Posts tagged cells
Posts tagged cells
A novel imaging technique provides insights into the role of redox signaling and reactive oxygen species in living neurons, in real time. Scientists of the Technische Universität München (TUM) and the Ludwig-Maximilians-Universität München (LMU) have developed a new optical microscopy technique to unravel the role of “oxidative stress” in healthy as well as injured nervous systems. The work is reported in the latest issue of Nature Medicine.
Reactive oxygen species are important intracellular signaling molecules, but their mode of action is complex: In low concentrations they regulate key aspects of cellular function and behavior, while at high concentrations they can cause “oxidative stress”, which damages organelles, membranes and DNA. To analyze how redox signaling unfolds in single cells and organelles in real-time, an innovative optical microscopy technique has been developed jointly by the teams of LMU Professor Martin Kerschensteiner and TUM Professor Thomas Misgeld, both investigators of the Munich Cluster for Systems Neurology (SyNergy).
“Our new optical approach allows us to visualize the redox state of important cellular organelles, mitochondria, in real time in living tissue” Kerschensteiner says. Mitochondria are the cell’s power plants, which convert nutrients into usable energy. In earlier studies, Kerschensteiner and Misgeld had obtained evidence that oxidative damage of mitochondria might contribute to the destruction of axons in inflammatory diseases such as multiple sclerosis.
The new method allows them to record the oxidation states of individual mitochondria with high spatial and temporal resolution. Kerschensteiner explains the motivation behind the development of the technique: “Redox signals have important physiological functions, but can also cause damage, for example when present in high concentrations around immune cells.”
Kerschensteiner and Misgeld used redox-sensitive variants of the Green Fluorescent Protein (GFP) as visualization tools. “By combining these with other biosensors and vital dyes, we were able to establish an approach that permits us to simultaneously monitor redox signals together with mitochondrial calcium currents, as well as changes in the electrical potential and the proton (pH) gradient across the mitochondrial membrane,” says Thomas Misgeld.
The researchers have applied the technique to two experimental models, and have arrived at some unexpected insights. On the one hand, they have been able, for the first time, to study redox signal induction in response to neural damage – in this case, spinal cord injury – in the mammalian nervous system. The observations revealed that severance of an axon results in a wave of oxidation of the mitochondria, which begins at the site of damage and is propagated along the fiber. Furthermore, an influx of calcium at the site of axonal resection was shown to be essential for the ensuing functional damage to mitochondria.
Perhaps the most surprising outcome of the new study was that the study’s first author, graduate student Michael Breckwoldt, was able to image, also for the first time, spontaneous contractions of mitochondria that are accompanied by a rapid shift in the redox state of the organelle. As Misgeld explains, “This appears to be a fail-safe system that is activated in response to stress and temporarily attenuates mitochondrial activity. Under pathological conditions, the contractions are more prolonged and may become irreversible, and this can ultimately result in irreparable damage to the nerve process.”
A team led by scientists at The Scripps Research Institute (TSRI) has identified a long-sought protein that facilitates one of the most basic functions of cells: regulating their volume to keep from swelling excessively.
The identification of the protein, dubbed SWELL1, solves a decades-long mystery of cell biology and points to further discoveries about its roles in health and disease—including a serious immune deficiency that appears to result from its improper function.
“Knowing the identity of this protein and its gene opens up a broad new avenue of research,” said the study’s principal investigator Ardem Patapoutian, a Howard Hughes Medical Institute (HHMI) Investigator and professor at TSRI’s Dorris Neuroscience Center and Department of Molecular and Cellular Neuroscience.
The report appears as the cover story in the April 10, 2014 issue of the journal Cell.
Unraveling the Mystery
Water passes through the membrane of most cells with relative ease and tends to flow in a direction that evens out the concentration of dissolved molecules or “solutes.” “Water in effect follows the solutes,” explained Zhaozhu Qiu, a member of the Patapoutian laboratory who was first author of the study. “Any decrease in the solute concentration outside a cell or an increase within the cell will make the cell swell with water.”
For decades, experiments have demonstrated the existence of a key relief valve for this swelling: an unidentified ion channel in the cell membrane, dubbed VRAC (volume-regulated anion channel). VRAC opens in response to cell swelling and permits an outflow of chloride ions and some other negatively charged molecules—which water molecules follow, thus reducing the swelling.
“For the past 30 years, scientists have known that there is this VRAC channel, and yet they haven’t known its molecular identity,” said Patapoutian.
