A Bright Future: Using Bioluminescence as a Reporter of Mechanosensitivity

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The cover of Biophysical Journal (Volume 108, Issue 6) shows autofluorescence of a cell of the bioluminescent dinoflagellate Pyrocystis lunula. In this laser scanning confocal microscope image, blue is the fluorescence of luciferin, the substrate molecule for the bioluminescence reaction that originates from vesicles called scintillons, while red is chlorophyll fluorescence originating from the plastids. Dinoflagellate bioluminescence, which in nature functions in predator defense, here serves as a rapid whole-cell reporter of mechanosensitivity. Bioluminescence emission is mediated by a poorly understood but complex signaling pathway that involves activity at the plasma membrane, release of calcium from intracellular stores, depolarization of the vacuolar membrane, and acidification of the scintillons to activate the oxidation of luciferin. With a delay from mechanical stimulation to response of only 15-20 ms, dinoflagellate bioluminescence is one of the fastest known mechanosensitive cell systems. Thus dinoflagellate bioluminescence serves as an extremely rapid, whole cell noninvasive reporter of mechanosensitivity.

In our study, we used atomic force microscopy and a spherical probe to stimulate individual cells and to examine the relationship between cell mechanical properties and mechanosensitivity as assessed by intrinsic bioluminescence. The dinoflagellate flash, in this species lasting about 400 ms, is an all-or-nothing phenomenon that served as an indicator of cell response. By varying the parameters of the applied stimulation, we were able to determine a threshold force and velocity that was necessary to stimulate the cell. We observed that cells did not respond to a low indentation velocity. To explain this phenomenon we carried out stress relaxation experiments to measure the viscoeleastic properties of the cell. We formulated a simple viscoelastic model involving dashpots and springs to explain the velocity-dependent responses in terms of mechanosensor activation. At high rates of stimulation, stress accumulates in the cell membrane leading to a conformational change in mechanosensors, while at low stimulation rates the energy is dissipated due to relaxation. We are excited to develop our studies using dinoflagellate bioluminescence as a tool to investigate cellular mechanisms of rapid mechanosensing.

In nature, dinoflagellate bioluminescence is responsible for spectacular nighttime light displays when stimulated by mechanical stress associated with swimming animals, boat wakes, and breaking waves. In the laboratory, dinoflagellate bioluminescence is demonstrating its value to the physical sciences as a flow visualization tool for regions of increased mechanical stress, especially in applications not amenable to conventional measurement methods, such as shear stress within bioreactors, in breaking waves, above a rippled seabed, and associated with a moving dolphin.

Our use of dinoflagellate bioluminescence as a flow visualization tool was inspired by Leonardo da Vinci, who more than 500 years ago used grass seeds as particles to visualize flow patterns. Bioluminescence is a beautiful expression of nature, and it has been inspiring to collaborate with artists to express that beauty in photographs and video, for example http://www.erikablumenfeld.com/artworks/gallery/bioluminescence/. Dinoflagellate bioluminescence also serves as a tool in education and public outreach, that, along with its artistic value, is valuable in bringing science to the public. For more information about our research and dinoflagellate bioluminescence, visit http://siobiolum.ucsd.edu

– Benoit Tesson, Michael Latz

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Pi, Pie, Knotted Structures, and Biophysics

Biophysicists have always found themselves closely connected to many allied disciplines and conducting research that blurs the lines between these disciplines. With March 14 officially marking the celebration of Pi, the Biophysical Society thought it was a good time to shed some light on the amazing discoveries mathematics research (and Pi!) has contributed to the field of biophysics.

The Society was fortunate to co-sponsor a meeting on the significance of knotted structures for functions of proteins and nucleic acids that included among the speakers many mathematicians.  We have asked three speakers from that meeting to share some thoughts on their work, their career trajectory, and of course, Pi, with us in honor of Pi day.

 Stu Whittington
University of Toronto

Stuart (Stu) Whittington’s interest in Biophysics was sparked when he realized that circular DNA molecules from bacteria could be knotted and that these knots could interfere with cellular processes such as replication. He says that he is “definitely not a biologist — indeed I never took a biology course in high school or in university.” His main research interests are in rigorous statistical mechanics, especially of models of polymers, and he likes models with a combinatorial (so that he can count instead of integrating) or topological flavor. Whittington first became interested in knotting in ring polymers when he heard De Witt Sumners talk about it in about 1986. Whittingon and Sumners have been working together on random knotting and linking ever since. They have proved some results about the inevitability of knotting in long flexible objects (like hose pipes and DNA molecules) and also about the inevitability of writhe. These ideas are easily understood by the general public (who hasn’t found random knots in an extension cord?) and are a useful way to engage the average person about the utility of mathematics in biophysics, as well as in chemistry and physics.

