The Membrane Metro

by Kathleen M. Wong

Randy Schekman is also a Howard Hughes Medical Investigator and editor in chief of the scientific journal Proceedings of the National Academy of Sciences. Photo credit: courtesy Randy Schekman

Cells are nature's protein factories. They build enzymes, antibodies, neurotransmitters, and a host of other critical molecules on a nanoscale assembly line. The moment a new protein is completed, it is swept into an intricate packing and shipping system that rivals the U.S. Postal Service.

First, a bubble-like membrane forms around the new proteins and ferries them to their next destination. Upon arrival, the molecules of each transport vesicle then merge with the membrane surrounding the cell itself or an organelle. In doing so, they pass their protein cargo to the other side of the oily wall.

Randy Schekman, a Berkeley professor of cell and developmental biology, has devoted his career to describing this transport system in great detail. His findings are helping to explain a host of human maladies such as diabetes, hemophilia, and Alzheimer's disease.

Upon joining the Berkeley faculty in 1976, Schekman began studying protein transport and secretion in yeast. This modest organism, he says, was key to pinpointing each step of the protein trafficking process. "Yeast has much of the complexity of a mammalian cell, but you can clone them and do genetics with great precision. It's possible to isolate mutations and study their effects."

A schematic of the protein secretion pathway. COPI and COPII are among the many proteins Schekman studies that are required for vesicle budding. Image credit: courtesy Randy Schekman

In the ensuing years, Schekman isolated mutant yeasts with hang-ups at many stages of the protein shipping system. In this way, he was able to identify almost 50 genes critical in protein transport and secretion. These are responsible for the scaffolding that captures new proteins for transport; channels that direct proteins into an organelle called the endoplasmic reticulum, or ER; budding vesicles from existing membranes; and many other functions. Other researchers have confirmed that these secretory genes are nearly identical to those that control protein secretion in humans and other mammals. For this work, Schekman was named a 2002 winner of the Lasker Award, considered the U.S. equivalent of the Nobel Prize.

He has now expanded his research into protein secretion malfunctions in human diseases. "There are a lot of weird maladies you'd never connect to a core process like protein secretion. We've been able to figure them out because once we know where the mutation is, we can go in and see how it screws things up and reproduce the steps in a test tube."

One of these is called cranio-lenticulo-sutural dysplasia, or CLSD. Found in a Bedouin family from Saudi Arabia, CLSD prevents the soft spot on the top of a baby's skull from fusing properly in adulthood. The disease is caused by a mutation that distorts the structure of the ER, preventing the protein capturing scaffolding from assembling properly.

Schekman has now turned his attention to Alzheimer's disease. This devastating disorder typically appears in elderly patients. But some families begin developing the memory loss and brain plaques characteristic of Alzheimer's as early as their 40s and 50s.

Schekman suspects the problem stems from a backup in their secretory pathways. Affected families have mutated versions of an enzyme called gamma secretase, a kind of molecular shears that snips proteins. The mutations appear to delay the enzyme's departure from organelles like the ER. While waiting, the mutant enzyme trims a membrane protein called amyloid precursor protein, or APP, in a slightly different place than the normal edition. "It generates a section only two amino acids longer. But that difference makes it much worse. It has a much higher propensity to aggregate into plaques," Schekman says.

Electron micrographs of thin sections of skin cells from a patient with the craniofacial disorder CLSD. The disease distorts the structure of the endoplasmic reticulum (ER), disrupting protein transport and ultimately preventing the soft fontanels atop a baby's skull from fusing at maturity. Image credit: courtesy Randy Schekman

Schekman also serves as co-director of the Berkeley Stem Cell Center, a group of faculty studying the science and societal implications of stem cell technologies. Stem cells may be somewhat removed from protein secretion, but his interest in the subject is intensely personal. Schekman's wife has Parkinson's disease, a condition scientists hope someday to treat with stem cell therapies.

The Center includes not only scientists but also legal and humanities scholars. "I have been keen all along to have a program that includes not just hard science but also involves the humanities and social scientists, and takes advantage of Berkeley's strengths," Schekman says.

Schekman has helped obtain support for the Center from the California Institute for Regenerative Medicine, or CIRM, and from private donors. This enables the Center to sponsor monthly round tables, an annual retreat, and seminars. One to two floors of the Li Ka Shing Center for Biomedical and Health Sciences, slated to open in 2011, will be dedicated to the labs and offices of stem cell scholars.

Related Web Sites

• Schekman Lab
• Berkeley Stem Cell Center
• "Wit and Wisdom: One researcher's approach to science and service has a lasting impact on many," by Evelyn Strauss, HHMI Bulletin, February 2008
• Protein trafficking, Virtual Cell Animation Collection

Habitat for Human Stem Cells

by Kathleen M. Wong

David Schaffer is also codirector of the Berkeley Stem Cell Center.