Finding the proteins that make VRAC and their genes was a goal that had eluded prior attempts because of the technical hurdles involved. However, in the new study, Qiu and his colleagues were able to set up a rapid, “high-throughput” screening test based on fluorescence. They engineered human cells to produce a fluorescent protein whose glow would be quenched when the cells became swollen and VRAC channels opened.
With the help of automated screening specialists at the La Jolla-based Genomics Institute of the Novartis Research Foundation (GNF), which recently began a broad new collaboration agreement with TSRI, the team cultured large arrays of the cells and, using a technique known as RNA interference, blocked the activity of a different gene for each clump of cells.
The idea was to watch for the groups of cells that continued to glow—indicating that the gene inactivation had disrupted VRAC.
In this way, with several rounds of tests, the team sifted through the human genome and ultimately found one gene whose disruption reliably terminated VRAC activity. It was a gene that had been discovered in 2003 and catalogued as “LRRC8.” Although it appeared to code for a cell-membrane-spanning protein—as one would expect for an ion channel—almost nothing else was known about it.
The team renamed it SWELL1.
Potential Roles in Disease
Investigating further, the researchers showed that SWELL1 does indeed localize to the cell membrane as an ion channel protein would. Experiments by Adrienne Dubin, a staff scientist at TSRI, showed that certain mutations of SWELL1 alter the VRAC channel’s ion-passing properties—indicating that SWELL1 is a central feature of the ion channel itself.
“It is at least a major part of the VRAC channel for which cell biologists have been searching all this time,” said Patapoutian.
Patapoutian, Qiu and their colleagues now will study SWELL1 further, including an examination of what happens to lab mice that lack the protein in various cell types.
Curiously, the gene for SWELL1 was first noted by scientists because a mutant, dysfunctional form of it causes a very rare type of agammaglobulinemia—a lack of antibody-producing B cells, which leaves a person unusually vulnerable to infections. That suggests that SWELL1 is somehow required for normal B-cell development.
“There also have been suggestions from prior studies that this volume-sensitive ion channel is involved in stroke because of the brain-tissue swelling associated with stroke and that it may be involved as well in the secretion of insulin by pancreatic cells,” said Patapoutian. “So there are lots of hints out there about its relevance to disease—we just have to go and figure it all out now.”
From the neurons that enable thought to the keratinocytes that make toenails grow-a complex canopy of sugar molecules, commonly known as glycans, envelop every living cell in the human body.
These complex carbohydrate chains perform a host of vital functions, providing the necessary machinery for cells to communicate, replicate and survive. It stands to reason, then, that when something goes wrong with a person’s glycans, something goes wrong with them.
Now, researchers at the University of Georgia are learning how changes in normal glycan behavior are related to a rare but fatal lysosomal disease known as Niemann-Pick type C (NPC), a genetic disorder that prevents the body from metabolizing cholesterol properly. The findings were published recently in the PNAS Early Edition.
"We are learning that the problems associated with cholesterol trafficking in the cell lead to problems with glycans on the cell’s surface, and that causes a multitude of negative effects," said Geert-Jan Boons, professor of chemistry in the Franklin College of Arts and Sciences and researcher at UGA’s Complex Carbohydrate Research Center. "Now, for the first time, we can see what these problems are, which we hope will lead to a new understanding of diseases like NPC."
Because NPC patients are unable to metabolize cholesterol, the waxy substance begins to accumulate in the brain. This can lead to a host of serious problems, including neurodegeneration, which the researchers hypothesize may be caused by improper recycling of glycans on the surface of an NPC patient’s cells.
Glycans normally undergo a kind of recycling process when they enter the cell only to be returned to the surface recharged and ready to work. The researchers discovered that glycans in NPC cells do not do this.
"One of the secondary effects of NPC is the disruption of traffic pathways within the cell, and this can lead to altered recycling of glycans," said Richard Steet, associate professor of biochemistry and molecular biology and CCRC researcher. "The glycans come into the cell, but they won’t recycle back up to the cell’s surface where they must exist to function as receptors or ion channels."
"Basically, the machinery gets clogged up," Boons said.
Like downed phone lines and flooded roads in a thunderstorm, glycans get stuck inside the cell making communication and travel for these cells difficult or impossible. Without these basic abilities, the body’s motor, sensory and cognitive functions begin to suffer. This might explain why NPC patients suffer from such a wide variety of neurological and psychiatric disorders, such as uncoordinated limb movements, slurred speech, epilepsy, paralysis, psychosis, dementia and hallucinations.
