Scientists uncover the nuclear life of actin

ORIGINAL: Physorg

First discovered in muscle cells more than 70 years ago, actin has a well-established identity as a cytoplasmic protein that works by linking itself in chains to form filaments. Fibers formed by these actin polymers are crucial to muscle contraction.

Figure 1: F- actin, a controller of organism motion. F-actin acts as a motor for movement in one direction. As the filament pushes the cell membrane from inside the cell, cell projections are formed, and cells move. ORIGINAL: Riken Research

Figure 3: F-actin structure. G-actin consists of two twisted domains. When constructed into F-actin, the domains of G-actin untwist into a flat comformation.
Figure 4: Structural change in actin and hydrolysis of ATP. The hydrolysis of ATP is hypothesized as follows. G-actin (upper) polymerizes to form F-actin and assumes a flat comformation (lower). A region around the amino acid anchoring a water molecule deforms to allow the water molecule to approach the ATP. Upon rotation of the ATP-bound domain (yellow), the ATP approaches the water molecule, allowing the ATP and water molecule to react leading to ATP hydrolysis. ORIGINAL: Riken Research

So it came as a surprise when scientists discovered actin in the nucleus. Labs have been working for the past few decades to figure out exactly what it's doing there.

A new study published this week in Nature Structural & Molecular Biology reveals that actin has a new and fundamental nuclear function, and that surprisingly, it accomplishes this task in its single-molecule (monomeric) form – not through polymerization.

Senior author of the study Xuetong "Snow" Shen, Ph.D., associate professor in The University of Texas MD Anderson Cancer Center Department of Molecular Carcinogenesis, has been fascinated by the mystery of nuclear actin. In collaboration with researchers from Colorado State University, his lab developed a unique model system to nail down actin's function in the nucleus by studying the actin-containing INO80 chromatin remodeling complex.

In 2000, as a postdoc at NIH in Carl Wu's lab, Shen identified actin as a component of the INO80 complex, adding to the growing list of evidence that actin indeed has a life in the nucleus. However, how actin actually works in the nucleus remains fuzzy due to lack of clear experimental systems.

"Our model system opened up a new opportunity to look in depth at the function of nuclear actin as it relates to gene regulation, genome stability, and ultimately cancer," Snow said.

A nuclear role for monomeric actin

Because yeast have only a single actin gene, the authors reasoned that studying INO80 in yeast cells would allow a direct assessment of the protein's nuclear function. In contrast, mammals have at least six forms of actin coded by separate genes, making their study more difficult.

The researchers used both genetic and biochemical methods to dissect actin's role in the INO80 complex. The INO80 complex normally functions in the nucleus to rearrange chromatin ¬– the intertwined proteins and DNA that are packaged into chromosomes – regulating the expression of many different genes.

The authors found that a mutant form of actin impairs the ability of INO80 to function correctly, implicating actin in the process of chromatin remodeling – an exploding field of research with applications in cancer diagnosis and treatment.

In the cytoplasm, actin functions primarily as a polymer. Cytoplasmic actin is a component of the cytoskeleton and the muscle contractile machinery, and is essential for cell mobility, including cancer metastasis. Actin inside the INO80 complex is arranged in a clever way such that it cannot polymerize; instead, actin's monomeric form appears to interact with chromatin.

"Our study challenges the dogma that actin functions through polymerization, revealing a novel and likely a fundamental mechanism for monomeric nuclear actin," Shen said.

New findings for an ancient complex

Because actin and several of the other INO80 components are so highly conserved, even in human cells, this mechanism likely represents an ancient, fundamental role of actin, which has been preserved through evolution.

Shen's group is now teasing out the exact mechanism by which nuclear actin interacts with chromatin. They also hope to extend the results to human cells and to identify potential ways by which nuclear actin could be involved in cancer.

Chromatin is critical for maintaining the delicate balance between gene activation and repression, Shen said. "Disrupting this regulation can lead to cancer, and it remains to be seen whether nuclear actin has a role in this process."

Journal reference: Nature Structural & Molecular Biology

Provided by University of Texas M. D. Anderson Cancer Center

Scientists discover a periodic and ringed cytoskeleton within axons

ORIGINAL: HHMI

JANUARY 25, 2013 

3D STORM images of spectrin in the axons of hippocampal neurons. The color codes for z-position information. Image: Zhuang lab

The plasma membranes that give cells their shapes are typically upheld by linear meshworks of the protein actin. In contrast, Howard Hughes Medical Institute scientists have now discovered that periodic ring-shaped actin arrangements encircle the long axonal fibers of nerve cells.

The actin structures loop around axons at perfect intervals, which are connected by an associated cytoskeletal protein, spectrin. “No one has ever seen periodic rings like this formed by actin before,” says HHMI investigator Xiaowei Zhuang, a biophysicist at Harvard University who led the study. The authors, who published their findings in the January 25, 2013, issue of the journal Science, suggest that these periodically distributed actin rings, connected by flexible spectrin tetramers, lend a flexible yet robust support for the skinny nerve fibers.

“No one has ever seen periodic rings like this formed by actin before.”