Chris Soteros
University of Saskatchewan

Chris Soteros focuses on lattice models of polymers and biopolymers.  She uses combinatorics, probability, and asymptotic analysis to study the models as well as exact enumeration and Monte Carlo computer simulation methods. In addition, analyzing the computer simulation data involves statistical methods. By using these mathematical approaches, Soteros and her colleagues can identify the minimal ingredients needed to incorporate into a model in order to observe trends similar to those obtained in polymer and biopolymer experiments.  Sometimes it is possible to prove results about the models.  This can lead to new insights into the experimental results or can significantly strengthen the evidence for what was previously conjectured.

Soteros, who identifies herself as a mathematician, got her start in biophysical research in 1988 when she was a postdoc working with Stuart Whittington.  At that time she had the opportunity to study a mathematical problem that was motivated by a DNA topology question. This work was in collaboration with De Witt Sumners and resulted in a paper:  C.E. Soteros, D.W. Sumners and S.G. Whittington, 1992. Entanglement Complexity of Graphs in Z3. Math. Proc. Camb. Phil. Soc., 111, 75-91.  Soteros has been studying related problems every since then.

One challenge Soteros finds in working at this interface is learning enough terminology from another discipline, such as molecular biology, to read journal articles in that discipline. It can also be a challenge to keep on top of the latest advances in more than one discipline.  On the other hand, these barriers are greatly reduced when one collaborates with others who have complementary expertise and who are open to bridging the communication gap.  Soteros feels fortunate to have been involved in many such collaborations.

Soteros is especially grateful for having many opportunities to learn from others at interdisciplinary conferences involving molecular biologists, physicists, chemists, mathematicians, and computer scientists. The September 2014 Biophysical Society’s thematic meeting, “Significance of Knotted Structures for Functions of Proteins and Nucleic Acids’’ Conference in Warsaw, Poland was a prime example.

From the perspective of other researchers, Soteros thinks her work is interesting when she and her colleagues prove or find strong evidence for hypotheses that were previously only conjectures, or when they propose a novel hypothesis or conjecture.  She offers an example, “We were able to prove for a model of polymers confined to a tube, that knotting is inevitable for very large polymers, regardless of whether they are stretched or compressed (M. Atapour, C. Soteros and S. Whittington, 2009. Stretched polygons in a lattice tube. J. Phys. A:  Math. Theor., vol. 42, 322002 (9pp)).

From the perspective of the public, Soteros’ work demonstrates that fairly simple mathematical models can be used to gain insights into DNA experiments. More broadly, improved understanding of enzyme action on DNA through collaborative efforts involving mathematicians, physicists and molecular biologists is expected to lead to improved cancer treatments.

As for Pi, Soteros does use it in her work.  She says, “Some simple examples come immediately to mind. We studied how knot reduction in a model of topoisomerase action on DNA depends on the opening angle at the strand-passage site (M. L. Szafron and C. E. Soteros, 2011. The effect of juxtaposition angle on knot reduction in a lattice polygon model of strand passage. Fast Track Communication, J. of Phys. A: Math. Theor., Vol. 44 (322001), (11 pp)).   In the calculations, the angles were calculated in radians where 2Pi radians corresponds to 360 degrees.  Also, in the statistical analysis of our computer simulation data, we use the central limit theorem; this uses the standard Normal (or Gaussian) distribution that has density function function 1/√2π e^(-〖x〗^2/2).  Also, we use polygons on the simple-cubic lattice to model polymer and biopolymer configurations.  The bond angles on this lattice are all either Pi/2 or Pi.”

Soteros looks forward to celebrating Pi day with some PIE!

Note:  Soteros wanted BPS blog readers  to be aware of two upcoming meetings focused on the type of research she has described in this post: May 18-29, 2015 Graduate Summer School in Applied Combinatorics  and June 1-4, 2015 The Canadian Discrete and Algorithmic Mathematics Conference (CanaDAM)

Soteros

A planar projection of a 3-dimensional 5100-edge simple-cubic lattice polygon sampled from computer simulations for the paper Cheston M., McGregor K., Soteros C., and Szafron M., 2014. New evidence on the asymptotics of knotted lattice polygons via local strand-passage models. J. Stat. Mech.: Theor. Exp., 2014(2): P02014. It is a polygon with knot-type 5_1 where the “knotted part’’ is in the bright green part of the polygon at the right of the image. This is a randomly chosen 5_1 lattice polygon that illustrates what we expect to occur most often for large polygons, namely that the “knotted part” is relatively localized within the polygon. The image was created by M. Szafron using Rob Scharein’s KnotPlot software and the colour of an edge indicates its depth in the direction perpendicular to the plane of the image.