The researchers made these observations in fibroblasts taken from diseased patients. These cells are most commonly found in connective tissues, and they play a vital role in wound healing. However, they hope to continue their investigation into the effects of NPC by studying glycan behavior in neural cells, which make up the human brain.
While they caution that much more work must be done, they hope that an improved understanding of the roles that glycans play in neural cells will lead to new therapeutics for NPC and other diseases like it.
"It is exciting to work on projects like these, because we believe glycobiology is the next frontier, the next level of complexity," Boons said. "The time is right for new discovery."
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.”
In a paper published in Cell yesterday, scientists from the US and Thailand have, for the first time, successfully produced embryonic stem cells from human skin cells.
That sounds interesting, but what are stem cells and where do they come from?
If you take a limb from a rose tree, and put it in soil, it will grow into a thriving bush.
There is a special type of flatworm that can be cut in half, again and again hundreds of times, and each half grows back into a full worm.
Similarly, if you cut out half a human liver, it will grow back. The story of Prometheus, whose liver was eaten away by eagles and regrew each day, suggests that the Greeks of ancient times knew about regeneration of organs.
This sort of regeneration is attributed to special cells called “stem cells”.
Reprogramming the workers
Most of our cells are like many professional workers – they are hardened in their ways and can’t manage career changes.
Blood cells carry oxygen or fight disease, muscle cells expand and contract to move us around, nerve cells carry signals, skin cells form a protective layer over our bodies, and structures made up of kidney cells filter our blood.
The cells of most organs or tissues are referred to as “terminally differentiated” cells. They have specialised, and many won’t divide again. If they are damaged or die they will disappear. This is very important.
Although we feel like we grow a lot after we are born, we really only double in size two or three times and most of our cells don’t divide much.
If they did we would be at great risk from cancer – the uncontrolled doubling of cells at the wrong time.
We have a lot of cells and it is important that none of them run out of control.
But some cells can double to renew themselves and can also differentiate and give rise to specialised progeny.
These are the stem cells. We need them to produce new skin to replace damaged skin cells. Similarly, we need them in our guts to replace damaged cells on the surface of our intestines.
Our blood cells also get worn out as they race around our bodies so we have blood stem cells that divide and replace themselves. They also differentiate to form the different types of white and red blood cells we need.
Australian researchers identified stem cells in the breast that can proliferate and form a complete functioning breast. There are also stem cells in the brain and in the heart.
While stem cells tend to be very rare, they exist in many of our organs.
Types of stem cells
The ultimate stem cells are embryonic stem cells.
These cells are found in the inner cell mass of the early embryo and are referred to as “totipotent” since they have the ability to form every cell that is needed in the growing embryo.
They can be extracted from the early embryo and grown in culture dishes.
They can also be genetically modified by the addition of DNA, then injected back into other embryos or into adult animals where find their way into localities that suit them and replace themselves by duplication or differentiate into other cell types that may be needed. For a long time this type of work had been done primarily in laboratory mice.
The techniques in yesterday’s Cell paper involved injecting the nucleus from a human skin cell into a human egg (the nucleus of which has been destroyed) then growing the resulting embryo until the inner cell mass cells could be harvested.
The method may still be controversial because it uses unfertilised eggs, but many people will regard it as preferable to using human embryos. And there are other interesting methods for making stem cells.
Somatic cells to stem cells
It is also possible to convert skin cells, and indeed many different terminally differentiated cells, back into what are called “induced pluripotent stem cells” or iPS cells.
One uses the “magic four” or “OKSM” set of DNA-binding proteins that govern normal stem cell biology:
In 2012 Shinya Yamanaka won the Nobel Prize for discovering how to convert normal cells into iPS cells using the OKSM regulators to turn on and off the right genes and convert skin cells into stem cells.
Researchers are continuing to investigate whether iPS cells have the same therapeutic potential as embryo derived stem cells.
It is hoped that stem cells may provide therapies for people suffering from degenerative diseases.
Skin cells could be taken from a patient, converted to stem cells, and then these could be injected back into the damaged organ.
Ideally, they would repopulate the damaged organ with new cells.
So why doesn’t this happen in normal biology? Why aren’t our own heart stem cells busy trying to repair broken hearts?
They may be but our natural supply of stem cells is limited and presumably insufficient to tackle severe disease.
So why don’t we just have more stem cells in our bodies?
The down side of having too many stem cells may be cancer.
Stem cells share a number of features with cancer cells – both are able to self-renew and double without limit.
