Researchers explain how railways within cells are built in order to transport essential cargos

Posted by Biomechanism

“Complex system transports essential cargoes such as proteins and membrane vesicles.”

Every cell in the human body contains a complex system to transport essential cargoes such as proteins and membrane vesicles, from point A to point B. These tiny molecular motor proteins move at blistering speeds on miniature railways carrying components of the cell to their proper destinations. But just how cells construct these transport railways to fit precisely inside of confined spaces of the individual cells has been a complex question, as it is critical that these railways do not grow too long or come up too short, as that would cause a misdirection of the proteins being transported.

Nine years ago, Goode and his colleagues discovered that a family of proteins called formins stimulate the rapid growth of actin filaments.

Bruce Goode, professor of biology, working in collaboration with the labs of Laurent Blanchoin (Grenoble, France) and Roland Wedlich-Soldner (Munich, Germany), have come one step closer to understanding the elusive mechanics of this process.

In a recent paper published in Developmental Cell, a team led by Goode’s Ph.D. student Melissa Chesarone-Cataldo shows that the length of the railways is controlled by one of its “passengers,” which pauses during the journey to communicate with the machinery that is building the railways.

“The frequency of these chats between the passengers and builders may provide the feedback necessary to say a railway is long enough, and construction should now slow down,” says Goode.

Much like a real construction site, a system must be in place with roadways and transporters to move the building materials. In this case, cellular proteins called actin cables act as the roadways, and the transporters are myosin molecules, nanoscale motor proteins that rapidly deliver critical cargoes to one end of a cell. Each cable is assembled from hundreds or thousands of copies of the actin, which is called a helical filament.

Nine years ago, Goode and his colleagues discovered that a family of proteins called formins stimulate the rapid growth of actin filaments. Recently, the team began to question how a cell controls the power of formins, which tell them when to speed up, when to slow down, when to stop altogether.

Enter Smy1, a myosin-passenger protein.

Goode and his colleagues hypothesized that a passenger protein like Smy1 would provide the perfect mechanism for slowing down formins when roadways are longer and would be carrying more passengers. They tested their theory in yeast cells, where formins construct actin cables that transport building materials essential for cell growth and division. As Goode says, they struck gold.

When they deleted the gene for Smy1, cables grew abnormally fast and hit the back of the cell, buckling and misdirecting transport. When they purified Smy1 and placed it in a test tube with formins they discovered that Smy1 slows down actin filament growth.

To further explore, they tagged Smy1 in living cells and learned that Smy1 molecules are carried on cables by myosin to the formin, where they pause for 1-2 seconds to give formins the message to slow down.

Goode says their working model illustrates that as a cable grows longer, it loads up more and more Smy1 molecules, which are transported on the cable to send a message to the formin to slow down.

“This prevents overgrowth of longer cables that are nearing the back of the cell, but allows rapid growth of the shorter cables,” says Goode.

This paper will help scientists understand the general mechanisms that are used for directing cell shape and division. The next challenge says Goode, is “to find out whether related mechanisms are used to control formins in mammalian cells and understand the physiological consequences of disrupting those mechanisms.”

Scientists discover secret life of chromatin

Posted by Biomechanism 

Chromatin – the intertwined histone proteins and DNA that make up chromosomes – constantly receives messages that pour in from a cell’s intricate signaling networks: Turn that gene on. Stifle that one.

Chromsome spread. Photo: NIH

But chromatin also talks back, scientists at The University of Texas MD Anderson Cancer Center report today in the journalCell, issuing orders affecting a protein that has nothing to do with chromatin’s central role in gene transcription – the first step in protein formation.

“Our findings indicate chromatin might have another life as a direct signaling molecule, that it can signal back to other proteins irrespective of gene transcription,” senior author Sharon Dent, Ph.D., professor and chair of MD Anderson’s Department of Molecular Carcinogenesis and director of the Center for Cancer Epigenetics.

In a series of yeast experiments, Dent and colleagues show that a signal through a histone protein regulates another protein called Dam1 that is involved in the separation of chromosomes during cell division.

Signaling cascades don’t dead-end at DNA

“It’s a basic change in our way of thinking about cell signaling – that all signals go into the nucleus and dead-end at DNA, that they point to chromatin and stop,” Dent said. “Our data show that’s not the case. We have a new fundamental aspect of cellular regulation that we need to now explore.” DNA is tightly intertwined with histones and assembled in histone/DNA units called nucleosomes along the connecting length of a string of DNA. This structure is often described as being like beads on a string.

