amudu: January 2013

Axons' Unexpected Cytoskeleton Structure

A3D STORM images of spectrin in the axons of hippocampal neurons. The color codes for z-position information. Credit: Zhuang lab

(Phys.org)—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.

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."
More information: www.hhmi.org/research/investigators/zhuang.html

Provided by Howard Hughes Medical Institute

"Axons' unexpected cytoskeleton structure." January 28th, 2013. http://phys.org/news/2013-01-axons-unexpected-cytoskeleton.html

Posted by
Robert Karl Stonjek

“She arose from the surface of the earth when it was pierced by a ploughshare. She was covered with auspicious dust from the field that resembled the pollen from a lotus.” (Hanuman, Valmiki Ramayana, Sundara Kand, 16.16)

utthitā medinīm bhittvā kṣetre hala mukha kṣate |
padma reṇu nibhaiḥ kīrṇā śubhaiḥ kedāra pāṃsubhiḥ ||

Lord Chaitanya Mahaprabhu has declared that just as Krishna is worshipable, so is His land. Therefore places like Vrindavana, Mathura and Dvaraka are popular pilgrimage destinations. The earth of these areas is considered sacred because Shri Krishna, the Supreme Personality of Godhead, placed His feet on it. In the same way, the land of Tirahuta is also sacred, for the Supreme Lord’s eternal consort, Lakshmi Devi, arose from underneath its ground one time when a famous king was ploughing the field for a sacrifice. That earth was auspicious before Janaka’s daughter arose, and afterwards it became even more important.

The idea that certain sections of land would be more important than others should make sense if we think about it. In the United States, there are tourist areas relating to the Founding Fathers, the group of men who founded the country. These landmarks include the family estates of the famous men, along with other areas of interest. There is also the famous Graceland, which relates to the rock n roll singer Elvis Presley. These areas sport ordinary homes built on ordinary dirt, but since they relate to famous personalities, they take on an added significance.

In one sense, this whole world can be thought of as sacred, since it emanated from the Supreme Lord. There is an original creator, though we can’t get our minds around the concept of eternal time. Keep going back in time as far as you can and you still won’t reach the beginning. Travel into the future as far as you can think of and you still won’t reach the end of time. Yet from our own experiences we know that the things we see had to be created at some point. They don’t just appear from nowhere. We can speculate that the unknown source is a collection of chemicals, an impersonal force, or a personality that is a supreme being.

The wise know it to be a supreme being because only through life can we get life. Chemicals don’t combine together to create life; it is just not possible. Through logical deduction alone, we can assume that a person was responsible for this vast cosmos. In the Vedas, more information is given about that person. It is said that He is all-attractive. He is also all-pervading. Hence two ways to address Him are Krishna and Vishnu. He is the origin of matter and spirit, and while the material creation is a representation of His external potency, sometimes He personally arrives to walk upon that which He created. Those visits are documented in the sacred texts like the Ramayana and Shrimad Bhagavatam.

It is said in the Vedas that Krishna is the original form of Godhead and that He appears in Mathura during the Dvapara Yuga, or the third time period of the creation. He then spends His childhood years in the neighboring town of Vrindavana. These areas still exist today, so one can walk on the same land that Krishna walked on. Krishna’s body is non-different from Him. His body does not undergo change like ours do. His body is transcendental, immune to the effects of karma.

“Unintelligent men, who know Me not, think that I have assumed this form and personality. Due to their small knowledge, they do not know My higher nature, which is changeless and supreme.” (Lord Krishna, Bhagavad-gita, 7.24)

Krishna’s name is also non-different from Him. Therefore the easiest and most effective way to stay in God’s company is to always chant His names, “Hare Krishna Hare Krishna, Krishna Krishna, Hare Hare, Hare Rama Hare Rama, Rama Rama, Hare Hare.” Food that has been offered to the Lord is also non-different from Him. Therefore devotees regularly prepare items in the mode of goodness for offering and then eat the remnants known as prasadam, or the Lord’s mercy. The devotee who always thinks of and glorifies Krishna is also non-different from the Lord. Therefore the wise souls always crave the association of devotees, as this is like being in God’s company.
A long time back, in the Treta Yuga, the second time period of creation, a famous king named Janaka ruled over the sacred land of Tirahuta. In his Janaki Mangala, Goswami Tulsidas refers to this area as the tilaka of the earth, or its sacred mark. The tilaka on the forehead represents Lord Vishnu, who is the same Krishna. Tirahuta thus also represents Vishnu with its earth. Its leader back then also represented Vishnu, as he was a devotee.

“That country is looking so beautiful, and the Vedas have described its purity. Known in the three worlds, Tirahuta [Janakpur] is the tilaka of the earth.” (Janaki Mangala, 4)

Not surprisingly, that devotee welcomed Vishnu’s wife, Lakshmi Devi, into his family when she appeared on earth to accompany her husband, who appeared as Lord Rama from the Raghu dynasty. In the above referenced verse from the Ramayana, Shri Hanuman mentions the unique circumstances of Sita’s appearance. She arose from the earth as it was being pierced by a ploughshare. As a pious king, Janaka was preparing for a sacrifice, and wouldn’t you know it, the fruit of that religious act arrived before the ceremony officially began. A beautiful baby was found in the ground, and Janaka named her Sita because of where she came from. The dust covering baby Sita resembled the pollen from a lotus flower. Originally, Sita is Lakshmi, who is very fond of lotuses. Therefore it wasn’t surprising that she would be covered by this kind of dirt when she appeared in Tirahuta.

Hanuman remembered this event as part of an identification process. He finally found Sita after an extensive search. Her husband Rama started looking for her after she went missing from the Dandaka forest. Hanuman had to find her based only on descriptions of her given by others. In this Ashoka grove inside of the kingdom of Lanka, Hanuman saw Rama’s wife, and to glorify her further, he reviewed some of her noteworthy features, such as her relation to the sacred earth in Tirahuta. And just as that land is worshipable, so is Sita.

In Closing:

When Supreme Lord walks on the land,

Earth same as Him understand.

The same for His holy names go,

Thus the devotees chant them feverishly so.

God’s wife to arrive in Janaka’s family chose,

From sacred earth of Tirahuta she arose.

To protect her as daughter Janaka avowed,

Gave her the name Sita, new father proud.

When Hanuman saw her in the Ashoka grove,

Her divine vision to that history drove.