One theory about cancer holds that the disease most often originates not from terminally differentiated cells but from one of the small number of stem cells in the relevant tissues.
The obvious concern about using stem cells for therapy is that injecting too many could increase the chances that some of these cells would proliferate beyond control, and ultimately give rise to cancer.
Stem cell therapy for regenerative medicine is an exciting idea.
Every day we are learning more about stem cells – how to purify or make them, and how to grow them in culture and direct them down particular pathways to repopulate different organs.
Future research will assess the risks and how effective they can be in experimental systems and ultimately in human patients.
A study led by researchers from Plymouth University Peninsula Schools of Medicine and Dentistry has for the first time revealed how the loss of a particular tumour suppressing protein leads to the abnormal growth of tumours of the brain and nervous system.
The study is published in Brain: A Journal of Neurology.
Tumour suppressors exist in cells to prevent abnormal cell division in our bodies. The loss of a tumour suppressor called Merlin leads to tumours in many cell types within our nervous systems. There are two copies of a tumour suppressor, one on each chromosome that we inherit from our parents. The loss of Merlin can be caused by random loss of both copies in a single cell, causing sporadic tumours, or by inheriting one abnormal copy and losing the second copy throughout our lifetime as is seen in the inherited condition of neurofibromatosis type 2 (NF2).
With either sporadic loss or inherited NF2, these tumours lacking the Merlin protein develop in the Schwann cells that form the sheaths that surround and electrically insulate neurons. These tumours are called schwannomas, but tumours can also arise in the cells that form the membrane around the brain and spinal cord, and the cells that line the ventricles of the brain.
Although the schwannomas are slow-growing and benign, they are frequent and come in numbers. The sheer number of tumours caused by this gene defect can overwhelm a patient, often leading to hearing loss, disability and eventually death. Patients can suffer from 20 to 30 tumours at any one time, and the condition typically manifests in the teenage years and through into adulthood.
No effective therapy for these tumours exists, other than repeated invasive surgery or radiotherapy aiming at a single tumour at a time and which is unlikely to eradicate the full extent of the tumours.
The Brain study investigated how loss of a protein called Sox10 functions in causing these tumours. Sox10 is known to play a major role in the development of Schwann cells, but this is the first time it has been shown to be involved in the growth of schwannoma tumour cells. By understanding the mechanism, the research team has opened the way for new therapies to be developed that will provide a viable to alternative to surgery or radiotherapy.
The study, undertaken by researchers from Plymouth University Peninsula Schools of Medicine and Dentistry with colleagues from the State University of New York and Universitat Erlangen-Nurmberg, was led by Professor David Parkinson.
He said: “We have for the first time shown that human schwannoma cells have reduced expression of Sox10 protein and messenger RNA. By identifying this correlation and gaining an understanding of the mechanism of this process, we hope that drug-based therapies may in time be created and introduced that will reduce or negate the need for multiple surgery or radiotherapy.”
When Charles Babbage prototyped the first computing machine in the 19th century, he imagined using mechanical gears and latches to control information. ENIAC, the first modern computer developed in the 1940s, used vacuum tubes and electricity. Today, computers use transistors made from highly engineered semiconducting materials to carry out their logical operations.
And now a team of Stanford University bioengineers has taken computing beyond mechanics and electronics into the living realm of biology. In a paper published March 28 in Science, the team details a biological transistor made from genetic material — DNA and RNA — in place of gears or electrons. The team calls its biological transistor the “transcriptor.”
“Transcriptors are the key component behind amplifying genetic logic — akin to the transistor and electronics,” said Jerome Bonnet, PhD, a postdoctoral scholar in bioengineering and the paper’s lead author.
The creation of the transcriptor allows engineers to compute inside living cells to record, for instance, when cells have been exposed to certain external stimuli or environmental factors, or even to turn on and off cell reproduction as needed.
“Biological computers can be used to study and reprogram living systems, monitor environments and improve cellular therapeutics,” said Drew Endy, PhD, assistant professor of bioengineering and the paper’s senior author.
The biological computer
In electronics, a transistor controls the flow of electrons along a circuit. Similarly, in biologics, a transcriptor controls the flow of a specific protein, RNA polymerase, as it travels along a strand of DNA.
“We have repurposed a group of natural proteins, called integrases, to realize digital control over the flow of RNA polymerase along DNA, which in turn allowed us to engineer amplifying genetic logic,” said Endy.