Genes are turned on by transcription factors, proteins that attach to the gene’s promoter region and order the gene to make an RNA copy of its DNA that can be translated into a protein. Histone proteins regulate access to genes, blocking or facilitating transcription.

Histones and other proteins are modified by the attachment of chemical groups to specific spots on the protein. Attachment of a methyl group (a carbon atom joined to three hydrogen atoms) to a histone can help or hinder gene transcription depending on where the methylation occurs on the histone, Dent said.

Crucial cross-talk between proteins

In a 2005 Cell paper, Dent and colleagues reported that a methyl group-transferring protein called Set1 methylates the protein Dam1, which is part of a structure that assists in the orderly separation of chromosomes during cell division.

Set1 is part of a protein complex that works along with multiple regulatory factors to facilitate transcription by attaching methyl groups to a specific histone, H3, which was the only previously known target of Set1.

Dent’s team set out to discover the exact mechanism by which Set1 methylates Dam1. To their surprise, they found that Dam1 methylation does not depend on gene transcription, revealing news roles for proteins formerly thought to be involved only in that process.

Rather, the crucial step is the attachment of a single signaling molecule called ubiquitin to a histone protein called H2B. This event was known to direct addition of methyl groups to histone H3, but Dent’s work indicates it is also required for methylation of Dam1.

Communication between H2B and Dam1 is the first such instance of cross-talk between histone and non-histone proteins, the authors report. The signaling connection between a chromatin change and a non-DNA-templated process such as chromosome separation is also new.

Connections between histone ubquitination and histone methylation also occur in human cells, and mutations in a protein highly related to Set1, called MLL, are involved in leukemia. Dent’s work raises the possibility that histones can signal to non-histone proteins in human cells and that mismanagement of these events caused by MLL mutations might contribute to leukemia development.

Dent’s group is looking for other proteins that might be affected by histone modifications in both yeast and human cells. And they are studying the details of Dam1 methylation and its function in chromosome separation.

Engineered Stem Cells

The cell factory: James Thomson (above) and Junying Yu first transformed adult cells into stem cells called iPS cells in 2007.
Credit: Kevin Miyazaki

MULTIMEDIA

• Transforming Adult Cells into Stem Cells

James Thompson
(Cellular Dynamics) Engineered stem cells could revolutionize drug discovery

OTHERS WORKING ON ENGINEERED STEM CELLS
Fate Therapeutics, San Diego, CA
iPierian, South San Francisco, CA
George Daley, Children's Hospital Boston, Boston, MA
Shinya Yamanaka, Kyoto University, Kyoto, Japan

 Engineered Stem Cells

Mimicking human disease in a dish.

• BY EMILY SINGER

Audio »

This article is part of an annual list of what we believe are the 10 most important emerging technologies. See the full list here.

The small plastic vial in James ­Thomson's hand contains more than 1.5 billion carefully coddled heart cells grown at Cellular Dynamics, a startup based in Madison, WI. They are derived from a new type of stem cell that ­Thomson, a cofounder of the company, hopes will improve our models of human diseases and transform the way drugs are developed and tested.

Thomson, director of regenerative biology at the Morgridge Institute at the University of Wisconsin, first isolated human embryonic stem cells in 1998. Isolating these cells, which are capable of maturing into any other type of cell, marked a landmark in biology--but a controversial one, since the process destroys a human embryo. A decade later, ­Thomson and Junying Yu, then a Wisconsin postdoc, reached another milestone: they developed a way to make stem cells from adult cells by adding just four genes that are normally active only in embryos. (Japanese researcher Shinya Yamanaka simultaneously published a similar approach.) Dubbed induced pluripotent stem cells (iPS cells), they have the two defining characteristics of embryonic stem cells: they can reproduce themselves many times over, and they can develop into any cell type in the human body. Because no human embryos are used to create them, iPS cells solve two problems that had long plagued researchers: political protest and shortages of material.

Much of the excitement over iPS cells, and stem cells in general, arises from the possibility that they could replace damaged or diseased tissue. But Thomson thinks their most important contribution will be to provide an unprecedented window on human development and disease. Scientists can create stem cells from the adult cells of people with different disorders, such as diabetes, and induce them to differentiate into the types of cells damaged by the disease. This could allow researchers to watch the disease as it unfolds and trace the molecular processes that have gone awry.

