In a new study in the journal Development, University of Wisconsin-Madison researchers identified structures of neurons crucial in guiding axons through tissue in the developing nervous system. Those structures function much like structures cancer cells use to migrate throughout the body.
Scientists at the UW School of Medicine and Public Health used several forms of fluorescence microscopy, including cutting-edge super-resolution microscopy, to visualize the pathfinding activity of growth cones of Xenopus (African Clawed Frog) neurons in tadpoles and in human neurons in vitro developed from induced pluripotent stem cells. They discovered that growth cones form specialized structures called invadopodia that are home to proteins that degrade the extracellular matrix (ECM), a crucial step in allowing developing neurons to grow outward through tissues toward their eventual targets.
Cancer cells degrade the ECM the same way. Similar to a dense and sticky webbing, the ECM provides structural support for cells, helping related cells communicate and stay together. Cancer cells can be restricted to the ECM’s structure.
How Neurons Become a Network
Cell protrusions called neurites link neurons together so they can communicate, but to reach out to their neighbors and make those connections, they must stretch out away from the cell body. Random growth would be at best ineffective to create the 100 trillion specific connections between neurons in the body, so neurons rely on special sensory-motor structures called growth cones to guide the process.
Growth cones house sensitive molecular receptors to identify what is surrounding the developing neuron and provide key navigational cues to guide axon growth.
“As new neurons are first born, unspecialized cell bodies develop their characteristic complex shapes, or morphologies, over many months of development to build the nervous system,” said Timothy Gomez, professor of neuroscience at the UW and senior author of the study. “These cells become motor neurons or cortical neurons or others, as they extend an axon and dendrites (the cell structures that allow neurons to send and receive signals to other neurons), taking on complex morphologies and often growing long distances to reach their targets. That’s what the growth cones do; they navigate using this extracellular guidance cues to build the nervous system.”
The sensitive structures of the growth cone guide the developing neurons through three- dimensional tissues. In some instances, though, said Gomez, some neurons have to abandon a common extracellular roadway and extend in new directions. For example, motor neuron axons start in the spinal cord, but turn outward toward the developing muscle. At times it even appears they are escaping by force, using the MMPs (metalloproteinases, proteins that degrade ECMs as well as other varied roles) available on and secreted by invadopodia.
“There is a thick matrix of proteins around the spinal cord, which developing axons extend within, and we believe that motor neuron growth cones form invadopodia on their surface to escape out,” said Gomez. “There are many places neurons have to make these three-dimensional choices, but nobody is really studying this. People have shown that MMPs are important for axon guidance, but they haven’t shown how they’re being targeted and released. We believe it’s through a tightly regulated use of invadopodia, which are most commonly understood in pathology.”
Invadopodia have a highly deleterious function when associated with cancer cells. Highly metastatic cancers can invade other parts of the body more effectively when they make matrix degrading invadopodia.
Former UW graduate student Miguel Santiago-Medina, first author of the study, initially suggested to Gomez that the structures they were seeing in growth cones looked similar to structures described in cancer cells. They began reviewing the cancer literature and found many commonalities between what they observed and invadopodia. Many of the experiments they subsequently planned were guided by work performed first in cancer cells.
By leveraging a super-resolution technique called Structured Illumination Microscopy (SIM), which roughly doubles the resolution of traditional light microscopes from 250 nanometers to about 125 nanometers along the x and y axis, Gomez generated some of the clearest visuals of invadopodia to date. SIM also helped produce better three-dimensional images, cutting resolution along the z axis from 600 to about 300 nanometers.
“Super resolution imaging allowed us to see cellular details we could never see before with our standard techniques available to us at the time,” said Gomez. “Fine details appear blurred especially along the z axis, so we were always missing the true cellular structure. People have been studying invadopodia for quite some time in cancer cells, but I don’t believe they have shown this level of detail in three-dimensional images. Seeing the structures both in cultured cells and within tissues provides an added level of validation you would otherwise not have.”
“The big picture impact is that researchers have largely ignored how cells change their tissue environments,” said Gomez. “If you look at growth cones, they’re largely planer structures. But then how do they migrate through these 3-D tissues? It’s an untapped area of research.”
University of Wisconsin School of Medicine and Public Health