When cells migrate, they need guidance to reach their final destination. Nerve cells, in particular do not migrate. Instead, they have a specialized extension, known as an axon that reaches out and navigates a precise pathway in order to build connections to form the neuronal circuitry in the brain. Vascular endothelial cells alternatively detect starving tissue and grow new blood vessels. These processes are both controlled by guidance cues that interact with cell surface receptors that accumulate on the tip of the migrating cells or on the tip of growth cones of an axon. Cells can detect a guidance cue gradient through their receptors, and this subsequently leads to the cells becoming attracted to the guidance cue source, or repelling away from it. One universal guidance cue is netrin-1, and it stimulates axon navigation and blood vessel growth. Depending on the receptors present on the surface of the cell, netrin-1 can trigger either attraction or repulsion.

Yan Zhang’s study showed how the ligand netrin-1 is able to switch between the attraction and repulsion of an axon. In the current study, structural data reveals netrin-1 in complex with one of its receptors, DCC. The complex structure shows that netrin-1 can bind with two molecules of DCC simultaneously and that although one of these binding sites is specific to DCC, the other is a unique generic binding site that is tuned by environmental factors. The scientists further demonstrated by functional experiments that when another receptor called UNC5A is present on the growth cone besides DCC, it binds to the tunable generic site on netrin-1, leading to repulsion. The molecular understanding of this guidance switch can one day be used to alter the behavior of migrating cells in processes such as neurogenes is and tumor metastasis.

Nerve cells, otherwise known as neurons, are specialized cells and key components of the nervous system encompassing the majority of species in the animal kingdom. During the developmental process, a special cellular extension called an axon, projecting from the cell body of a neuron moves and grows in response to chemical signals released from target cells, either moving towards, or away from a concentration gradient of these specific chemicals. These chemicals are a small group of proteins, which act as guidance cues. Receptors that recognize these guidance cues sit on the tip of the neuron in the growth cone. The receptors then translate these chemical signals into movement. Netrins are a class of proteins involved in neuronal guidance and were the first group of guidance proteins identified in both vertebrates and invertebrates. Netrin-1 can trigger either an attraction or repulsion effect, depending on the receptors present on the growth cone of an axon of neurons.

The switch made from attraction to repulsion is related to the presence of these two receptors: DCC and UNC5. DCC (Deleted in colorectal cancer) was first discovered in an entirely different context: as a marker for colon cancer. Netrin-1/DCC is in fact, in addition to axon guidance, functions in many other cell biological processes, including in cancer biology. The deletion of DCC has been linked to the propensity of cancerous cells to metastasize, which is another form of cell migration. UNC5 (un-coordinated 5) was discovered in C. Elegans, which left a worm that lacked this receptor to make uncoordinated movements. Netrin-1 interacts with these two receptors – interaction with DCC alone causes an attraction response and interaction with the UNC-5 receptor together with DCC causes repulsion.

Scientists at Peking University teamed up with groups from Harvard and EMBL Hamburg to resolve how these interactions work on a molecular level. The Wang Laboratory from Peking University and Harvard Medical School provided DCC fragments that were combined with netrin-1 produced by the Meijers laboratory at EMBL to make a crystal of the netrin/DCC complex. The EMBL Hamburg beamlines situated at the PETRA synchrotron operated by DESY were used to determine the crystal structure and to study the behavior of the complex in solution by Small Angle X-ray Scattering. Careful analyses of the complex structure by the scientists led to the prediction of the key structural elements between netrin-1 and its receptor, DCC. To verify the structural observation, the Zhang laboratory from Peking University performed repulsion and attraction assays on neurons that contained DCC and UNC5 receptors. “The collaboration between the three groups from three different time zones was very intensive, and the project moved forward through a twenty-four hour cycle”, says Rob Meijers who led the research in Hamburg. “It is very exciting to see that you can translate the abstract observations made from a protein structure into the manipulation of neuronal cell migration patterns.”

When the crystal structure was determined, it became clear that it contained a unique protein-protein interaction site between netrin-1 and DCC, which needed the presence of sulfate ions to be maintained. In the context of a cellular environment, these sulfate ions are probably replaced by larger sugar-like molecule known as heparan sulfates. It was already known that these molecules influence neuronal wiring, and now it appears that heparan sulfates could help to determine which receptor binds to netrin, and consequently how the cell will respond to netrin. The scientists are now investigating how the so-called sugar code is related to netrin/receptor interactions. “It is very challenging to target protein-protein interactions with small molecule drugs, but this particular mode of interaction could be exploited to fine tune netrin biology”, says Rob. “In this way, we can target cells with a certain receptor repertoire to undergo proliferation or apoptosis”.

Lorenzo I. Finci1, Nina Krüger, Xiaqin Sun, Jie Zhang1, Magda Chegkazi, Yu Wu1, Gundolf Schenk, Haydyn D.T. Mertens, Dmitri I. Svergun, Yan Zhang, Jia-huai Wang, Rob Meijers,(2014) The Crystal Structure of Netrin-1 in Complex with DCC Reveals the Bifunctionality of Netrin-1 As a Guidance Cue,  Neuron , DOI: 10.1016/j.neuron.2014.07.010