News Article | March 8, 2016
Ordinarily, of course a fly does not shimmer green. Here, researchers - with the help of genetic tricks - succeeded in making a muscle protein glow, which they can then locate under a fluorescence microscope. Credit: MPI for Biochemistry The human genome codes for more than 20,000 different proteins, however the molecular role for many of these proteins is not known. As most proteins are conserved from fly to humans, understanding the molecular role of a protein in flies can be the first step towards a therapy against a variety of human diseases that are often caused by aberrantly behaving proteins. A consortium of scientists from the Max Planck Institutes of Biochemistry in Martinsried and Molecular Cell Biology and Genetics in Dresden, and the National Centre for Biological Sciences (NCBS) in Bangalore have now reached a milestone towards understanding the function of these proteins by using the fruit fly. The human body is built by many hundreds of different cell types; each one has a very particular function in the body. Red blood cells transport oxygen, nerve cells exchange signals and muscle cells generate mechanical forces. The majority of cellular functions is produced by the action of 20,000-25,000 proteins coded in the human genome. Although sequencing and annotation of human genome were completed in 2004, to date the function of many thousands of these proteins is still mysterious. It is often unknown, which cell types produce which proteins, and particularly where these proteins are located within the cells. Are they in the nucleus or within membranous vesicles, are they within neuronal dendrites or synapses, or are they within the contractile machinery of muscles? Protein localisation is an important piece of information, as it is the first step towards identifying a molecular function for a protein. Unravelling the function of a protein is often started in simpler model organisms such as worms or flies. Like humans, fruit flies have muscles, neurons, oocytes, sperm and many other essential cells types. The fly genome contains about 13,000 protein coding genes, which are responsible for building and maintaining all fly organs. Importantly, many of these proteins are very similar to the human proteins, thus studying a protein in flies will teach us about its role in the human body. To boost these protein studies onto a systematic level, groups headed by Frank Schnorrer at the Max-Planck Institute in Martinsried, Pavel Tomancak and Mihail Sarov at the Max-Planck Institute in Dresden and K VijayRaghavan at the NCBS in Bangalore have generated a large resource for visualizing proteins in Drosophila melanogaster. By using modern molecular biology tricks, the scientists have attached a green fluorescent protein (GFP) tag to 10,000 of these protein coding genes in the test tube. Each tagged gene can then be re-introduced into the fly genome as a 'transgene', creating the fly 'TransgeneOme'. "Together, we thus far generated 880 different fly strains, each of which expresses a different fluorescently tagged protein", explains Frank Schnorrer, "these proteins can then be observed by fluorescent video microscopy in various cell types of the developing fruit fly". For more than 200 proteins, the scientists documented where they are located during fly development, starting with an oocyte that develops into an embryo and finally into the mature fly. The Tomancak group used the so-called light sheet microscopy to film how proteins emerge in cells of the embryo during the first day of its development. The Schnorrer group used this resource to study the localisation of proteins in muscles. As in human skeletal muscles, fly muscles contain complex mini-machines called sarcomeres that produce the mechanical forces enabling animal movements. "We have looked so far at only 200 of these transgenic lines. The future challenge lies in systematically imaging the localization of these proteins in many fly tissues and this is best achieved by involving the powerful Drosophila research community" predicts Pavel Tomancak. The resource will have enormous impact on the understanding of not only fly biology but also on the understanding of protein function in the different human cell types." More information: Mihail Sarov et al. A genome-wide resource for the analysis of protein localisation in , eLife (2016). DOI: 10.7554/eLife.12068
News Article | December 28, 2016
