By reexamining 3D images used to map the connections between brain cells, researchers are uncovering new information about a small, elusive, and often overlooked cellular appendage.
Many cells in our body have a single primary cilium, a micrometer-long, hair-like organelle protruding from the cell surface that transmits cellular signals. Cilia are important for regulating cellular processes, but because of their small size and number, it has been difficult for scientists to explore cilia in brain cells with traditional techniques, leaving their organization and function unclear.
In a new series of work, researchers at HHMI's Janelia Research Campus, the Allen Institute, the University of Texas Southwestern Medical Center, and Harvard Medical School used super high-resolution 3D electron microscopy images of mouse brain tissue generated for creating connectomes to get the best look yet at primary cilia.
Their research uncovers new insights about cilia in these critical brain regions that could help scientists better understand their role and potential contribution to disease.
"Repurposing these large-volume EM datasets to look at the cell biology has given us a view that you couldn't get before," says Carolyn Ott, a senior scientist in the Lippincott-Schwartz Lab who led the projects.
Revealing new biology
Cilia are notoriously difficult to study. In brain cells, a single cilium, only tens of microns long, extends from a cell's surface into a jumble of other cells. Using traditional microscopy methods to find and image cilia is like looking for needles in a haystack, and the sample sizes are often too small to draw conclusions. Researchers have gained some knowledge from studying isolated cilia in cultured cells, but these techniques miss information about how the organelle behaves in its natural environment.
In recent years, scientists have developed new ways to obtain ultra high-resolution 3D images of tissues, enabling researchers to see the inner workings of cells in fine detail. Collectively called volume electron microscopy, or volume EM, these methods use electron microscopes to image thin layers of a specimen and then stitch the layers together to create ultra high-resolution 3D pictures. Researchers have used this technology to create connectomes that provide maps of individual neurons and their connections in the brain.
But the volume EM images used to create connectomes don't just show neurons. They provide nanometer-scale views of all the cells and structures in a volume of tissue – including cilia.
"Every time you look into these large-scale volume data sets, you are seeing new biology," says Janelia Senior Group Leader Jennifer Lippincott-Schwartz, head of Janelia's 4D Cellular Physiology research area and a senior author on the new papers. "There is a lot of biology out there that we still don't have our hands on that is now possible because of this emerging technology."
Investigating cilia
Ott, who has been studying cilia for nearly two decades, first became intrigued with the idea of using volume EM to investigate the organelles when a visitor to the lab pointed out cilia in 3D images of mouse brain tissue. She was captivated by the ability to see dozens of cilia enmeshed in a network of cells and other structures.
"I was just fascinated to see the cilium in the context of the brain tissue, to see where it went and think about what was outside of it," Ott says. "With volume EM, you get a whole different kind of context."
Ott started looking deeper into the 3D EM images from Janelia, Harvard, and the Allen Institute that were used to generate connectomes. Realizing that they could see different types of cilia on different cell types, a team co-led by Ott and Russel Torres, a scientific programmer at the Allen Institute, decided to do a quantitative study of cilia in the cells of the mouse visual cortex, the brain region responsible for visual processing.
The result is the most detailed and comprehensive description to date of the cilia in this part of the mouse brain, including which cell types do or do not have cilia, structural differences in cilia on different cell types, and the location of cilia near synapses, where signaling between neurons occurs.
Together, these findings provide new insights and raise new questions about how these differences and locations affect cilia function, Ott says.
"We know that diseases that involve cilia can have different symptoms, and having a better understanding that not all cilia are the same could help us understand why all disease phenotypes are not the same," she says.
Studying cilia development
After hearing about Ott's work on the visual cortex, Saikat Mukhopadhyay, an associate professor at UT Southwestern, approached her about using volume EM data to examine cilia on granule cells in the mouse cerebellum.
Cerebellar granule cells are the most abundant cell type in the mouse brain. These cells contain cilia while they are developing, which helps the cells detect a protein that controls proliferation and differentiation. But adult granule cells don't have cilia, which likely helps these mature neurons to stop growing and changing. Why and how the cells lose their cilia had intrigued Mukhopadhyay since he started working on cerebellum development, but traditional techniques didn't provide a clear picture of what was happening.
A team led by Ott and Sandii Constable, a former postdoc in the Mukhopadhyay Lab, was able to track down volume EM images of developing tissue in the mouse cerebellum, which allowed them to see cells and cilia in various stages of development . They saw that many of the cilia on intermediary cells are enclosed and concealed, ensuring that the cilia aren't exposed on the cell surface and limiting their ability to detect the protein that causes proliferation. In mature cells, the cilia are disassembled and go away completely. However, the centriole, the structure from which the cilium grows, remains docked on the surface of these mature cells.
Through additional analysis , the team found that genes important for maintaining cilia turn off as the cells mature, suggesting that cells get rid of their cilia by stopping the synthesis of proteins needed to maintain them. The centriole on the cell's surface contains a capping complex that prevents the cilium from regrowing.
The new findings could help scientists understand a type of brain tumor in which mature granule cells abnormally retain cilia that are responsive to the protein that causes proliferation, allowing the tumor to grow. Understanding what is going wrong in the tumor cells could help scientists better understand the disease.
Mukhopadhyay says these findings would not have been possible without the volume EM images, which provide unprecedented views of the granule cells' cilia. Mukhopadhyay, who has studied cilia his entire career, says more researchers are paying attention to the importance of cilia than they were a decade ago.
"Now the frontier is: How is this causing disease, and can we do something to treat these patients," he says.