With the power to transform into any tissue, stem cells hold great promise for the future of medicine. Scientists hope someday to be able to transplant stem cells into the body to repair injured nerve cells, regenerate diseased organs, and replace defective tissues. But stem cells are also prima donnas. They're tricky to keep alive in the lab, difficult to multiply, and must be coaxed with a series of largely unknown cues before they will differentiate on demand.

Once stem cells are removed from their habitat within the body, "they lose the signals they need to mature and repair tissues," says David Schaffer, a Berkeley professor of chemical engineering. "To control their behavior, we need to learn the way in which their natural microenvironment, or niche, feeds signals to cells to regulate their behavior."

Schaffer is identifying the elements that constitute good stem cell habitat. By examining the roles of small molecules, proteins, mechanical stressors, and other factors, he hopes to learn how the body controls stem cell behavior. He is now developing synthetic ways to mimic these factors, to speed the day when stem cell therapies are a trusted part of modern medicine.

The adult brain adds new neurons throughout human life. Here, stem cells in the adult hippocampus (blue) divide and differentiate into mature neurons (green), whose function is supported by neighboring astrocytes (red). Image credit: David Schaffer and Karen Lai, Schaffer Lab

The environments in which stem cells live are rich indeed. Resting atop a cushion of membrane proteins, stem cells are also surrounded by a matrix of neighboring cells and bathed in a mixture of proteins and other small molecules. Each communicates developmental messages via the cell's surface receptors.

"It sounds like a complex recipe to reconstitute outside the body, but we've come a long way in trying to reassemble niches from elemental pieces to create environments that can really regulate cell behavior," Schaffer says.

For example, like Goldilocks, stem cells care about surface hardness or softness. Schaffer found that by growing neural stem cells atop surfaces of varying stiffness, he could alter their fate. Those raised on soft surfaces developed into neurons. Those raised on hard surfaces matured into supporting glial cells. And cells on surfaces of intermediate rigidity multiplied while still in their immature state.

Neural stem cells isolated from the adult hippocampus can be grown in cell culture and differentiated into the three major cell types of the nervous system, including astrocytes (red) and oligodendrocytes (green). Nuclei are labeled blue. Image credit: Linda Hinh and David Schaffer, Schaffer Lab

But exterior cues tell only half the story. The rest occurs inside stem cells themselves. Environmental inputs such as surface hardness get filtered through elaborate networks of surface receptors and signaling pathways. This processed message is ultimately what turns genes on and off in the nucleus, and then guides cells into making important decisions like continuing to divide, or converting into specialized cell types that can potentially be used to treat disease.

Schaffer is untangling stem cell signaling networks to write a truly complete stem cell control manual. Using a combination of laboratory experiments and computer models, he is refining ideas of how certain signals affect molecular pathways and gene transcription. "We view these networks as nothing more than computational devices. They take in information, compute or process it, and the cell switches behavior as a result," he says.

Schaffer is now applying what he knows about stem cell behavior to engineer artificial environments for them in the laboratory. At present, most human embryonic stem cells are cultured on a commercial cocktail of hundreds of proteins derived from mouse tumor cells. But this concoction is hardly suited for medical use, Schaffer says. "Its contents are poorly defined, so it raises concerns for reproducibility, safety, virus infection and other issues that will be important to consider when using stem cells in patients."

Viruses can be used to deliver genes to the adult brain, revealing gene function in the nervous system and neural stem cells. Here, a lentivirus carrying the gene for green fluorescent protein (GFP) has been injected into the adult brain. Nuclei are blue. Image credit: Julie Yu, Schaffer Lab

To address this problem, Schaffer and fellow Berkeley engineering professor Kevin Healy are developing a 100-percent synthetic, chemically defined medium for embryonic stem cell growth. Instead of using complex whole proteins that can only be manufactured by live cells, they plan to use only snippets in direct contact with cell receptors. These pieces are small enough to manufacture and can be attached to an artificial surface. Their artificial matrix would be cheaper and easier to produce on a large scale for clinical use, and could keep the costs of future stem cell treatments affordable.

So far, this effort has identified a handful of protein bits that stimulate critical stem cell receptors. The present version of his matrix can keep human embryonic stem cells dividing for several weeks at a time. Future versions might help guide stem cells to differentiate into specific cell types.

"We know stem cells have all these incredible properties. The challenge is now to control that process better to turn stem cells into powerful, affordable and viable therapies," Schaffer says.

Related Web Sites

• Schaffer Lab
• David Schaffer, Berkeley Stem Cell Center
• "'Evolved' virus may improve gene therapy for cystic fibrosis," by Robert Sanders, Berkeley NewsCenter, 17 February 2009
• "Scientists force virus to evolve as better delivery vehicle for gene therapy," by Robert Sanders, Berkeley NewsCenter, 07 February 2006
• "Scientists exploit HIV's noisy genetics to force virus into latency," by Robert Sanders, Berkeley NewsCenter, 08 August 2005

The Making of T Cells

by Kathleen M. Wong

Ellen Robey is also a faculty member of the Berkeley Stem Cell Institute.