Mrs. M would never forget that day. She was walking along a busy road next to the vegetable market when two goons zipped past on a bike. One man's hand shot out and grabbed the chain around her neck. The next instant, she had stumbled to her knees, and was dragged along in the wake of the bike. Thankfully, the chain snapped, and she got away with a mildly bruised neck. Though dazed by the incident, Mrs. M was fine until a week after the incident. She would struggle and yell and fight in her sleep every night with phantom chain snatchers. Every bout left her charged with anger and often left her depressed. The episodes continued for several months until they finally stopped. How could a single stressful event have such extended consequences? A new study by Indian scientists has gained insights into how a single instance of severe stress can lead to delayed and long-term psychological trauma. The work pinpoints key molecular and physiological processes that could be driving changes in brain architecture. The team, led by Sumantra Chattarji from the National Centre for Biological Sciences (NCBS) and the Institute for Stem Cell Biology and Regenerative Medicine (inStem), Bangalore, have shown that a single stressful incident can lead to increased electrical activity in a brain region known as the amygdala. This activity sets in late, occurring ten days after a single stressful episode, and is dependent on a molecule known as the N-Methyl-D-Aspartate Receptor (NMDA-R), an ion channel protein on nerve cells known to be crucial for memory functions. The amygdala is a small, almond-shaped groups of nerve cells that is located deep within the temporal lobe of the brain. This region of the brain is known to play key roles in emotional reactions, memory and making decisions. Changes in the amygdala are linked to the development of Post-Traumatic Stress Disorder (PTSD), a mental condition that develops in a delayed fashion after a harrowing experience. Previously, Chattarji's group had shown that a single instance of acute stress had no immediate effects on the amygdala of rats. But ten days later, these animals began to show increased anxiety, and delayed changes in the architecture of their brains, especially the amygdala. "We showed that our study system is applicable to PTSD. This delayed effect after a single episode of stress was reminiscent of what happens in PTSD patients," says Chattarji. "We know that the amygdala is hyperactive in PTSD patients. But no one knows as of now, what is going on in there," he adds. Investigations revealed major changes in the microscopic structure of the nerve cells in the amygdala. Stress seems to have caused the formation of new nerve connections called synapses in this region of the brain. However, until now, the physiological effects of these new connections were unknown. In their recent study, Chattarji's team has established that the new nerve connections in the amygdala lead to heightened electrical activity in this region of the brain. "Most studies on stress are done on a chronic stress paradigm with repeated stress, or with a single stress episode where changes are looked at immediately afterwards - like a day after the stress," says Farhana Yasmin, one of the Chattarji's students. "So, our work is unique in that we show a reaction to a single instance of stress, but at a delayed time point," she adds. Furthermore, a well-known protein involved in memory and learning, called NMDA-R has been recognised as one of the agents that bring about these changes. Blocking the NMDA-R during the stressful period not only stopped the formation of new synapses, it also blocked the increase in electrical activity at these synapses. "So we have for the first time, a molecular mechanism that shows what is required for the culmination of events ten days after a single stress," says Chattarji. "In this study, we have blocked the NMDA Receptor during stress. But we would like to know if blocking the molecule after stress can also block the delayed effects of the stress. And if so, how long after the stress can we block the receptor to define a window for therapy," he adds. Chattarji's group first began their investigations into how stress affects the amygdala and other regions of the brain around ten years ago. The work has required the team to employ an array of highly specialised and diverse procedures that range from observing behaviour to recording electrical signals from single brain cells and using an assortment of microscopy techniques. "To do this, we have needed to use a variety of techniques, for which we required collaborations with people who have expertise in such techniques," says Chattarji. "And the glue for such collaborations especially in terms of training is vital. We are very grateful to the Wadhwani Foundation that supports our collaborative efforts and to the DBT and DAE for funding this work," he adds.
