Following the often-quoted advice of Yogi Berra — "You can observe a lot by just watching" — Rockefeller University scientists show that nerve cells in the developing brains of humans and other mammals move in a two-part "step" led by a structure within the cell called the centrosome.
Once the centrosome, the key organizing point for the cell's internal skeleton, moves forward, the cell nucleus follows. The Rockefeller scientists produced time-lapse movies that show nerve cell migration in unprecedented clarity and detail.
The finding, reported in the October 10 online issue of Nature Neuroscience, overturns a widely accepted view of how nerve cells, or neurons, move in the young developing brain. Until now, researchers generally thought that the explanation for how a neuron migrates to its destination in the brain lies in the way the cell adheres to, and later releases, thread-like cells called radial glia. The researchers, led by Mary E. Hatten, Frederick P. Rose Professor and head of the Laboratory of Developmental Neurobiology at Rockefeller, also discovered that a protein called Par6-alpha plays an important role in spurring the centrosome to action.
"Scientists have spent the last 15 years focusing on adhesion as the most important aspect of cell migration," says Hatten. "These experiments illustrate that we've been looking in the wrong place. Adhesion is necessary, but not sufficient for neuronal migration."
In humans, the migration of young neurons to the brain's cerebellar cortex, which controls movement and balance, continues until a child reaches the age of two. Serious disorders ranging from the epilepsies to cortical malformations result if the process goes awry, and the brain's proper architecture does not develop.
Neurons are produced in the center of the brain as a human or other mammal develops from an embryo into an infant or young animal. They then travel outward to form the brain's outer layer, or cortex, and other brain structures. Long fibers of cells called glia guide the neurons in their journey. Under the microscope, a migrating neuron looks like a round cell perched on a rope, the glial fiber leading to its destination. Rather than gliding smoothly along the fiber, the neuron takes a "step." First it stretches out along the fiber, extending what scientists call a leading process in the direction it wants to move. About three minutes later the cell body catches up.
"It's almost like a little inchworm in the way it moves," says Hatten, whose time-lapse movies first revealed this motion in 1987. With each step the cells travel a little more than a micron, or about half the width of a hair over the course of an hour. The new study shows that a surprising mechanism underlies neuronal migration.
The Rockefeller scientists suspected that the cytoskeleton, the scaffold of elements called microtubules that support a cell's three-dimensional shape, was important to neuron migration. They knew that disrupting the cytoskeleton with chemicals prevents the cell from moving. In addition, problems with the cytoskeleton are implicated in human disorders in which nerve cells fail to migrate properly, such as Miller-Deiker syndrome. But no one had previously watched the cytoskeleton in living neurons in real time.
The researchers took a new type of fluorescent dye called Venus, which glows 20 times brighter than other dyes, and tagged the microtubules of a type of mouse neuron called a granule cell. Rockefeller scientist Tarun Kapoor, Ph.D., an expert on the centrosome and on techniques for visualizing microtubules, collaborated with Hatten and her lab colleagues to create images showing these structures in extraordinary detail. Kapoor is head of Rockefeller's Laboratory of Chemistry and Cell Biology.
When the scientists looked at the cells with a spinning disk confocal microscope, the dye revealed a cage-like structure around the cell's nucleus. "Its like a bingo cage," says Hatten. "It holds the nucleus." The experiment confirmed the existence of this cage, which had been controversial. Although the cage is essential for the cell to migrate, further experiments showed that it does not initiate cell movement. Comparing the elements of the cage in migrating and stationary cells, the researchers found no difference.
Next the researchers looked at the protein Par6-alpha, referred to as mPar6-alpha in mouse cells. Recent studies in other laboratories led the scientists to believe that mPar6-alpha helps give a cell polarity — in a migrating cell, a leading and a trailing end. Because migrating neurons are highly polarized, Hatten and her coworkers suspected that mPar6-alpha was active in them.
Again, the researchers used Venus, this time to label mPar6-alpha protein. Its bright yellow glow concentrated in the centrosome, the organelle located just in front of the nucleus in migrating cells, which plays a role in organizing the cytoskeleton.
This in itself was an interesting discovery. "The centrosome has a large number of proteins that make up its structure. Most other known proteins in the centrosome are structural," says Hatten. "mPar6 is the first signaling protein found there."
Watching the cells with labeled mPar6-alpha as they moved along the glial fibers, the researchers could see a two-step process to every advance the cells made. First the centrosome slided forward, and then three minutes later the nucleus followed. "The timing of the advance of the centrosome and then of the nucleus was exactly the same as the timing we measured 15 years ago that it takes for the nerve cell to adhere, let go and take a new step along the glial monorail," says Hatten. Additional experiments that tagged a different component of the centrosome confirmed these results.
To further investigate the function of mPar6-alpha, the researchers created cells with either too much or too little of the protein. In both cases the cells sat motionless on their glial fibers. "By two different methods, overexpression and physically getting rid of the protein, we get a similar result," says David Solecki, a postdoctoral fellow and first author of the paper.
In addition, "There was a tremendous change in the morphology of the cell," Solecki says. In cells with too much mPar6-alpha, the nuclear cage disintegrated.
"In response to mPar6-alpha, the centrosome is playing a signaling role in setting the cadence for migration of the cell," says Hatten. "These results also suggest that the coordinated movement of the centrosome and the nucleus are necessary for migration."
"As many signaling pathways in the young neuron could talk to the Par6 signaling center, it is likely that discovering those signaling networks will lead us to novel classes of cell surface receptors that regulate migration, and thereby the formation of cortical areas of the brain," says Hatten.