Biophysicists measure mechanism that determines fate of living cells

Cells in the human body do not function in isolation. Living cells rely on communication with their environment—neighboring cells and the surrounding matrix—to activate a wide range of cellular functions, including reproduction of new cells, differentiation of stem cells into distinct cell types, cell adhesion, and migration of white blood cells to fight bodily infections. This cellular communication occurs on the molecular level and it is reciprocal: a cell receives cues from and also transmits function-activating cues to its neighbors.

The mechanics of this type of cellular interaction have been studied extensively: receptors extending through the cell membrane are activated when they form a bond to specific molecules. Now for the first time, University of Illinois biophysicists at the Center for the Physics of Living Cells and the Institute for Genomic Biology have measured the molecular force required to mechanically transmit function-regulating signals within a cell.

The new laboratory method, named the tension gauge tether (TGT) approach, developed by Taekjip Ha with postdoctoral researcher Xuefeng Wang, and reported in the May 24, 2013, issue of the journal Science, has made it possible to detect and measure the mechanics of the single-molecule interaction by which human cell receptors are activated. The researchers used integrin, a cell membrane receptor protein that is activated when it bonds to a ligand molecule.

In the TGT approach, Ha and Wang repurposed DNA strands, using them as tethers for ligand molecules, to test the tension required to activate cell adhesion through integrin. The integrin bonds to the tethered ligand, and adhesion is activated only if the DNA tether does not rupture (See video animation).

Taking advantage of the geometric characteristics of DNA’s double helix form, the researchers were able to tune the strands to rupture at discrete tension levels: by varying the attachment points along the DNA strands, the force required for rupture was either low (unzipping the helix), high (shearing the strands), or intermediary (combination of unzipping and shearing).

“If you went fishing and a fish broke your 30-lb fishing line but not the 40-lb one, you would know that its strength was in the range of 30­–40 pounds,” explained Wang. “Here we applied the same strategy to measure the molecular tension applied by cells (the fish). Mammalian cells apply a force to activate cell membrane proteins called integrins which mediate cell adhesion. We immobilized ligand molecules (the bait) on a surface through molecular tethers (the fishing line) with defined tension tolerances, tunable from 10 pico Newton (pN) to 60 pN). After integrin-ligand binding, cells apply a force on the bonds, and we compare this force to the molecular tether strength by observing cell adhesion status.”

Since single-molecule interactions are difficult to monitor, the researchers observed the receptor-regulated cellular function, to gauge whether the integrin was activated. Ha and Wang discovered that integrin experiences a well-defined “quantum of force,” about 40 pico-Newton (pN), to activate cell’s adhesion to a surface.

“We observed that mammalian cells adhere on the culture surface with 43 pN tension tolerance of ligands, but not on 33 pN surface. Therefore we deduced that single molecular tension is around 40 pN on integrin cell-membrane receptors during cell adhesion,” Wang added.

“This is a very exciting result,” commented Ha, an Edward William and Jane Marr Gutgsell Endowed Professor at Illinois. “With the ability to define the single molecular forces required to make living cells behave as desired, we may be one step closer to a remedy for certain hard-to-cure diseases. We know that the behavior of cancer cells and stem cells can be controlled by how stiff or soft their environments are. Understanding and manipulating molecular conversation through defined forces has huge implications for the development of future medical interventions. We expect the TGT approach will have broad applications in laboratory studies of cell differentiation, cancer metastasis, as well as immunology and infectious disease.”

Finding a family for a pair of orphan receptors in the brain

Researchers at Emory University have identified a protein that stimulates a pair of “orphan receptors” found in the brain, solving a long-standing biological puzzle and possibly leading to future treatments for neurological diseases.

The results are published in the Proceedings of the National Academy of Sciences, Early Edition.

The human genome is littered with orphans: proteins that look like they will bind and respond to a hormone or a brain chemical, based on the similarity of their sequences to other proteins. However, scientists haven’t figured out what each orphan’s partner chemical is yet.