News Article | April 26, 2016
Many tropical forest trees depend on large fruit-eating animals such as elephants, tapirs, monkeys and hornbills for dispersing their sizeable seeds. Declines of these large mammals and birds due to hunting and forest disturbance, and consequent declines of tree species that they disperse, constitute a global conservation problem. Now, an international consortium of researchers predicts that such losses can cause substantial changes in the potential for tropical forests across the globe to store carbon, and thereby alter their ability to regulate our world's climate. Plants convert atmospheric carbon-dioxide to carbon stored in their living tissues through the process of photosynthesis. Trees in tropical forests form a large terrestrial 'carbon sink', which store carbon and play a crucial role in regulating atmospheric carbon-dioxide concentrations. However, when it comes to storing carbon, not all tree species are the same. In a recent study, reported in the journal Nature Communications, researchers find that large-seeded tree species which depend on big animals for seed dispersal, grow to greater sizes as adults and thus have higher carbon storage potential than species with smaller seeds in tropical forests worldwide. Losses of large seed dispersers can therefore reduce carbon storage by the Earth's tropical forests by decreasing the volume of vegetation biomass in these forests. "Scientists are only just beginning to understand the numerous ways in which animals affect the carbon cycle of tropical forests, and the consequences of declines of these animals -- also termed 'defaunation' -- for terrestrial carbon storage ", says Anand M Osuri, a member of Mahesh Sankaran's group at NCBS and the study's lead author. "Although defaunation is a problem affecting tropical forests the world over, our understanding of its consequences for carbon storage relies heavily on patterns seen in one part of the tropics -- the forests of South America", he adds. The study predicts that the forests of the Americas, Africa and South Asia -- which are primarily composed of tree species dependant on animals for seed dispersal -- will face the most severe reductions of carbon storage due to declines of large seed dispersers. In contrast, carbon storage may be less sensitive to losses of large seed dispersers in the forests of Southeast Asia, where a number of large tree species depend on wind and gravity, rather than animals, for seed dispersal."Insights into these regional differences, which arise due to the unique evolutionary histories of trees in each region, are among the most interesting contributions of this study", says Jayashree Ratnam, one of the co-authors of this publication. "Even while tropical forests across the globe seem to look alike, they are functionally different -- and we are reminded once again that a one-size-fits-all model for a biome may often hide critically important differences in form and function", she adds. Through extensive computer simulations, the study predicts that if 50% of all trees dispersed by large animals were replaced over time by trees with other modes of seed dispersal, carbon storage in tropical forests of the Americas, Africa and South Asia would be reduced by 2%. This is roughly equivalent to 14 years' worth of Amazonian deforestation. Apart from these predictions, the study also highlights an urgent need for more research on understanding the complex dynamics of forests that have lost their animal inhabitants. "A number of factors contribute to determining which tree species will ultimately succeed in defaunated forests. Many of these factors are not understood very well at present", says Mahesh Sankaran. "Long term observations and experiments are essential to predict the exact magnitudes of, and time scales over which, community-level responses to defaunation may play out", he adds. At present, policies such as REDD+ that are aimed at mitigating terrestrial carbon emissions primarily focus on reducing carbon losses by protecting tropical forests from deforestation and logging. This study shows that in addition to protecting the forests themselves, policies that give importance to conserving their fauna could also result in significant benefits for carbon storage in tropical forests.
News Article | December 6, 2016
