Nerve Cells On The Go

February 26, 1999

Harvard Researchers Tie Axon Pathfinding to Cytoskeleton Research

Boston, MA--February 25, 1999--One of the hottest fields in cell biology aims to understand the molecules that drive the cytoskeleton, the gel-like inner scaffold that allows a cell to 'morph' into different shapes as it responds to important changes in its environment.

And one of the hottest fields in neurobiology aims to understand how the hand-shaped end of a growing neuron, called the growth cone, explores the territory it traverses on its way to its target tissue.

The twain now meet in two NIH-funded studies published in the February Neuron. In them, David Van Vactor, HMS assistant professor of cell biology, describes how his team found an uninterrupted chain of signaling events that neurons in fly embryos use to transmit outside information from the membrane all the way to the actin cytoskeleton.

In previous research, cytoskeleton researchers have worked from the bottom up, tracing their way backward from actin. They have found a bewildering number of partial connections but have not yet made the leap to the membrane receptor. Working from the top down, neuron guidance researchers traditionally study which external cues the growth cone encounters and which receptors it uses to recognize them. But this research has not yet completed the link to actin.

Van Vactor's work bridges that gap by describing the first continuous line of communication linking a receptor in a growth cone's membrane to actin, the final agent of change.

The cell's cytoskeleton is a continuously changing fabric of protein filaments underneath the cell membrane. Actin is one of its major components. Beyond giving a cell mechanical strength, the cytoskeleton is the actual executor of most biological events that require a change in a cell's shape or motility. Examples include the development of an organism with different types of cells arranged in the proper places, or the rogue travels of metastatic cancer cells.

Van Vactor started this research as a postdoctoral fellow in the lab of Corey Goodman at the University of California, Berkeley, who is a co-author on the first paper.

Using the fruit fly, Van Vactor analyzed two types of mutant phenotypes with derailed motor nerve development. In one set of mutants--dubbed stop short--a motor nerve called intersegmental nerve b (ISNb) arrested its growth before reaching its target muscles, suggesting the disrupted genes were essential for the growth cones to proceed. In mutants dubbed bypass, ISNb neurons miss their exit, growing straight past the muscle instead of turning sharply toward it.

When he cloned the genes underlying stop short and bypass, Van Vactor expected the genes would operate in different contexts. But once he analyzed them a puzzle fell into place, and the genes turned out to belong to the same pathway. The story starts at the bottom of the pathway.

The first gene he cloned caused the stop short phenotype. It turned out to be profilin, a much-studied protein actin known bind and control actin. That made sense but was not really surprising, Van Vactor says.

Unexpected, however, was his finding that the stop short phenotype also arose in embryos lacking the gene for the protein kinase Abl. (Protein kinases are enzymes that tack phosphate groups onto other kinds of proteins.) When analyzing mutant embryos that lacked both profilin genes and one copy of the Abl gene, the scientists found that cutting the amount of Abl protein in half dramatically worsened the embryos' stunted nerve growth.

This genetic way of asking whether one protein is sensitive to the dose of another helps scientists find out whether two proteins cooperate in the same pathway. Profilin and Abl clearly seemed to do so.

Abl provided an intriguing step up the pathway, since its substrate--a protein called Ena--was known from other systems to bind profilin and affect actin.

The second paper exposes the other half of the pathway from the membrane downwards. It begins with Dlar, a gene causing the bypass phenotype. Dlar is a member of the receptor tyrosine phosphatase family-- membrane-spanning proteins that slice phosphate groups off other proteins inside the cell. Three years ago, Van Vactor and Haruo Saito, HMS professor of biological chemistry and molecular pharmacology, first implicated Dlar in axon guidance.

Trying to understand how Dlar signals, Van Vactor, working with graduate student Zachary Wills and others, discovered that a triumvirate of proteins--Dlar, Abl, and Ena--is bound together in intimate, antagonistic relationships. The trio makes key decisions about what information is transmitted to the cytoskeleton, Van Vactor says.

Evidence supporting that idea also comes from experiments testing the sensitivity of one protein to the dose of another. The scientists found that Dlar is as sensitive to the amount of Abl as is profilin. Halving the amount of Abl protein suppresses the damage wrought by the Dlar mutation--that is, fewer ISNb nerves bypass their targets. Conversely, increasing the amount of Abl protein beyond normal levels overwhelmed Dlar and produced the bypass phenotype in normal fly embryos, just as if they had a Dlar mutation.

This key experiment shows that the kinase Abl and the phosphatase Dlar are opposing enzymes locked in a balance of power, and each one can tip the scale. Bringing the research full circle, the scientists show that an object of this competition was the phosphate recipient Ena, the protein known to interact with profilin.

One reason this work appears complicated is that even though the researchers have established a sequence of players, they still do not fully understand the precise relationships among them.

It is not as if a valve opened at the membrane, passing information all the way to actin like water flowing smoothly down a tube. On the contrary, there seems to be plenty of turbulence, with members of the pathway frequently opposing their binding partners, as if they were haggling over the final outcome every step of the way. Take these examples: Ena suppresses Abl, Abl fights Dlar, Ena and Dlar work together, but Ena seems to inhibit profilin, and profilin curbs the polymerization of actin.

"Even so, a coherent picture is finally beginning to drop out of all this madness," Van Vactor says. Indeed, these kinds of checks and balances, combined with input from multiple tributaries to this flow that have yet to be established, may represent the molecular basis of a 'thinking' signal transduction system that integrates multifaceted, and sometimes conflicting, information to come up with the appropriate biological response.

Many questions remain unanswered, Van Vactor says, but it seems clear that these interlocking relationships are well suited to make the cytoskeleton as dynamic as it is. The adding and removing of small phosphate groups to signal transduction proteins in the growth cone probably serves as a fast and reversible mechanism enabling the growth cone to weigh attractive versus repellent cues as it travels the ever-changing landscape of the developing embryo.

Harvard Medical School

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