Xiaowei Zhuang

The cytoskeletal protein actin is found in all but the simplest cells. Its function is strongly influenced by the forms created by the organization of actin molecules. For example, a branched network of actin at the leading edge of motile cells helps them crawl and the actin-spectrin lattice of hexagons and pentagons in red blood cells helps the cells withstand compression as they circulate through vessels. Although experiments have shown that brain cells rely on actin for growth and stabilization of axons, among other things, the way in which actin is organized in the axon fibers has remained unknown.

Visualizing actin’s form inside cells can help researchers better understand its function. However, individual actin filaments are so thin and so densely packed in cells that researchers have had difficulty distinguishing them using standard optical imaging techniques. But in 2006, Zhuang invented a method for imaging cells with resolution that far exceeds that achieved with conventional light microscopes. Last year, she and her colleagues directed this method at actin in the brain.

One of several methods of “super-resolution” imaging developed in the last decade, Zhuang’s STORM (stochastic optical reconstruction microscopy) allows researchers to overcome limits imposed on convention light microscopy due to the diffraction of light. STORM works by turning on only a subset of fluorescent molecules in the sample at a time, and taking snapshots of these tiny glowing molecules at different times, so that their positions can be determined with very high precision. The coordinates of these fluorescent molecules are used to reconstruct an image of the sample. Zhuang describes it as a pointillistic approach.

Zhuang and her team decided to use the technique to examine fluorescently labeled actin within rat and mouse neurons. With STORM, the team was able to resolve structures as small as 10 nanometers (a ten-millionth of a millimeter) – about 20 times smaller than high-power confocal scanning microscopes can manage.

When Zhuang asked her team to image actin in neurons, their original target was the junctions where axons transmit messages to their neighboring cells – the synapses. However, post-doctoral researcher Ke Xu paused when he noticed regularly spaced fluorescent dots along the axons, instead of the expected continuous glowing lines. Xu and Guisheng Zhong, another postdoctoral research in the lab, investigated further and eventually revealed the novel cytoskeleton structure.

“When they first noticed the seemingly periodic pattern, it was not so obvious at all,” says Zhuang. “These talented guys were so persistent in overcoming one difficulty after another to optimize the experimental conditions and eventually generate these beautiful and convincing images. I really love that they did not let go of this unexpected observation and simply go back to imaging synapses, as I asked them to.”

Unveiling a new structure for actin proves STORM’s worth, Zhuang says. “Making a scientific discovery is extremely rewarding for people like us who develop new methods. It is the ultimate validation of a method.”

The dots turned out to be individual rings of actin encircling axons just below the cell membrane, along their length. Each actin ring consists of small actin filaments. The rings occur between spectrin spacers about 190 nanometers in length, which give the structure its periodicity. The team thinks that this ribbed structure might endow long axons with an ability to weave through the nervous system without snapping, as well as endure mechanical stress when an animal moves.

For comparison, the team also imaged actin within dendrites, neurons’ branched projections. There, actin formed long filaments that ran along the dendritic shaft.

Next, the team discovered a sign that the periodic actin and spectrin rings might influence how nerve cells communicate with each other. Neurons communicate through action potentials, in which ion channels embedded in their membranes generate electrical charge that rises and falls quickly. The team found a periodic pattern among sodium ion channels in the axon membranes that corresponded to the underlying ringed lattice. “One can’t help but wonder whether this periodicity influences the action potential,” Zhuang says.

Just as the ultimate effect of ion channel periodicity remains mysterious, so too does the development of the actin and spectrin rings, and whether they indeed confer flexibility and durability as the team hypothesizes. “This is just the beginning.” says Zhuang. “We discovered something so novel that we have opened up more questions than we answered.”

HHMI INVESTIGATOR 

Xiaowei Zhuang

Single-Molecule Imaging of Biomolecular and Cellular Processes

Growing implant tissue on 3-D scaffolds

ORIGINAL: MIT News

Anne Trafton, MIT News Office

Cells grown on different types of scaffolds vary in their ability to help repair damaged blood vessels.

Laura Indolfi, left, holds up a sample of a sponge-like scaffold that she and Elazer Edelman, right, used to show that implanted cells’ therapeutic properties depend on their shape. Photo: Patrick Gillooly

Tissue implants made of cells grown on a sponge-like scaffold have been shown in clinical trials to help heal arteries scarred by atherosclerosis and other vascular diseases. However, it has been unclear why some implants work better than others.

MIT researchers led by Elazer Edelman, the Thomas D. and Virginia W. Cabot Professor of Health Sciences and Technology, have now shown that implanted cells’ therapeutic properties depend on their shape, which is determined by the type of scaffold on which they are grown. The work could allow scientists to develop even more effective implants and also target many other diseases, including cancer.

“The goal is to design a material that can engineer the cBells to release whatever we think is most appropriate to fight a specific disease. Then we can implant the cells and use them as an incubator,” says Laura Indolfi, a postdoc in Edelman’s lab and lead author of a paper on the research recently published online in the journal Biomaterials.

Aaron Baker, a former postdoc in Edelman’s lab and now an assistant professor at the University of Texas at Austin, is also an author of the paper.