Once triggered by light, rhodopsin molecules on the surface membrane must be 'reset' in order to sense light again, a process that occurs within the cell. This requires rhodopsin to be moved into the cell and for 'reset' rhodopsin to be recycled back onto the surfaces. Therefore, to function normally, the light-sensing membranes in these cells undergo constant recycling to restore the light-detectors they carry. Scientists from the National Centre for Biological Sciences (NCBS), Bangalore, and the Babraham Institute in UK have recently found a critical player essential for proper membrane recycling. Using the light-sensitive membranes of fruit-fly eyes as a model system, the researchers have discovered that the enzyme Phospholipase D or PLD is necessary for membrane recycling to sustain normal sight. The resetting of rhodopsin molecules begins with a process called endocytosis, where the cell pinches off parts of its surface membranes into structures called endosomes. The rhodopsin in these endosomes is eventually recycled back onto the cell surface for further events of light detection. Since a photoreceptor's sensitivity depends on how many rhodopsin molecules it has on its surface, membrane turnover in these cells is critical in preserving normal eyesight. "You can think of endocytosis and membrane recycling as two arms of the membrane turnover process," says Raghu Padinjat from NCBS. "There needs to be a balance between the two, or else the size of the membrane will shrink - a condition that could lead to retinal degeneration in the eyes. This is actually seen in an inherited genetic disease, a rare disorder called Best's macular degeneration," he adds. However, the connection between endocytosis and the eventual recycling process to maintain photo-sensitive surface membranes have remained unclear - until now. A collaborative study from Padinjat's laboratory at NCBS and the Babraham Institute in UK, has found that a well-known enzyme called Phospholipase D or PLD plays a central role in linking endocytosis to membrane recycling. Using fruit fly photoreceptor cells as a model system, the team has found that when these cells are exposed to light, PLD is switched on, and that its activity is essential in coupling endocytosis with recycling of rhodopsin back to the cell surface. In mutant flies that lack PLD in their photoreceptors, the endocytosis process is unlinked from membrane recycling. When exposed continuously to light, the cell surface of photoreceptors in mutant flies gradually shrinks, with reducing rhodopsin levels that make it progressively less sensitive to light. Without PLD activity, the retina gradually degenerates and mutant flies go blind. Padinjat's team chose to use the fruit fly eye as a model system since it has many ideal characteristics for studying membrane turnover. This is because the light-sensing parts of fruit fly photoreceptors are highly expanded, forming a structure called the rhabdomere. When exposed to light, changes in the size of this membrane can be distinctly visualised through electron microscopy. Furthermore, due to the genetic tractability of the system, the researchers were able to clearly identify PLD as an essential component regulating membrane turnover. "The enzyme PLD converts a molecule called phosphatidylcholine into phosphatidic acid or PA, which is implicated in membrane turnover. However, PA is also produced by other enzymes, and our study conclusively shows that the PA regulating membrane turnover was produced by PLD," says Rajan Thakur, a researcher from Padijat's group and the primary author of a publication in the journal eLife that reports these results. Despite identifying a key player in the membrane turnover process, Padinjat's team believe that many more gaps need filling in understanding the phenomenon. For example, the work shows that the PLD activity in photoreceptors is light-activated, but how this happens is still unresolved. "The light receptor, rhodopsin is on the membrane surface, and can detect light when the surface of the cell is illuminated. But PLD, which is also light-activated, is somewhere inside the cell. So how is the information about perceived light conveyed to PLD for it to get activated?" asks Thakur. "We also need to fill in blanks about how PA actually affects membrane recycling and the turnover process. Our finding has opened up more questions to answer in the membrane turnover process," he adds. But the results from this study are not limited only to membrane turnover in the light-sensitive membranes of the eyes. Membrane turnover is a critical mechanism that maintains cell surface area. In cells that have expanded cell surfaces such as those lining the airways of the lungs or the nutrition-absorbing cells of the gut, maintaining cell surface area is essential for their normal function. Even processes such as cell migration have extensive endocytosis and membrane recycling events that must be tightly regulated. "Therefore, regardless of what the cell type is, there need to be mechanisms to couple endocytosis with recycling of membrane," says Padinjat. "And that is the importance of our work - we define a mechanism by which cell membrane size is regulated," he adds. More information: Rajan Thakur et al, Phospholipase D activity couples plasma membrane endocytosis with retromer dependent recycling, eLife (2016). DOI: 10.7554/eLife.18515
News Article | November 4, 2016
GREEN BAY, Wis., Nov. 4, 2016 /PRNewswire/ -- Nicolet Bankshares, Inc. (NASDAQ: NCBS) ("Nicolet") and First Menasha Bancshares, Inc. (OTCQX: FMBJ) ("First Menasha") today jointly announce the execution of a definitive merger agreement, pursuant to which Nicolet will acquire First Menasha...