T cells are the linchpins of the human immune system. They nab cells gone bad due to infection or cancer, can rekindle the fight against familiar pathogens, and ensure immune tolerance for the body's own cells.

For an immature T cell, getting a chance to join this elite corps is rare indeed. Ninety-nine percent of those that begin the maturation process don't make the grade. The receptors they carry, which someday might recognize an invader or other foreign substance, must first be tested to ensure they won't elicit an autoimmune response. Only those that pass muster eventually enter the bloodstream as mature T cells.

"We're trying to understand that maturation and winnowing process in the thymus that allows only useful and not dangerous T cells to emerge," says Ellen Robey. A UC Berkeley professor of immunology and pathogenesis, Robey is uncovering how T cells mature and go on to manage infections. She is now working to culture T cells from mouse and human stem cells, laying the foundation needed to manufacture these immune cells for medicine.

Some blood cells, such as T cells and neutrophils, specialize in finding cells that have fallen prey to infection. To observe this process, Robey is studying how immune cells interact with the protozoan Toxoplasma gondii, which causes largely symptomless infections in up to 50 percent of Americans. Here, neutrophils (green) are responding to Toxoplasma (red/orange) within a lymph node. Source: Chtanova et al (2008) Immunity Sep; 29(3):487

All the action in T cell formation is hidden behind the walls of the thymus. But Robey has found a way around this hurdle. Instead of studying just fixed, stained tissue slices, Robey has helped pioneer the use of a new technique that lets scientists observe the three-dimensional movements of cells within an intact, living organ.

Robey's videos have revealed how dynamic the T cell maturation process is. Using two-photon laser-scanning microscopy, she has observed immature T cells meandering around in search of cells while they undergo their first survival test in the outer region of the thymus. Developing T cells that pass this test then make a beeline toward the central region of the thymus, where they undergo a second, self-compatibility test. The one percent of cells that pass both exams eventually enter the bloodstream to join the pool of mature T cells.

"Before we started these studies, we had no idea how developing T cells traveled between different regions of the thymus, or even that they were motile. It was a surprise to see how actively they were migrating around, sampling their environment by random walk, and then undergoing rapid directional migration toward the next destination once they received the appropriate signals," Robey says.

T cells (blue cells, white tracks) migrating around Toxoplasma cysts (red) and components of the immune system called dendritic cells (green) in the brain. Source: Schaeffer et al (2009) J. Immunol. in press.

She is now identifying which types of cells the new T cells contact, quantifying how long those contacts last, and determining the signaling molecules they are receiving at each stage of development. Some of her newest work reveals how T cells that flunk their tests get the message to self-destruct.

Robey's findings are what's needed to grow T cells for research and, someday, patient therapies. Such treatments could revolutionize the treatment of ailments ranging from HIV to infections to the management of tissue grafts. At present, people receiving organ transplants must now take powerful immunosuppressive drugs. Regulatory T cells which normally provide tolerance for body tissues might be engineered to prevent transplant rejection, too.

One way to manufacture such therapeutic T cells is to start with human embryonic stem cells, or hESC. In theory, hESC have the potential to differentiate into every type of cell in the body. In practice, current hESC lines tend to develop into certain types of cells and not others. Robey has been analyzing more than a dozen available lines to find those most likely to grow into blood cells and T cells.

Until this spring, the use of federal funds was banned for most stem cell lines. So Robey turned to more local sources instead. "Funding from the California Institute of Regenerative Medicine and the Berkeley Stem Cell Center has allowed us to have a space where we can grow and test stem cell lines side by side, to evaluate their different potentials," Robey says.

Like any good gardener, Robey is trying to optimize her harvest by testing out a wide range of growing regimens. But instead of test driving different types of fertilizers and soils, she experiments with culture surfaces, growth factors, and cytokines.

Developing T cells (green cells, colored tracks) in the thymus interacting with the dendritic cells (red) that help specify immune system targeting. Source: Ladi et al (2008) J. Immunol, Nov 15;181(10):7014

The potential to use stem cells to treat patients has profoundly changed Robey's view of her own work. "Until recently I felt there was a big gap between what we do in lab, and, say, how to cure cancer or other human diseases," Robey says. "But I don't feel that way as much now because of the promise of regenerative medicine. And that's made me shift from being a pure basic scientist to focus more on the potential clinical relevance of our work. If we can make this work the way we want it to, we may be able to really do something important for people."

Related Web Sites

• The Robey Lab
• Ellen Robey, Department of Molecular and Cell Biology
• Ellen Robey, Berkeley Stem Cell Center
• Stem cell research: A new era
• "New tool reveals secrets of migrating cells," by Robert Sanders, Berkeley NewsCenter, 03 May 2005
• "Parasite vaccines within reach," by Rachel Tompa, Berkeley NewsCenter, 02 July 2008

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