By revisiting 3D images that map the connections among brain cells, scientists are discovering new insights about a small, elusive, and frequently ignored cellular structure.
By revisiting 3D images that map the connections among brain cells, scientists are discovering new insights about a small, elusive, and frequently ignored cellular structure.
In our bodies, many cells feature a single primary cilium, a tiny, hair-like structure that extends from the cell surface and plays a role in transmitting signals. Cilia are crucial for controlling various cellular processes, but their small size and limited quantity make it challenging for researchers to study them in brain cells using conventional methods, leading to a lack of clarity regarding their organization and functionality.
In recent research, teams from HHMI’s Janelia Research Campus, the Allen Institute, the University of Texas Southwestern Medical Center, and Harvard Medical School have utilized exceptionally high-resolution 3D electron microscopy images of mouse brain tissue, previously made for mapping connectomes, to closely examine primary cilia.
Their findings reveal new information about cilia in key brain areas, which could enhance scientists’ understanding of these structures’ roles and their potential links to diseases.
“Reusing these extensive electron microscopy datasets for studying cell biology has provided us with insights that were previously unattainable,” explains Carolyn Ott, a senior scientist at the Lippincott-Schwartz Lab, who led the projects.
Uncovering new biological insights
Cilia are notoriously challenging to investigate. In brain cells, a single cilium, just a few microns in length, protrudes from the cell into a complex network of other cells. Utilizing traditional microscopy methods to locate and image cilia is akin to searching for needles in a haystack, and the often small sample sizes make drawing firm conclusions difficult. While researchers have learned some information from examining isolated cilia in lab-cultured cells, these techniques overlook how the organelle functions within its natural context.
Recently, scientists have developed advanced techniques for producing ultra high-resolution 3D images of tissues, allowing researchers to explore the intricate details of cellular processes. Collectively known as volume electron microscopy, or volume EM, these methods involve imaging thin layers of a specimen with electron microscopes and then assembling the layers to construct ultra high-resolution 3D images. Researchers have employed this technology to build connectomes, providing maps of individual neurons and their interconnections within the brain.
However, the volume EM images used for constructing connectomes don’t solely depict neurons. They offer nanometer-scale perspectives of all cells and structures within a tissue sample, including cilia.
“Each time you delve into these extensive volume datasets, you’re uncovering new biological information,” states Janelia Senior Group Leader Jennifer Lippincott-Schwartz, who leads Janelia’s 4D Cellular Physiology research area and is a co-author of the recently published papers. “There is a vast amount of biology that remains unexplored, and this new technology is making that exploration possible.”
Exploring cilia further
After hearing about Ott’s work with the visual cortex, Saikat Mukhopadhyay, an associate professor at UT Southwestern, reached out to her regarding using volume EM data to look at cilia on granule cells located in the mouse cerebellum.
Cerebellar granule cells make up the most prominent cell type in the mouse brain. These cells feature cilia during their developmental phase, which aids in detecting a protein essential for their growth and maturation. However, adult granule cells lack cilia, likely contributing to the cessation of growth and alterations in these mature neurons. Mukhopadhyay has been fascinated by why and how these cells lose their cilia since he began studying cerebellum development, but conventional methods did not provide a clear insight.
A coalition led by Ott and Sandii Constable, a former postdoctoral researcher in Mukhopadhyay’s lab, successfully located volume EM images of developing mouse cerebellum tissues, enabling them to observe cilia at various developmental stages. They discovered that many cilia on intermediary cells are enclosed and hidden, preventing exposure on the cell surface and restricting their ability to sense the protein responsible for proliferation. In mature cells, the cilia disassemble and disappear entirely, although the centriole, the structure from which a cilium emerges, remains anchored to the surface of these fully matured cells.
Additional examination indicated that the genes essential for cilia maintenance become inactive as the cells develop, implying that cells eliminate their cilia by ceasing the production of necessary maintenance proteins. The centriole found on the cell surface contains a capping complex that inhibits the regrowth of cilia.
These new findings may help scientists comprehend a type of brain tumor where mature granule cells retain cilia anomalously responsive to the protein causing cell proliferation, allowing tumor growth. Understanding what goes awry in these tumor cells could shed light on the disease as a whole.
Mukhopadhyay believes these discoveries wouldn’t have been achievable without the volume EM images, which offer unparalleled views of the cilia on granule cells. He observes that there is now heightened interest among researchers in the significance of cilia compared to a decade ago.
“The current challenge is: How does this lead to disease, and can we find ways to treat these patients?” he remarks.