In the nearer term, iPS cells may revolutionize toxicity testing for drugs. The cells are "the first unlimited source of any type of human tissue," says Thomson, who founded Cellular Dynamics to put stem cells to practical use. The company sells heart muscle cells derived from its iPS cells to pharmaceutical giants such as Roche, which are using them to screen experimental drugs for harmful side effects. Thomson hopes those cells will help uncover problems early in the drug development process, saving billions of dollars on research and testing. For instance, since the iPS-derived heart cells will beat in a dish, scientists should be able to detect which drugs alter the heart's rhythm. Scientists can also use the cells to study how the heart functions at a molecular level. And the company is developing other cell types, including brain and liver cells. The latter are of particular interest to pharmaceutical researchers, since drug toxicity often shows up in the liver. "Having a model that would predict toxicity before going into humans is incredibly valuable," says Chris Parker, vice president and chief commercial officer of Cellular Dynamics.

By generating iPS cells from people with diverse ethnic backgrounds and genetic conditions, and from those who have reacted poorly to certain drugs, scientists can also gain a better picture of how compounds will affect different people. Thomson and others have already created iPS cells from people with ALS, Down syndrome, and spinal muscular atrophy, among other disorders. While it's not yet clear how well those cells reflect the specific diseases, early research is promising. If it succeeds, researchers hope to use iPS cells to study other disorders and develop drugs to treat them. "That's the thing that would fundamentally change the way drug development happens," says Kyle Kolaja, director of early safety and investigative toxicology at Roche, which has partnered with Cellular Dynamics.

The last decade brought many difficult years for Thomson. His work on embryonic stem cells was a breakthrough, but it also brought intense controversy and media attention, turning him somewhat reclusive. With the rise of iPS cells and Cellular Dynamics, Thomson is beginning to come back to the limelight. "I think the legacy of embryonic stem cells will be that they gave rise to iPS cells," he says. "These cells will be used in creative ways we can't even imagine." 

Solar Fuel

Designing the perfect renewable fuel.

• BY KEVIN BULLIS

Fuel for the future: Joule Biotechnologies' genetically engineered microörganisms can turn sunlight into ethanol or diesel.
Credit: Bob O'Connor

The answer seems to be yes, according to Joule Biotechnologies, the company that Afeyan founded (also in Cambridge) to design this new fuel. By manipulating and designing genes, Joule has created photosynthetic microörganisms that use sunlight to efficiently convert carbon dioxide into ethanol or diesel--the first time this has ever been done, the company says. Joule grows the microbes in photobioreactors that need no fresh water and occupy only a fraction of the land needed for biomass-based approaches. The creatures secrete fuel continuously, so it's easy to collect. Lab tests and small trials lead Afeyan to estimate that the process will yield 100 times as much fuel per hectare as fermenting corn to produce ethanol, and 10 times as much as making it from sources such as agricultural waste. He says costs could be competitive with those of fossil fuels.
If Afeyan is right, biofuels could become an alternative to petroleum on a much broader scale than has ever seemed possible. The supply of conventional biofuels, such as those made from corn, is constrained by the vast amount of water and agricultural land needed to grow the plants they're made from. And while advanced biofuels require less water and don't need high-quality land, their potential is limited by the expensive, multistep processes needed to make them. As a result, the International Energy Agency estimates that in 2050, biodiesel and ethanol will meet only 26 percent of world demand for transportation fuel.

Joule's bioengineers have equipped their microörganisms with a genetic switch that limits growth. The scientists allow them to multiply for only a couple of days before flipping that switch to divert the organisms' energy from growth into fuel production. While other companies try to grow as much biomass as possible, Afeyan says, "I want to make as little biomass as I can." In retrospect, the approach might seem obvious. Indeed, the startup Synthetic Genomics and an academic group at the BioTechnology Institute at the University of Minnesota are also working on making fuels directly from carbon dioxide. Joule hopes to succeed by developing both its organisms and its photobioreactor from scratch, so that they work perfectly together. Still, it's a risky strategy, since it departs from established processes. Usually, a startup sets out determined to do something novel, says James Collins, a professor of biomedical engineering at Boston University and a member of Joule's scientific advisory board, "and it falls quickly back on trying to find something that works ... an old thing that's been well established." Afeyan, however, has pushed the company to stay innovative. This summer, it will move beyond lab-scale tinkering; an outdoor pilot plant is currently under construction in Leander, TX.

As both a venture capitalist and a technologist--he received his PhD in chemical engineering from MIT in 1987--Afeyan is keenly aware of the challenges in demonstrating that a novel process can operate economically and make fuel in large volumes. To minimize the financial risks, he steered Joule toward a modular process that doesn't require large and expensive demonstration plants.