Orphans that look like GPCRs (G protein-coupled receptors) currently number about 100. GPCRs are the targets of many drugs and are involved in vision, smell and brain cells’ responses to a host of hormones and neurotransmitters. One orphan GPCR, called GPR37, has attracted interest from researchers because it is connected with an inherited form of Parkinson’s disease. It is abundant in the dopamine-producing neurons that degenerate in Parkinson’s. But its partner chemical, or “ligand,” has not been found.

"We reasoned that GPR37 had to be doing something important, besides becoming misfolded in some forms of Parkinson’s," says senior author Randy Hall, PhD, professor of pharmacology at Emory University School of Medicine.

Working with Hall, graduate student Rebecca Meyer devised a way to detect when cells producing GPR37 were reacting with GPR37’s ligand.

"Usually, cells remove GPCRs from their surfaces when they encounter their ligand," Meyer says. "So we set things up so that GPR37 would be labeled red on the surface of the cell, but would appear green once internalized."

They discovered that cells producing GPR37 – and also a close relative, GPR37L1 — respond to a protein known as prosaposin, which was discovered by John O’Brien of University of California San Diego in the 1990s.

Prosaposin is a growth factor for brain cells and protects them from stress. Scientists studying it had worked out that it stimulates cells via a GPCR – but which one was unclear until now. In animal models, prosaposin has shown potential for treating conditions such as stroke, Parkinson’s and neuropathic pain. An artificial fragment of prosaposin called prosaptide has been tested in clinical studies, but it quickly breaks down in the body.

"That’s the reason why it was so important to find the receptor," Hall says. "Then we can actually do some pharmacology."

Now, Hall’s laboratory is planning to look for other compounds that can activate GPR37 as well. These could be more stable in the body than the previously studied protein fragment and thus better potential drugs.

Doctors have reported a few cases of genetic deficiency in prosaposin, leading to severe neurodegeneration. Mice engineered to lack GPR37 have more subtle brain perturbations, so Hall also plans to test the hypothesis that prosaposin acts by both GPR37 and GPR37L1, by “knocking out” both in mice, potentially duplicating the same severe effects seen in the human cases of prosaposin deficiency.

Drugs found to both prevent and treat Alzheimer’s disease in mice

Researchers at USC have found that a class of pharmaceuticals can both prevent and treat Alzheimer’s Disease in mice.

The drugs, known as “TSPO ligands,” are currently used for certain types of neuroimaging.

"We looked at the effects of TSPO ligand in young adult mice when pathology was at an early stage, and in aged mice when pathology was quite severe," said lead researcher Christian Pike of the USC Davis School of Gerontology. "TSPO ligand reduced measures of pathology and improved behavior at both ages."

The team’s findings were published online by the Journal of Neuroscience on May 15. Pike’s coauthors include USC postdoctoral scientists Anna M. Barron, Anusha Jayaraman and Joo-Won Lee; as well as Donatella Caruso and Roberto C. Melcangi of the University of Milan and Luis M. Garcia-Segura of the Instituto Cajal in Spain.

The most surprising finding for Pike and his team was the effect of TSPO ligand in the aged mice. Four treatments—once per week over four weeks—in older mice resulted in a significant decrease of Alzheimer’s-related symptoms and improvements in memory – meaning that TSPO ligands may actually reverse some elements of Alzheimer’s disease.

"Our data suggests the possibility of drugs that can prevent and treat Alzheimer’s," Pike said. "It’s just mouse data, but extremely encouraging mouse data. There is a strong possibility that TSPO ligands similar to the ones used in our study could be evaluated for therapeutic efficacy in Alzheimer’s patients within the next few years."

Next, the team will next focus on understanding how TSPO ligands reduce Alzheimer’s disease pathology. Building on the established knowledge that TSPO ligands can reduce inflammation—shielding nerve cells from injury and increasing the production of neuroactive hormones in the brain—the team will study which of these actions is the most significant in fighting Alzheimer’s disease so they can develop newer TSPO ligands accordingly.