Using transcriptors, the team has created what are known in electrical engineering as logic gates that can derive true-false answers to virtually any biochemical question that might be posed within a cell.
They refer to their transcriptor-based logic gates as “Boolean Integrase Logic,” or “BIL gates” for short.
Transcriptor-based gates alone do not constitute a computer, but they are the third and final component of a biological computer that could operate within individual living cells.
Despite their outward differences, all modern computers, from ENIAC to Apple, share three basic functions: storing, transmitting and performing logical operations on information.
Last year, Endy and his team made news in delivering the other two core components of a fully functional genetic computer. The first was a type of rewritable digital data storage within DNA. They also developed a mechanism for transmitting genetic information from cell to cell, a sort of biological Internet.
It all adds up to creating a computer inside a living cell.
Digital logic is often referred to as “Boolean logic,” after George Boole, the mathematician who proposed the system in 1854. Today, Boolean logic typically takes the form of 1s and 0s within a computer. Answer true, gate open; answer false, gate closed. Open. Closed. On. Off. 1. 0. It’s that basic. But it turns out that with just these simple tools and ways of thinking you can accomplish quite a lot.
“AND” and “OR” are just two of the most basic Boolean logic gates. An “AND” gate, for instance, is “true” when both of its inputs are true — when “a” and “b” are true. An “OR” gate, on the other hand, is true when either or both of its inputs are true.
In a biological setting, the possibilities for logic are as limitless as in electronics, Bonnet explained. “You could test whether a given cell had been exposed to any number of external stimuli — the presence of glucose and caffeine, for instance. BIL gates would allow you to make that determination and to store that information so you could easily identify those which had been exposed and which had not,” he said.
By the same token, you could tell the cell to start or stop reproducing if certain factors were present. And, by coupling BIL gates with the team’s biological Internet, it is possible to communicate genetic information from cell to cell to orchestrate the behavior of a group of cells.
“The potential applications are limited only by the imagination of the researcher,” said co-author Monica Ortiz, a PhD candidate in bioengineering who demonstrated autonomous cell-to-cell communication of DNA encoding various BIL gates.
Building a transcriptor
To create transcriptors and logic gates, the team used carefully calibrated combinations of enzymes — the integrases mentioned earlier — that control the flow of RNA polymerase along strands of DNA. If this were electronics, DNA is the wire and RNA polymerase is the electron.
“The choice of enzymes is important,” Bonnet said. “We have been careful to select enzymes that function in bacteria, fungi, plants and animals, so that bio-computers can be engineered within a variety of organisms.”
On the technical side, the transcriptor achieves a key similarity between the biological transistor and its semiconducting cousin: signal amplification.
With transcriptors, a very small change in the expression of an integrase can create a very large change in the expression of any two other genes.
To understand the importance of amplification, consider that the transistor was first conceived as a way to replace expensive, inefficient and unreliable vacuum tubes in the amplification of telephone signals for transcontinental phone calls. Electrical signals traveling along wires get weaker the farther they travel, but if you put an amplifier every so often along the way, you can relay the signal across a great distance. The same would hold in biological systems as signals get transmitted among a group of cells.
“It is a concept similar to transistor radios,” said Pakpoom Subsoontorn, a PhD candidate in bioengineering and co-author of the study who developed theoretical models to predict the behavior of BIL gates. “Relatively weak radio waves traveling through the air can get amplified into sound.”
To bring the age of the biological computer to a much speedier reality, Endy and his team have contributed all of BIL gates to the public domain so that others can immediately harness and improve upon the tools.
“Most of biotechnology has not yet been imagined, let alone made true. By freely sharing important basic tools everyone can work better together,” Bonnet said.
The research was funded by the National Science Foundation and the Townshend Lamarre Foundation.
Like tiny, crawling compass needles, whole living cells and cell fragments orient and move in response to electric fields — but in opposite directions, scientists at the University of California, Davis, have found. Their results, published April 8 in the journal Current Biology, could ultimately lead to new ways to heal wounds and deliver stem cell therapies.
When cells crawl into wounded flesh to heal it, they follow an electric field. In healthy tissue there’s a flux of charged particles between layers. Damage to tissue sets up a “short circuit,” changing the flux direction and creating an electrical field that leads cells into the wound. But exactly how and why does this happen? That’s unclear.
"We know that cells can respond to a weak electrical field, but we don’t know how they sense it," said Min Zhao, professor of dermatology and ophthalmology and a researcher at UC Davis’ stem cell center, the Institute for Regenerative Cures. "If we can understand the process better, we can make wound healing and tissue regeneration more effective.”