News Article | February 28, 2017
After much deliberation and anxiety, the family finally sought psychiatric help for their son. And the results were in a way, a relief. The doctors' verdict was that their child, their teenage son, was suffering from bipolar disorder. His wild mood swings between hyper-enthusiastic activity and deep depression were treatable. Once therapy started, however, it became apparent that standard drugs - effective in most cases - were not working here. And so, doctors turned to a less preferred drug called Clozapine. Though effective in controlling the manic-depressive cycles, one of the many side effects of the drug were very apparent - sedation - extreme sleepiness and fatigue. This side effect can severely hamper a recovering patient's integration back into normal society. As of now, the mechanisms by which such side effects occur are poorly understood, and options for treating them are limited. But, a recent study, on the drug Clozapine from the National Centre for Biological Sciences (NCBS), Bangalore holds out new hope for such cases. The work by Radhika Joshi from Mitradas Panicker's group at NCBS has proven that the molecule 5-HT2A in the brain is partially responsible for the sedative effects of Clozapine. Furthermore, the team has also discovered that the drug-induced sedation can be affected by environmental factors. The drug Clozapine helps manage symptoms of depression and anxiety by binding to receptor molecules in the brain that respond to neurotransmitters - chemicals used by nerve cells to communicate with each other. Clozapine mainly targets receptors for the neurotransmitters serotonin and dopamine. Amongst these, 5-HT2A is a serotonin receptor that was identified as one that could be partially responsible for the sedative effects of Clozapine. Radhika in collaboration with Dr. Rupasri Ain and co-workers generated and characterized a genetically modified mice to test if the 5-HT2A receptor was involved in the sedative side effects of Clozapine. When given Clozapine, mutant mice lacking the 5-HT2A receptor were found to be more active and less sensitive to sedation than normal mice. But while conducting these studies, the researchers also noticed that the environment played a role in modulating these effects. "While most work on drugs focuses on improving their therapeutic effects, side effects are often ignored - and sometimes side effects can be severe enough to result in non-compliance and require secondary medication," says Radhika Joshi, a doctoral student in Panicker's laboratory. "Our observations indicated that the environment can modulate the side effect of an antipsychotic. This has not been reported before, and so we investigated." she adds. When either normal mice or mice without the 5-HT2A receptor were given Clozapine in their "home" environments - in cages where the mice are normally housed - the animals were less active and more sensitive to sedation. However, when the drug was given in a "novel" cage or if a new object such as a toy was put in their home cages, the mice were less somnolent, particularly the 5-HT2A knockout mice. Having something different in their surroundings served to decrease the drowsy effects of Clozapine and this effect was more pronounced in the mice lacking the 5-HT2A receptor. Further investigations also revealed that Clozapine sedation could be reduced if the mice were given caffeine before the drug. "We believe that a novel environment is often stimulating to organisms and this stimulation helps overcome the sedation induced by an antipsychotic, as does caffeine," says Joshi. At the same time, she advises caution in interpreting the results of this study. "Our experiments were conducted on mice and a direct extrapolation to human contexts must be done with utmost caution," she says. Despite her warning, Joshi is hopeful that their results on environmental context and caffeine can potentially help doctors develop real life strategies for managing the side effects of antipsychotics without secondary medication. "Our results are one of the first few steps towards designing of drugs that are safer and with lesser side effects though a lot more work is needed before we can expect better drugs, we are getting there" she adds.
News Article | December 30, 2016
A team of researchers from India have discovered how a single incident of trauma causes a delayed onset of debilitating psychological stress, which is experienced for a long period of time. The researchers also unraveled the psychological and molecular events associated with the process. It is noted in the study that the stressful event increases the electrical activity of a specific region of the brain called as amygdala. The onset of the symptoms usually begin in about 10 days after the incident and a compound known as N-Methyl-D-Aspartate Receptor (NMDA-R) plays a key role in the process. NMDA-R, which is a protein in the ion-channel of the neurons, is responsible for functions such as memory. Amygdala, which is found deep inside the temporal lobe, is made of small group of neurons. This part of brain is known to control functions such as decision making, memory, and emotions. Post-Traumatic Stress Disorder (PTSD), a psychiatric condition experienced by a person after a traumatic incident develops due to alternations in amygdala. Sumantra Chattarji, the lead author of the study at National Centre for Biological Sciences (NCBS), said that the current study is applicable for PTSD since the activity of amygdala is intense in patients suffering from it. However, the mechanism involved in the process is not found by far, added Chattarji. After experiencing a trauma, minute structures in the neurons of amygdala region of a person's brain undergo major alternations. As a result, new nerve connections known as synapses are formed in this area of the brain. Farhana Yasmin, who is also a part of the study, said that studies conducted by far focused on stress experienced on repetitive basis and single stressful episode that inflict changes immediately, for instance a day or two. However, the current study deals with a single episode of trauma that causes changes after a week or so, added Yasmin. The researchers, who identified that NMDA-R is the key component in causing the changes, tested it on the ailment. It was found that when NMDA-R was blocked it not only put an end to the formation of synapses but also controlled the electrical activity taking place in the synapses. "So we have for the first time, a molecular mechanism that shows what is required for the culmination of events ten days after a single stress," said Chattarji in a press release. Chattarji also added that in the current study, the protein NMDA-R was blocked during stress; however, it is necessary to check if blocking the compound immediately after the stressful incident would help manage the problem. In that case, how long after the incident the protein can be blocked for effective intervention needs to be studied. The study is published in the journal Physiological Reports. © 2017 Tech Times, All rights reserved. Do not reproduce without permission.
