March 15, 2002

VUMC’s probe into dual role of protein could alter study of human diseases

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Peter A. Kolodziej, Ph.D., is leading the research at VUMC.

VUMC’s probe into dual role of protein could alter study of human diseases

Sections of fly embryos stained to show network of branching tracheae. (C) Branching in normal, wild type (WT) embryos is continuous at points indicated by arrows. (D) Arrows indicate areas of incomplete branching in short stop mutant embryos (shot) where lumen formation is discontinuous.

Sections of fly embryos stained to show network of branching tracheae. (C) Branching in normal, wild type (WT) embryos is continuous at points indicated by arrows. (D) Arrows indicate areas of incomplete branching in short stop mutant embryos (shot) where lumen formation is discontinuous.

As an embryo develops from a single cell to an organism with fully formed organs and structures, how do the cells know what shape to take and where to go?

Research from the laboratory of Peter A. Kolodziej, Ph.D., assistant professor of Cell and Developmental Biology, yields some clues that might help answer this question. The findings are presented in two papers co-authored by Kolodziej and Seungbok Lee, Ph.D., a post-doctoral fellow, to be published this month in the journal Development.

The studies focused on development in the fruit fly—Drosophila melanogaster—and the role that a large cytoskeletal protein, called Short Stop, plays in generating two different cell shapes or, you might say, shape changes. Kolodziej and Lee determined how Short Stop uses one mechanism to enable axon growth during development of the nerve system and a second mechanism to join tubular structures, such as the branching tracheae of the respiratory system.

Insights gained by working with this model organism may shed light on how human cytoskeletal proteins contribute to normal development, as well as to certain abnormal conditions or diseases.

The components of the cytoskeleton in the fly are, according to Kolodziej, “like a collection of tinker toys.” As in tinker toys, there is a rod-like structure—the Short Stop protein—having ends that interact with other elements, including F-actin and microtubules. These two proteins are dynamic filamentous polymers that lengthen and shorten in response to cellular signaling, and they can be crosslinked and organized into three-dimensional shapes in a wide variety of ways. Actin and microtubules are required not only to give cells their static shape, but also for cells to change shape.

The first paper to be published addresses Drosophila neuron formation. During development, neurons make specific connections with other cells via extensions called axons and dendrites. At the tip of each is a growth cone, a complex migratory organelle that responds to a variety of cues causing growth either forward or backward. In much the same way that we might navigate through an unfamiliar neighborhood, growth cones follow a trajectory determined using combinations of chemical landmarks and avoidance signals.

Despite the movement that occurs as the growth cone travels, the relative organization of actin and microtubules within the cell remains constant, with actin at the periphery and microtubules in the core. Lee and Kolodziej demonstrated that Short Stop is central to this arrangement. Actin is bound at one of its ends and microtubules at the other, and both binding domains are necessary to maintain cell shape and motility. They also discovered that Short Stop’s function in the growth cone requires a calcium-binding domain that lies just next to the microtubule-binding domain, suggesting that calcium may help regulate crosslinking activity.

“Seungbok Lee, who essentially did all of this work, was able to manipulate these different domains and show that Short Stop is, in fact, the ‘missing link’ that connects actin to the microtubules,” Kolodziej said. “There are probably other ways these filaments interact, but this shows that Short Stop links them physically, not just in an independent way, as through a signal.”

The researchers’ second paper reports on Short Stop’s role in tracheal tube fusion in Drosophila. The tracheal system is the network of tubes in the fly essential to respiration, and serves as a valuable model for understanding the development of tubular networks in human organs, such as the lung and vasculature.

In the fruit fly’s tracheal system, Short Stop behaves in a completely different way. Though it doesn’t function in this cell type by crosslinking actin and microtubules, it still must interact with them to do its job.

The tracheal tubes originate in specific places in the fly’s segmented abdomen and eventually form a network of hollow branches. These branches must fuse at segment boundaries so that the lumen—the central cavity of the tube—is continuous throughout the body.

The cells lining the lumen are wedge-shaped, so that the narrower apical end of the cell faces the lumen and the wider basal end contacts the extracellular matrix. So, a cross-sectional slice from the tracheal tube would look somewhat like a slice through a cored grapefruit.

These lumenal cells are joined at their sides by junctional components, including the adhesion molecule cadherin, critical to determining which end of the cell is apical. During development, the apical surface and the junction signal one another to ensure that everything is in place.

“It’s a sort of self-reinforcing dynamic process at work here,” Kolodziej said. “There are specialized proteins on the apical surface. If you mess with those, you don’t get junctions. If you mess with junctions, the apical surface proteins don’t have a clear destination.”

At the tip of the tubular tracheal branches lies a different kind of cell, called the fusion cell, which takes on a donut shape during tracheal development. When two branches fuse, the initially separate lumens in the joining branches meet via the central “donut holes” in the fusion cells. It is when two of these fusion cells meet that Short Stop comes into play.

“What we found is that if you delete the Short Stop protein, you don’t get fusion,” Kolodziej said. “Short Stop is required for cadherin contacts to form, but only new ones. The other cells already packed around the lumen aren’t affected.”

First, a filamentous actin track—which also contains Short Stop—is formed that radiates outward from the cadherin contact between the fusion cells. The track appears to guide lumen formation and the deposition of components needed to establish apical polarity. The researchers showed that if Short Stop is missing, the track is not formed and, typically, no cadherin contact is made.

“The interesting thing about this,” Kolodziej said, “is that in this cell type, you can delete either the actin or the microtubule-binding domain [in Short Stop] and the system still works. You can delete the calcium-binding domain, and it still works. But if you delete both the actin and microtubule domains, it doesn’t work.”

So, for the successful formation of the tracheal system, Short Stop requires at least one site of cytoskeleton interaction, but not necessarily both. Though F-actin and microtubules are very different functionally in most cases, they appear to play redundant roles in this fusion process.

When they first started, Kolodziej said, it was to ask how the short stop gene affects the ability of the growth cone to migrate.

“But what we’ve learned is that proteins involved in making the cytoskeleton do one type of thing in one cell type—as with crosslinking that affects motility—and something else in another cell type—as with cell fusion in lumen formation,” he said. “The machinery in the cell behaves in a completely different way, almost like it’s a different protein at work. That’s the surprising twist.”

This new information could alter how developmental biologists approach the study of human diseases that involve defects in the cytoskeleton, including cancer.

“For the past 40 plus years, people have looked at actin and microtubules as distinct entities,” he said, “but in fact they are highly coordinated during developmental processes ranging from mitosis to moving around to changing shape, and also in physiological processes you probably wouldn’t think about, such as neurotransmission.”

Future research will address unanswered questions, such as those surrounding control of the Short Stop protein. For example, it’s not known how or what signaling molecules affect crosslinking during growth cone motility.

Though scientists have been using fruit fly genetics to study development for 100 years or so, Kolodziej feels there is much more this model organism can reveal about human biology.

“This just shows you the power of analyzing these things in the fly,” he said. “If we hadn’t had the ability to change these genes in the fly and test their function in their physiological context, we wouldn’t be able to say that Short Stop works as a crosslinker. It looked like a crosslinker, but we wouldn’t have known whether crosslinking was important or not without the genetics.

“Moreover, we can now use genetics and biochemistry in this powerful system to identify targets of short stop and its regulators that are physiologically relevant to developmental processes occurring in humans.”

The research was supported by funds from two grants from the National Institutes of Health and a discovery grant from Vanderbilt University Medical Center. Additional support for the research on axon extension was provided by the American Cancer Society. Additional support for the research on tracheal tube fusion was provided by the Howard Hughes Medical Institute.