vivo, but their precise function has remained unclear due to difficulty in studying their movements.

To circumvent this problem, the researchers exploited a common feature of structural proteins—a coiled coil domain. Vrabioiu fused the predicted C-terminal coiled coil domain of the septin proteins to the N-terminal alpha helix of GFP. The resulting fusion protein had a rigid linkage that constrained GFP’s fluorophore in a particular orientation relative to the septin portion of the fusion protein. “The technique worked amazingly well, much better than we expected,” said Vrabioiu. “The next step was to use this information to mathematically calculate the orientation of septin filaments in living yeast.”

When the fusion protein was expressed in vivo, the constrained GFP preferentially became excited by a particular direction of polarized light. Using polarized fluorescence microscopy, Vrabioiu and Mitchison were able to determine the orientation of the septin filaments in living yeast and calculate their movements during cell division. They found that during the budding stage, the filaments lined up longitudinally along the bud neck, followed by a 90-degree rotation in the plane of the membrane to form two ring structures around the circumference of the bud neck during the final stages of cytokinesis.

“This suggested to us that septins might play a more direct, mechanical role in cell division than has been believed,” said Vrabioiu. “What’s very exciting is that this technique should be able to work for other structural proteins, as long as they have an alpha helical domain.”

—Jillian Lokere

Disruption of Protein Modification System Shown to Cause Cleft Lip

“Sumoylation” has emerged as a common post-translational modification, the attachment of a small ubiquitin-related modifier (SUMO) to a protein substrate. But the process had never been shown to play a specific role in development. Now a team of HMS researchers led by Richard Maas, professor of medicine, and Cynthia Morton, the William Lambert Richardson professor of obstetrics, gynecology and reproductive biology, both at HMS and Brigham and Women’s Hospital, have shown that sumoylation “is a regulatory mechanism that governs how various gene products function in palate formation,” according to Maas. That, in turn, “provides the type of biochemical understanding ... that will be required to devise future preventive therapies,” he said. The research is published in the Sept. 22 Science.

The patient whose case led to the research was an otherwise normal Caucasian girl with cleft lip and palate (CL/P) and a chromosomal translocation that the researchers suspected caused the disorder by breaking a gene important for palate formation. The break disabled one of the patient’s two SUMO1 genes, “resulting in a 50 percent reduction in SUMO1 expression and sumoylation,” said Maas.

The researchers then created mice with reduced expression of Sumo1, the mouse homolog of the human gene, which exhibited cleft palate, demonstrating cause and effect.

The team then showed that combining reduced expression of Sumo1 with a mutant Eya1, another cleft palate gene, raises the phenotype’s frequency beyond that of the sum of each alone.

“The Eya1 protein is also sumoylated by Sumo1, so these results suggest sumoylation may regulate a network of genes that converge in palate morphogenesis,” explained Maas. Consistent with this regulatory model, which requires the proteins to be in the same cells concurrently, the genes are co-expressed at the site where palatal shelf fusion occurs. The death of embryos and newborns indicated that Sumo1 is required for other developmental functions, according to the paper.

“We are in the process of determining the overall frequency of SUMO1 mutations in patients with CL/P,” said Maas.

The research is part of the Developmental Genome Anatomy Project (DGAP), an NIH-supported program project grant that has the goal of using naturally occurring chromosomal rearrangements linked to human birth defects to identify the causal genes.

—David Holzman

Fat Cell Hormone May Regulate Addictive Behavior

People have long speculated that excessive feeding and addictive behavior might be related. Now Jeffrey Flier, the George C. Reisman professor of medicine, and Eleftheria Maratos-Flier, associate professor of medicine, both at Beth Israel Deaconess Medical Center and HMS, along with their collaborators, give evidence that this notion may be true.

An adipocyte hormone, leptin, regulates feeding by tracking stored energy in fat cells. Leptin-deficient mice are sluggish and eat constantly, with predictable results.

The hormone may also regulate pathways that mediate addiction. The leptin receptor is well known to exist in the hypothalamus. Leptin regulates secretion of dopamine, and the leptin receptor can also be found in the ventral tegmental area (VTA), part of the mesolimbic “reward” circuit of dopaminergic neurons. The flip side of reward is motivation, because it leads to reward-seeking behavior, the extreme of which is addiction. The VTA can be activated by psychostimulants, such as cocaine.

The researchers confirmed the existence of leptin receptors in the VTA. They traced the receptors’ dopaminergic neurons to the nucleus accumbens, showing that the cells were, indeed, part of the mesolimbic pathway.

The anatomy and biochemistry begged the question of whether “there was a behavioral component to what you are seeing at the biochemical level,” said Maratos-Flier, whose lab collaborates with her husband’s. She wanted to know if the sluggishness of leptin-deficient mice resulted from a lack of leptin. In normal mice, amphetamine triggers dopaminergic activity, stimulating locomotion. Just as she suspected, the weak response of leptin-deficient mice to amphetamine was corrected by replenishing leptin.

At this point, “We had the signaling, the behavioral data, and some anatomic data,” said Maratos-Flier. A difference in dopamine released by the appropriate brain areas of the deficient and nondeficient mice “would clinch the story,” she said.

And in fact, that is what their team observed. Electrically stimulating brain slices showed an average 90 percent reduction in dopamine signal from neurons arising from the VTA of leptin-deficient mice versus nondeficient mice.

The research “raises the intriguing possibility that the complex behavior of eating that goes beyond the need for calories is actually regulated, and that a hormone coming in from fat is playing a role in that regulation,” said Maratos-Flier. “It’s the same signal from the same place. In one case, it’s acting on replacing the calories you’ve used. In the other case, it’s also playing a role in controlling the hedonic consumption of food.… The leptin signal is playing a role in how successfully an animal will be able to recognize that it’s time to stop eating.”

—David Holzman

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