News Article | September 9, 2016
One set of such interlinked compartments - the Golgi complex - is essential for many cellular functions, and a question that has long puzzled scientists is: how did such a complex compartment and traffic system arise within a cell? Scientists from the National Centre for Biological Sciences have a possible answer to this question through a mathematical approach to explore how such organisation could have evolved. Somya Mani and Mukund Thattai from the Simons Centre at NCBS have shown that the Golgi complex with its attendant traffic system can emerge spontaneously from a simple model with no need for a special selection mechanism. Within cells, the Golgi complex is a set of compartments that is essential for processing, packaging and transporting proteins and other molecules. A key characteristic of the Golgi is its organisation as a 'maturation chain' with different compartments having variable molecular compositions. These compartments perform different processing and packaging functions, especially in the synthesis and transport of giant proteins like collagen. "Now, it's very strange that this maturation system developed in order to transport giant molecules. You can't transport large molecules if you don't have the whole system working, but if there was no function for the maturation system, what drove it's evolution?" asks Mukund Thattai. "It's a classic chicken-and-egg problem or what you call a catch-22," he says. The different compartments of the Golgi complex are connected to each other and to other cellular areas via mobile membrane-bound chambers called vesicles. Vesicles constantly bud off or fuse with compartments, forming a cell-wide transport system for different types of molecules. In order to investigate the origins of the Golgi complex, Mani and Thattai simulated this traffic system. Built on broad and simple rules, they modelled the stream of vesicles budding out of source compartments and fusing with target compartments within a cell. These events were specified by budding and fusion matrices to create a collection of simulated cellular traffic networks that had settled into a state of equilibrium. Now, Mani and Thattai did something unconventional - they filled up the budding and fusion matrices at random. Therefore, budding and fusion events were random, with no specific purpose guiding these events. But then, they got an astonishing result. In roughly 25% of their simulations, the researchers came across traffic networks that had developed distinct patterns closely resembling those of a Golgi complex. This means that despite the lack of a selection mechanism for budding or fusion, a vesicular traffic network in a cell could give rise to a functional 'maturation chain' of compartments purely by chance. In other words, Mani and Thattai's work shows that the evolution of the Golgi complex is likely to have been non-adaptive - no selection system need have pushed cells to develop a Golgi complex. "We could argue that the Golgi might have come about for some other purpose, a purpose that nobody has been able to figure out. But the essence of this work, is that there is no other purpose," says Thattai about the work that has been described in a recent publication in the journal eLife. "But once cells had the Golgi, fine! It's a great system for transporting giant molecules, and so cells used it," he continues. The scientists are now planning to use their model to study infectious systems like tuberculosis and HIV, which are caused by intracellular parasites that hijack a cell's vesicular traffic system for their own use. "The parameters of our model can be easily interpreted in terms of biological properties of traffic molecules. So, our model might give us clues into the molecular mechanisms that parasites like M. tuberculosis use to hijack the vesicular transport systems," says Mani. "We can also study mechanisms that the cell could use to reinstate its normal traffic system after such an infection," she adds. Explore further: Large-scale screen reveals how numerous signaling pathways intersect at the cell's primary protein-processing center
News Article | March 23, 2016
Three channel time lapse of myosin-induced actin contraction into polar asters. (Scale bar: 10 µm). Credit: Image credits: Through WikiCommons, William CrochotGIF copyright 2016 Köster et al., PNAS Like the phenomena of flocking birds and shoaling fish, the dance of molecules across a cell's surface has long fascinated theorists, physicists and biologists alike. Unlike bird and fish behaviour, however, cell surface dynamics cannot be observed and studied easily. However, it is important to understand these processes as they are crucial for cells to gain information about their environment and respond. So how does one understand the rules that govern movement of molecules across this arena? By reconstructing the cell surface from scratch, perhaps? Now, scientists from the National Centre for Biological Sciences (NCBS) in Bangalore have