Carnegie Mellon University

Cell Membranes from the Outside In

For a cell, membranes compartmentalize life. The cell membrane, which envelopes the entire cell, physically separates the interior of the cell from the extracellular space. But it is not just a partition - the cell membrane mediates the transport of ions that regulate neuronal firing, provides a docking site for signaling molecules that allow cells to communicate with one another and contains molecules that allow the immune system to identify a cell as "self" rather than "non-self." Within the cell's complex interior, membrane-bound compartments carry out critical biochemical processes, such as protein processing and energy production.

Despite their importance, there is still much to learn about membranes. Because the lipids and proteins that form membranes constantly move, shifting and rearranging themselves to serve the cell's needs, studying membranes is exceedingly difficult. This dynamism stymies scientists because traditional experimental techniques, like crystallography, don't work well with a fluid membrane, explains Physics Professor Mathias Lösche.

"Membranes are intrinsically disordered. If you want to study something using crystallography, you need to crystallize it to study it with X-rays. So you need an ordered structure that repeats itself over and over. That is something you cannot do with membranes, therefore you need to devise new characterization techniques and new approaches, both in experimental and theoretical physics and biology, in order to study these issues."

MCS scientists have invented and are using a suite of tools to gain a deeper knowledge of the molecular properties of membranes. This work has important implications for understanding the normal behavior of cells and what goes wrong in diseases like HIV, Alzheimer's disease and protein processing malfunctions that lead to cancer and neurological disorders.

Sneaking Past the Cellular Sentinel

The cell's outer membrane acts as a gate, preventing intruders from invading the cell. But viruses, like HIV, manage to sneak through the cell's protective barrier. Stephanie Tristram-Nagle, associate professor of research in biological physics, and her collaborator John Nagle, professor of physics and biological sciences, recently made an important discovery that aids the understanding of why HIV is able to gain access to immune cells with such apparent ease.

Scientists have known for more than 20 years that HIV fuses with immune cells via gp41, a protein located on the virus' surface. Although scientists have X-ray images of gp41 before and after it fuses with the cell membrane, understanding precisely what happens during fusion was a mystery, until now.

Tristram-Nagle and Nagle prepared stacks of thousands of fully hydrated, lipid bilayers using a novel method developed in their lab. The lipids at the bottom of the stack are attached to a solid support, giving the model membrane the necessary stability to be studied experimentally, while the lipid bilayers at the top of the stack retain their natural fluidity, a key requirement for any biologically-relevant model system. Tristram-Nagle seeded the artificial membranes with HIV fusion peptide 23 (FP-23), a short stretch of gp41 known to play a key role in viral fusion. Using the X-ray diffuse scattering technique they pioneered, Tristram-Nagle and Nagle quantified structural properties of the lipid bilayers in the presence of FP-23. After analyzing the diffuse X-ray data, they found that FP-23 dramatically decreases the energy needed to bend the membrane, making it much easier for the virus to fuse with and infect immune cells.

"In cells, membranes are bending all of the time, which requires energy," said Tristram-Nagle. "We found that the energy needed to bend the membrane is greatly decreased - by up to 13 fold - when we added FP-23. This should help explain, in part, how HIV infection occurs so readily."

A New Twist on a Century-Old Debate

In its role as gatekeeper, the cell membrane regulates molecular traffic into and out of the cell via specialized membrane proteins. Ion channels, proteins that span the cell membrane, are a prominent example. By regulating ions that enter and exit the cell, ion channels are a vital component in the initiation and propagation of electrical impulses in nerve cells. A dysfunction in either the ion channel itself or the membrane in which it resides can result in a variety of neurological disorders, including Alzheimer's Disease.

In the brains of those suffering from Alzheimer's Disease are insoluble plaques that contain misfolded peptides called amyloid beta (Aβ). The Aβ plaques build up between nerve cells and have been implicated in the disease since Alois Alzheimer first discovered them 100 years ago. In recent years, scientists have speculated that Aβ oligomers - aggregates of Aβ intermediate between the single peptide and the mature plaques - interact in some way with nerve cell membranes, but the actual mechanism of cell toxicity remains unclear.

"We know that Aβ oligomers interact strongly with membranes and interfere with their capability to preserve ion gradients between the inside and outside of the cell. However, it remains an enormous challenge to determine if Aβ oligomers actually make a hole in the membrane, or if they affect membrane properties just enough to alter critical properties of membrane channels" Lösche explains.

With neutron scattering techniques, Lösche and colleagues at the National Institute of Standards and Technology (NIST) are studying how Aβ interacts with synthetic membrane models called "tethered bilayer lipid membranes" (tBLMs). The tBLMs are made of a lipid bilayer that is chemically linked to a solid substrate, for example a silicon wafer, via polymer tethers.

"These tethered membranes are very powerful because they are extremely stable. We can manipulate and measure them over extended periods. That is extraordinary for an exquisitely fragile leaflet of fluid material of 5-nanometer thickness," said Lösche.