The researchers worked with cells that form fish scales, called keratocytes. These fish cells are commonly used to study cell motion, and they also readily shed cell fragments, wrapped in a cell membrane but lacking a nucleus, major organelles, DNA or much else in the way of other structures.
In a surprise discovery, whole cells and cell fragments moved in opposite directions in the same electric field, said Alex Mogilner, professor of mathematics and of neurobiology, physiology and behavior at UC Davis and co-senior author of the paper.
It’s the first time that such basic cell fragments have been shown to orient and move in an electric field, Mogilner said. That allowed the researchers to discover that the cells and cell fragments are oriented by a “tug of war” between two competing processes.
Think of a cell as a blob of fluid and protein gel wrapped in a membrane. Cells crawl along surfaces by sliding and ratcheting protein fibers inside the cell past each other, advancing the leading edge of the cell while withdrawing the trailing edge.
Assistant project scientist Yaohui Sun found that when whole cells were exposed to an electric field, actin protein fibers collected and grew on the side of the cell facing the negative electrode (cathode), while a mix of contracting actin and myosin fibers formed toward the positive electrode (anode). Both actin alone, and actin with myosin, can create motors that drive the cell forward.
The polarizing effect set up a tug-of-war between the two mechanisms. In whole cells, the actin mechanism won, and the cell crawled toward the cathode. But in cell fragments, the actin/myosin motor came out on top, got the rear of the cell oriented toward the cathode, and the cell fragment crawled in the opposite direction.
The results show that there are at least two distinct pathways through which cells respond to electric fields, Mogilner said. At least one of the pathways — leading to organized actin/myosin fibers — can work without a cell nucleus or any of the other organelles found in cells, beyond the cell membrane and proteins that make up the cytoskeleton.
Upstream of those two pathways is some kind of sensor that detects the electric field. In a separate paper to be published in the same journal issue, Mogilner and Stanford University researchers Greg Allen and Julie Theriot narrow down the possible mechanisms. The most likely explanation, they conclude, is that the electric field causes certain electrically charged proteins in the cell membrane to concentrate at the membrane edge, triggering a response.
New research, published in Neuron, gives insight into how single mutations in the VCP gene cause a range of neurological conditions including a form of dementia called Inclusion Body Myopathy, Paget’s Disease of the Bone and Frontotemporal Dementia (IBMPFD), and the motor neuron disease Amyotrophic Lateral Sclerosis (ALS).
Single mutations in one gene rarely cause such different diseases. This study shows that these mutations disrupt energy production in cells shedding new light on the role of VCP in these multiple disorders.
In healthy cells VCP helps remove damaged mitochondria, the energy-producing engines of cells. The mutant protein can’t do this and as a result, the dysfunctional mitochondria build up.
The new study led by Dr Fernando Bartolome, Dr Helene Plun-Favreau and Dr Andrey Abramov of the UCL Institute of Neurology, found that mitochondria are damaged in cells from patients with mutant VCP. Mitochondria generate a cell’s energy, and the study found these damaged mitochondria are less efficient, burning more nutrients but producing less energy. This reduction in available energy makes cells more vulnerable, which could explain why mutations in the VCP gene lead to neurological disorders.
Lead author Dr Fernando Bartolome said, “We have found that VCP mutations are associated with mitochondrial dysfunction. VCP had previously been shown to be important in the removal of damaged mitochondria and proteins, accumulation of which is potentially very toxic to cells. A single mutation in the VCP gene could cause multiple neurological diseases because a different type of protein is accumulating in each disorder”.
In the study, the researchers used live imaging techniques to examine the functioning of mitochondria in patient cells carrying three independent VCP mutations, and in nerve cells in which the amount of VCP has been reduced.
“The next step will be to find small molecules able to correct the mitochondrial dysfunction in the VCP deficient cells”, added Dr Bartolome .
Dr Brian Dickie, the Motor Neuron Disease Association’s Director of Research Development says: “Neurons - and motor neurons in particular - are incredibly energy hungry cells. These new findings from the team at UCL show that there is a significant interruption of energy supply in this hereditary form of MND, which has strong implications for understanding the degenerative process underpinning all forms of the disease.”
Therapeutic focus: Huntington’s disease
Description: Huntington’s stem cell derived oligodendrocyte precursors stained for phalloidin (green), vinculin (red) and DNA (blue).
Image credit: Anushree Balachandran (Genea, Australia) - High-content analysis winners