managed to do exactly that - construct a simplified cell surface from its constituent parts, namely, a mixture of fats and proteins. This reconstruction creates a crucial new tool that researchers can use to test theories on cell surface dynamics. Molecular movements on the cell surface are known to be non-random, incredibly complex, and do not seem to follow simple thermodynamic rules. Until recently, there were few experimental tools available to study such phenomena to really understand how the cell surface functioned. This has changed with the new experimental system that has been developed by a close collaboration between experimental biologists from Prof. Satyajit Mayor's group at NCBS, scientists from the University of California San Francisco (UCSF) and theoretical physicists from Prof. Madan Rao's group at NCBS and RRI (Raman Research Institute). The experimental system is a minimal model of the cell surface constructed from its basic components - purified fats and proteins known to be part of the cell surface. This tool could be the key to understanding how the surface of a living cell works. "This is just a beginning but an important one," says Prof Satyajit Mayor. "Important because it allows one to test ideas that have come from theory built around providing an explanation for active organization at the surface of a living cell. It's an exciting beginning since the feasibility of this simple minimal system opens up huge possibilities to explore the world of a living cell in a test tube system where every element is under our control. This work is inspired by the adage 'what we understand we should be able to build' and this is in trying to understand the principles behind how a living material, the cell surface, works," he adds. The 'active composite model' of the cell surface is one of the latest theories that attempts to explain the behaviour of cell surface molecules. This model visualises the cell surface as not just the cell membrane, but as an amalgamation of two elements - the cell membrane, made of fats and an interwoven mesh of the protein 'actin' that forms a thin layer just below the cell membrane. Another protein, 'myosin' that interacts with actin, behaves like a molecular motor and creates movement in the actin meshwork when supplied with energy. Many of the cell surface proteins whose movements have baffled scientists are often linked to the dynamic actin meshwork that lies just below the cell membrane. As proposed by the active composite model, the researchers decided to recreate a cell surface as an assembly of a fat-based membrane and an actin meshwork. This artificial cell surface was therefore constructed using a fat bilayer, actin and a fluorescent protein specially designed to be embedded in the membrane while also being linked to actin. Using various microscopic techniques, the group was able to study the behaviour of the construct via the patterns formed by the fluorescent proteins. As predicted by the 'active composite model', the dynamics of actin-bound fluorescent proteins were found to be dependent on the dynamics of the actin meshwork. When the molecular motor, myosin, was added and chemical energy provided, the forces generated by actin-myosin interactions drove the movements of these proteins. When the chemical energy was exhausted, the actin-bound proteins aggregated to form distinct bundle or aster-like structures based on the organisation of the actin meshwork. "The importance of active or energy consuming processes in understanding biological phenomena is becoming more and more evident. This is an emerging field in biology called 'active mechanics'. Often, the emerging organisation of biological molecules are not clear, and theoretical explanations for such observations are also far from complete. This makes it important to have proper experimental tools that go hand in hand with theory to test and improve our understanding of such systems. Our current study describes the creation of an experimental system that will serve us in this," says Darius Köster, the lead author of the study that was published in the leading journal PNAS (Proceedings of the Natural Academy of Sciences of the United States of America). "The motivation behind this work is to analyse mechanisms influencing the dynamics and organisation of molecules on the cell surface," says Kabir Husain, another author in this study. Processes like cell growth, division, immune recognition and many others are dependent on the organisation of protein receptors and other associated molecules on the cell surface. This implies that the ability of the cell to reliably control the organisation of its surface molecules is crucial to its survival and function. With the recreation of the cell surface in a testube, scientists have gained a solid experimental footing in the race to comprehend the mechanics of cell surface organisation. Explore further: Mitosis mystery solved as role of key protein is confirmed More information: Darius Vasco Köster et al. Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer, Proceedings of the National Academy of Sciences (2016). DOI: 10.1073/pnas.1514030113