In collaboration with chemists at the University of California at Irvine, Lösche's team incubated tBLMs with Aβ oligomers and studied the membrane's structural and functional response at the NIST Center for Neutron Research in Gaithersburg, Md. They observed a breakdown of the insulating properties of the lipid bilayer, which causes the membrane to leak ions. But the signature of the membrane leakage is distinct from that of other membrane dysfunctions, which Lösche's group has studied in detail. For example, some bacteria release a toxin, alpha-hemolysin, that inserts into host cell membranes, forming channels that cause water-filled ion leakage. In comparing alpha-hemolysin's mode of operation to that of Aβ, it is becoming clear that Aβ oligomers don't just "punch holes" in the membrane, according to Lösche. 

Lösche's group now extends this work in a collaboration with Markus Deserno, associate professor of physics, who develops computer models of cell membranes.

"You can do so much with experiments, but due to the intrinsic disorder of the biological membrane it is impossible to look at all the aspects of atomic detail or of molecular dynamics. These things can be done on a computer," explains Deserno.

Deserno and colleagues at the Max Planck Institute for Polymer Research in Mainz, Germany, created a computer simulation that follows the behavior of an artificial membrane consisting of 50,000 individual lipid molecules. Each lipid molecule is simply represented as three spheres.

"Our model is coarse-grained," explains Deserno. "You can think of it as an impressionist painting. At a distance, everything looks good. You can see water lilies or ballerinas. But up close, all the details are gone; you just see blotches of color. We're interested in what's happening with the water lilies, not the blotches of color," he says.

With this coarse-grained model, Deserno can capture important characteristics, like how the membrane bends and curves, which allows him to ask questions that are beyond the atomic level but less than the level on an entire cell. His model is also versatile; he can add specific proteins of interest to the lipid membrane and observe how they interact. The next step for Deserno and Lösche is to seed the artificial membrane in Deserno's computer model with Aβ proteins to obtain more clues about how Aβ damages the membrane.

Membrane Bound

The cell's outer membrane isn't the only membrane carrying out critical, life-sustaining processes. Cells compartmentalize their interiors into membrane-bound organelles, such as the endoplasmic reticulum (ER) and Golgi apparatus, to perform various tasks - such as protein production - more efficiently.

But an organelle's membrane is much more than just a barrier. Membranes play a key role in transporting proteins from the ER to the Golgi apparatus, within the Golgi and then from the Golgi to their final destination within the cell.

"Many diseases occur when there is a malfunction in membrane trafficking," said Adam Linstedt, professor of biological sciences. Researchers have discovered that dozens of human genetic disorders result from defects in membrane trafficking, including several neurodegenerative diseases and developmental disorders.

Linstedt, together with Christina Lee, assistant professor of biological sciences, is investigating the membrane trafficking pathways in the Golgi and the ER and learning a great deal about the organelles' structure along the way.

Linstedt has identified a group of proteins involved in forming the Golgi ribbon, a complex structure of Golgi subcompartments, or stacks, interconnected by tubules. The stacks, and the enzymes within them, function as an assembly line, processing thousands of newly synthesized proteins and lipids moving through the Golgi. As a newly synthesized protein moves within a stack, enzymes modify the protein by adding components like carbohydrates or phosphates. By the time the protein leaves the Golgi, it has been fully processed. Using a technique called RNA interference, Linstedt inhibited the expression of the Golgi proteins GM130 and GRASP65 and found that the Golgi stacks didn't come together into a ribbon. In cells without a ribbon, Linstedt found that some of the unlinked stacks had higher levels of enzymes while others had lower levels, unlike when the stacks are oriented in a ribbon and have an equal distribution of Golgi enzymes. Uniform enzyme levels could be critical, according to Linstedt, because cells with an unlinked Golgi ribbon had under-processed proteins. Impaired processing can lead to severe developmental defects in a variety of organisms, from mice to humans.

"No one knew what the ribbon was for," ex- plains Linstedt. "Now we have one explanation - the ribbon is important for equilibrating the enzyme concentration across the entire membrane network and is necessary for correct processing of proteins."

The Golgi plays an important role in the final processing of proteins, but proteins are initially assembled in the ER, a single, continuous membranous network that stretches from the nucleus to the cell membrane. Lee takes a biochemical approach to studying the ER, breaking open cells and washing with salt, which removes molecules electrostatically linked to the ER membrane. After testing these molecules one at a time to see their effect on the ER membrane, Lee identified a key factor involved in the formation of an extended ER network. The factor, a variant of the enzyme nucleoside diphosphate kinase (NDKB), was already known to function in cells but hadn't been connected to membrane morphology. It turns out that NDKB binds directly to acidic phospholipids in the ER membrane and may assemble to form a scaffold that stabilizes the extended membrane network.

"There is an enormous flux of membrane that starts in the ER and moves out to the cell surface," Linstedt explains. "Much of this membrane flux is generated by vesicles forming from one compartment and fusing with the next compartment. Conceivably, if we understood these processes better, we could target the membrane trafficking pathways to fight disease."

As MCS scientists use the tools of biology to understand basic cell function and the tools of physics to tease out the physical properties of membranes, they move one step closer to understanding how membranes function in health and disease.

"I believe that if biologists and physicists and engineers and mathematicians all puzzle together, we'll arrive at new insights much faster and with greater confidence," said Deserno.