"I'm not saying it's easy or around the corner, because I've done this for a long time," Afeyan says. But he does believe that Joule is onto something big: a renewable fuel that could compete with fossil fuels on both cost and scale. He says, "We have the elements of a potentially transformative technology." 

• MAY/JUNE 2010
• BY KEVIN BULLIS

Oil farm: Arrays of bioreactors filled with Joule Biotechnologies' microörganisms absorb sunlight. Supplied with carbon dioxide and nutrients, the organisms use photosynthesis to produce diesel. As they secrete it, the diesel fraction circulates to a separator that extracts the fuel and sends it to storage tanks.
Credit: Brown Bird Design

VIDEO

Stem-Cell Engineering Offers a Lifeline to Endangered Species

Back from beyond: Scientists have created stem cells from frozen skin samples of the endangered northern white rhinoceros.
Credit: San Diego Zoo

BIOMEDICINE

Stem-Cell Engineering Offers a Lifeline to Endangered Species

A technology used to develop new medical treatments might one day revive endangered or extinct species.

• BY EMILY SINGER

Audio »

In 1972, a group of forward-thinking conservationists in San Diego began freezing skin samples from endangered species. The hope was that science would eventually find a way to use the cells to help revive these fragile populations.

Jeanne Loring and collaborators at Scripps Research Institute have taken a key step toward fulfilling that hope by creating stem cells from frozen skin cells of two such species—the silver-maned drill monkey and the northern white rhinoceros.

In the near term, the researchers plan to build a frozen zoo of stem cells that scientists can use to study the animals' genomes, and perhaps to create stem-cell therapies for the animals. (Drill monkeys living in captivity often suffer from diabetes, a highly active area of research in the human stem-cell field.)

In the longer term, the researchers hope to be able to use the cells to create sperm and eggs, which would be incorporated into breeding programs to boost the genetic diversity of severely limited populations; the white rhinoceros is on the verge of extinction, with only seven animals alive today. They haven't bred in years.

"To think of the foresight they had in the 1970s to start this program," says Loring, director of the Center for Regenerative Medicine at Scripps. At that point, "no genome had been published, and the concept of this ever happening was science fiction." Her team aims to generate stem cells for at least 10 other species, including snow leopards and some species of elephants. San Diego's "Frozen Zoo" houses tissue samples from more than 800 species.

To create the stem cells, Inbar Friedrich Ben, a postdoctoral researcher in Loring's lab, used a technique first developed in 2007 called induced pluripotent stem (iPS) cell reprogramming. A handful of genes that are normally active in the developing embryo are expressed in a differentiated cell, such as a skin cell, causing that cell to revert back to its undifferentiated state.

Much to their surprise, the human genes that are typically used to reprogram human cells could also reprogram skin cells from both the monkey and the rhinoceros, though at a much lower efficiency. Still, the reprogrammed cells showed the defining characteristics of induced pluripotent stem cells; they could both differentiate into various cell types and generate more of themselves. The research was published this week in Nature Methods.

"This method paves the way to save endangered species such as the giant pandas, cheetahs, tigers, gorillas in East Africa, and even extinct species like the [now extinct] bucardo mountain goat," says Robert Lanza, chief scientific officer at Advanced Cell Technology. "It will open the way for new strategies to help maintain biodiversity and to respond to the challenges of large-scale extinctions ahead." Lanza was not involved in the study.

For animals like the white rhinoceros, each death represents a serious loss to the gene pool, which in turn weakens the population. By creating stem cells from animals that died, "their genes could be reintroduced to maintain the survival and genetic diversity of the species," says Lanza. "The bucardo mountain goat could be resurrected using this technology if combined with an ordinary goat breeding program."

Creating a new animal from a stem cell is likely a long way off. Researchers would need to first create sperm or eggs from the stem cells and then use them with sperm or eggs from a living animal to create an embryo. Fertility scientists are avidly searching for ways to develop sperm and eggs from stem cells in order to treat human infertility, and Loring hopes those technologies could be applied to these animals.

Prior to the development of iPS cell reprogramming, Lanza's group used cloning—the method used to create Dolly the sheep—to try to reproduce two species of wild cattle; the guar and the critically endangered banteng. But cloning is ill-suited to species conservation, since it is a technically challenging process that often results in sick or deformed animals.

Lanza says another way to use stem cells to propogate endangered species would be to inject the cells of a more common, related species into the embryo, and then use various experimental techniques to coax the cells descended from the endangered animal to grow into a fetus. His team has shown this approach works in mice.

The Avengers | Teaser Trailer | FULL HD

The Avengers (Teaser Trailer)

The Avengers Trailer 2012