Cellular transport vehicles caught on film

Specialized fluorescence microscopy movies show clathrin traveling on microtubules near surface of living cells

They look like soccer balls — only much smaller. They are tiny transport vehicles used by cells to import biological cargo and, for the first time, Rockefeller University researchers have caught them on film swimming across the surface of cells.

Known as “clathrin pits,” these microscopic vehicles consist of a cargo-filled vesicle and an outer geodesic coat made up of a protein called clathrin. They arise when cells engulf or “endocytose” molecular “foods” – the cargo. According to current thinking, the vesicles then shed their clathrin coats, before traveling through the cell to where the cargo is needed. Diseases associated with clathrin-mediated endocytosis include familial hypercholesterolemia, an inherited disorder that causes very high cholesterol levels, and certain viral infections such as adenovirus, a common cause of respiratory infections, and HIV, the virus that causes AIDS.

Until now, clathrin pits were thought to be confined to discrete spots on a cell’s surface. “If you look in any cell biology textbook, you’ll see clathrin pits represented as stationary points,” says final author Sanford Simon, Ph.D., a professor at Rockefeller and head of laboratory conducting the new research, which was reported in both the April issue of Journal of Cell Science and the July issue of Traffic.

But when the Rockefeller researchers pointed their microscopes at the surface of living human cells, in a technique called Total Internal Reflection Microscopy (TIRFM), they observed a subset of clathrin structures at the cell surface zigzagging back and forth like a swarm of bees.

“For the first time, we were able to characterize individual clathrin structures moving laterally across the surface of cells,” says Josh Rappoport, Ph.D., first author of the research papers and a postdoctoral fellow at Rockefeller.

And this wasn’t the only surprise. The researchers also watched the clathrin-coated vesicles travel along the route of structural highways called microtubules. Previously, scientists believed that microtubules operated primarily at the heart of cells, not near the surface, to guide biological cargo.

In another research paper, Simon and Jan Schmoranzer, Ph.D., a former visiting student at Rockefeller, reported in the April issue of Molecular Biology of the Cell that these microtubule highways also are responsible for exporting biological cargo, a process called exocytosis, all the way out of a cell. In the same paper, the researchers demonstrated that microtubules form an extensive labyrinth of roads across cellular surfaces (see news release at http://www.rockefeller.edu/pubinfo/041103b.php).

“It turns out that microtubule networks form a cortical network that runs parallel to the cell surface, something not at all expected,” says Rappoport.

The second author of the Traffic paper is Bushra Taha, a former high school student at The Dalton School in New York City who spent last summer in the Simon laboratory as part of Rockefeller’s Science Outreach program. The Science Outreach program provides high school students and K-12 teachers with the opportunity to experience scientific research first-hand. Taha continues to study science at Harvard University. This summer, however, she is back in the Simon laboratory working with Rappoport on a new project.

Hungry Cells

Like people, cells need to eat. They require nutrients, such as iron, as well as hormones, cholesterol and other external molecules to survive. They also must be able to internalize cell surface receptors to communicate with other cells. Most of these needs are met by one streamlined pathway – clathrin-mediated endocytosis.

In this process, biological cargo is drawn into the cell via clathrin-coated vesicles that pinch off then, after losing their coats, wind their way through the cell to various protein-processing locations. Shaped like geodesic domes, clathrin coats were first characterized by Barbara M.F. Pearse at the MRC Laboratory of Molecular Biology in 1975. Scientists now know that these cage-like structures, as well as similar coats worn by different types of transport vehicles, are required for the budding off of vesicles, yet the full details are not known. (The beautiful domes of clathrin coats are visible only via electron microscopy.)

Cells on celluloid

To further study the mechanisms underlying endocytosis, the Rockefeller researchers turned to Total Internal Reflection Microscopy, a refined fluorescence microscopy technique designed to peer into the surface layer of cells. In traditional fluorescence microscopy, scientists tag proteins of interest with different-colored fluorophores. Then, they have two choices. One option is to fix the relevant cell with a cross-linking agent, such as para-formaldehyde, before visualizing it under an epi-fluorescence microscope. But this method can only offer a snapshot of what takes place in a protein’s life.

A second option is real-time imaging of a living cell, but again there are limitations. To begin with, exposing the entire cell to continuous high energy radiation causes photo-damage, which essentially cooks the cell. Furthermore, when exciting fluorophores above and below the plane of focus, the image is often too blurry to be of use.

TIRFM overcomes both of these limitations by selectively exciting only those fluorophores that happen to be near the surface of living cells (within approximately 100 nanometers).

Caught in the act

The researchers used TIRFM to follow multiple proteins simultaneously. They fluorescently tagged both clathrin and microtubule proteins, and soon thereafter were watching the once inert clathrin pits ride along microtubules across the surface of cells.

“By monitoring multiple proteins over time, you can gain a more complete understanding of a biological mechanism,” says Simon. “Traditional snapshot methods of single proteins are analogous to trying to understand the rules of chess by following one piece per game without any knowledge of what the other pieces are doing.

“When we did this in our studies, we were surprised to see numerous proteins, such as clathrin, dynamin, adaptins and microtubules, come together or separate at steps that did not coincide with the cartoons in the textbooks.”

Taha adds, “By visualizing our subjects first-hand, we were able to obtain results in very little time.”

The findings immediately raise several questions. For example, at this point, it is not clear whether the clathrin pits’ movement is occurring before or after the vesicles have budded. Of course, the most obvious question remaining is “the why.” Why are the clathrin pits traveling across cellular surfaces? Though the researchers admit they really don’t know, according to Rappoport, they have already begun to use their favorite instrument – their eyes – to find out.