News Article | April 4, 2016
"A bend and a twist, then stretch and turn, now relax". What sounds like a series of exercise instructions, are also words that describe the various shapes a piece of DNA can assume. The classic double helix structure that one associates with DNA is but an extremely limited view of its physical 'shape'. The molecule that holds the codes of life is capable of further winding itself into myriad complex shapes called 'supercoils' that are capable of affecting gene expression patterns. Now, researchers from the National Centre for Biological Sciences (NCBS), Bangalore, and the National Institutes of Health (NIH), USA, have elucidated this pattern of supercoiling across the genome of the much studied bacterium E. coli. DNA molecules are wound and rewound into complex structures that condense their immense lengths to a fraction of their actual size in order to fit their long strings of information into microscopic cells. But this 'packed' DNA that fits neatly into a cell also needs to be 'unpacked' periodically for gene expression and replication. When a gene is expressed, it is 'read' by protein machineries to create a messenger transcript that codes for more proteins. This requires DNA to be unwound from its double helix - a process that causes further twisting and coiling or 'overwinding' in regions of DNA elsewhere on the genome. Similarly, unwinding and overwinding also occurs when the genome replicates during reproduction. Therefore, at any given time, a cell's genetic material is in a constant state of structural flux - coils, supercoils, bends, twists and turns are formed, lost and reformed depending on the cell's state of activity. A bacterial cell can be exposed to various environmental changes which include periods of starvation, lack of oxygen and unfavourable temperatures. Surviving these situations would require the bacterium to change its protein repertoire by altering the corresponding genetic expression profiles. Scientists have long thought that these changes could be effected through variations in the supercoiled structure of DNA. For example, the genomes of actively dividing cells under rich-nutrient conditions are known to be more underwound than the genomes of cells from the stationary phase when nutrients are scarce. In other words, supercoiling is likely to be sensitive to changes in the environment. Although recent advancements in methodology have allowed researchers to study DNA supercoiling in human and yeast cells at local scales, this methodology has never been applied to bacterial genomes. Researchers from Aswin S. N. Seshasayee's group at NCBS and Prof. Sankar Adhya's team at NIH have currently applied these methods to study DNA supercoiling in bacteria at a fine-scale level. Using the chemical trimethylpsoralen, exposure to UV light and microarray technology, the research team have gained information on section-specific variations in genomic supercoiling within bacteria exposed to different external conditions. "We have measured DNA supercoiling at a fine-scale resolution in bacteria for the first time. This study provides proof-of-concept that the supercoiling of a genome is not uniform and that it varies locally across genes. It also provides evidence to support the hypothesis that bacterial cells could be regulating gene expression and their own physiologies by altering the structure of their genomes," says Avantika Lal, the first author of the publication in the journal Nature Communications that details these findings. In order to study the effect of environmental stimuli on the supercoiling status of the bacterial genome, two populations of E. coli were used to simulate two different external conditions. One simulated a nutrient-rich situation where actively dividing cells represented a growing population; whereas the other represented a condition where a population had exhausted its nutrients and was in a 'stationary' phase. Since the binding of trimethylpsoralen to DNA is proportional to the amount of supercoiling in the DNA, one could study genome-wide patterns of winding under these two settings. The results have shown that E. coli cells in the 'stationary' phase display a gradient of supercoiling across their circular genomes. In actively dividing cells, however, this gradient was missing though the entire genome was more supercoiled than the genomes of cells from the 'stationary' phase. "It is very early days yet, but this work paves the way to understanding which genes' expression are affected by the environment," says Avantika. "This work can potentially teach us how we could control cell physiology by altering genetic expression via changes to DNA supercoiling by altering external conditions," she adds. More information: Avantika Lal et al. Genome scale patterns of supercoiling in a bacterial chromosome, Nature Communications (2016). DOI: 10.1038/ncomms11055