Caught on Camera

Pushing Boundaries

Like a person or a country, a cell has boundaries. Yet a cell’s thin, skin-like border doesn’t just define a living space. It also hosts an abundance of life-sustaining activity. Certain molecules embedded within the double-layered membrane control access to the cell’s interior, while others sense stimuli such as light, hormones or mechanical force. Still others package cargo, interact with its skeleton and much more.

To understand how membrane proteins carry out these tasks, one lab at Weill Cornell Medicine captures them in action on video.

Using an advanced imaging technique known as high-speed atomic force microscopy (HS-AFM), Dr. Simon Scheuring, a professor of physiology and biophysics, and his colleagues make the molecular equivalent of documentaries, capturing the behavior of these elusive proteins as it plays out across minuscule fractions of a second within their natural habitat, the membrane.

“When you look at single molecules with HS-AFM, it’s like you’re making movies of them going about their business, just like people walking in the street,” Dr. Scheuring says.

The insight his group gleans into these routines casts new light on fundamental processes crucial to health and implicated in disease.

Real World, Real Time

When seeking to see the form and function of proteins, scientists have a handful of options. With techniques known as X-ray crystallography and cryo-electron microscopy, they can create highly detailed snapshots of proteins’ architecture. But to make these images, they must remove the proteins from the membrane and turn them into crystals for crystallography, or flash freeze them for cryo-electron microscopy. To see how a protein moves, they can track fluorescent tags they have applied to the protein, but not the movement of the protein itself.

By comparison, HS-AFM allows researchers to see the proteins themselves at work within the membrane. This approach is uniquely suited to catching the fleeting, and sometimes unexpected, transitions these molecules can undergo.

Although HS-AFM produces imagery, it functions more like the sense of touch than sight. A finely tipped probe, measuring only a few atoms thick at its point, moves over a sample. The tip traces the contours of the surface below it, recording changes in the molecules’ topography as they move.

Events unfold quickly at the molecular scale, so HS-AFM must work rapidly to catch them. Dr. Scheuring and his colleagues are continuing to push the limits on its speed. At present, the fastest version can capture a channel protein opening or closing over approximately 50 millionths of a second.

High-speed Atomic Force Microscopy

This imaging technique uses a fine tip to trace the surface of molecules. A laser reports the position of the tip.

Dr. Scheuring’s lab has used HS-AFM to study many membrane structures, including two on opposing sides in cancer: the immune protein perforin, which punctures cells that pose a threat, and ESCRT, a set of proteins that forms spirals and heals wounds, among other functions.

Perforin

HS-AFM videos have shown that a break in a perforin ring triggers its segments to rotate and unfurl downward, attacking the membrane below. Typically, this transition produces an arc, not a full ring, above the hole it has punctured.

ESCRT

Dr. Scheuring’s lab has explored how this set of proteins forms rings, then spirals, which accumulate mechanical force like a spring and change the shape of the membrane, a necessary step for many processes within a cell.