Eric Rawdon
University of St. Thomas

Eric Rawdon studies the knotting and tangling that occurs in physical systems, e.g. with DNA, proteins, or subatomic glueballs. He says he has always been drawn to computers, and gravitates towards problems that have some computational aspect. Most recently, he and his colleagues have been studying knotting in proteins, trying to understand which proteins are knotted, how they are knotted, and why they are knotted.

From Rawdon’s perspective as a trained mathematician, he thinks the things that biologists are able to do in the lab are amazing.  He notes, “I am too clumsy or impatient to deal with such messy experiments.  So I try to understand certain knotting behavior in physical systems by stripping away less relevant details.  For example, proteins are chains of amino acids.  If you analyze proteins at the atomic level, it is a mess (or at least that is how it looks to me), there are atoms and bonds everywhere. So we simplify the situation and model the protein as a chain of line segments. This is a coarse model and gives you a sort of long distance view of how the system is behaving.”

Like Soteros, Rawdon got his start in biophysical research early in his career.  His PhD advisor, Jon Simon (retired mathematician, University of Iowa), seemed to be drawn to mathematical problems in the sciences.  At conferences, Simon introduced Rawdon to many different people working in many different fields.  Rawdon never expected to do “applied” mathematics, but the biologists, chemists, and physicists brought such interesting questions to the table that he couldn’t help himself.  Early in his career he started working with Ken Millett (mathematician, University of California Santa Barbara). Like Simon, Millett had done some hard math but was open to mathematical problems in the sciences. Millett and Rawdon then team up with Andrzej Stasiak (biologist, University of Lausanne, Switzerland).

When it comes to identifying himself, Rawdon isn’t really sure what to call himself.  “I was trained as a mathematician, but I tend to publish in biology, chemistry, and physics journals.  So honestly, I’m not sure what to call myself.  But deep down inside, I think I am more mathematician than anything.”

Rawdon has continued to attend the interdisciplinary conferences he was introduced to as a graduate student.  He says the meetings he goes to typically have researchers from a wide-variety of fields, and the researchers tend to be open to interdisciplinary work. He met Joanna Sulkowska (physicist, University of Warsaw and one of the organizers of the BPS thematic meeting on Knotted Structures), at one such meeting, and she is the one who interested him in knotted proteins.

When working with scientists trained in other disciplines, Rawdon finds the biggest barrier to be language He offers the following example:  “My collaborator Andrzej Stasiak and I occasionally have disagreements over email, only to discover later that we were not in disagreement at all.  We simply both had interpreted the others’ words in terms of the language of our own fields.  If I say “protein topology” to a biologist and a mathematician, they are likely to interpret the phrase very differently.”

When asked if he has had any surprise research findings, Rawdon recounts the following collaboration from 2012:

“My collaborators and I were searching for knots in proteins. For each protein, we would generate a picture that encoded the knotting.  We created a web page of these pictures and I put them together in groups because there were many pictures that looked the same.  I sent the web page to Joanna who noticed that the similar pictures were coming from proteins that performed common functions in different organisms.  These families of proteins had diverged over hundreds of millions of years of evolution, yet the knotting patterns stayed the same.  That suggests that the knot is there for a reason. For a knot guy like me, that was pretty cool.  Our results are in a paper titled “Conservation of complex knotting and slipknotting patterns in proteins” published in the Proceedings of the National Academy of Sciences in 2012 (with Joanna Sulkowska, Ken Millett, Jose Onuchic, and Andrzej Stasiak).”

For those very interested in the topic, Rawdon directs readers to visit their website KnotProt  that has all the information you would ever want about knotted proteins.

For a general audience, Rawdon thinks his work is appealing because of all of the places where knotting appears in the sciences.  Here are just a few examples:

  • A large number of cancer drugs attack topoisomerases, enzymes that are necessary for DNA untangling during replication.
  • The antibiotic Ciprofloxacin Hydrochloride, which also targets topoisomerases, is used to treat anthrax exposure.
  • Recently, Chunfeng Zhao, M.D., of the Mayo Clinic proposed that surgeons use a new type of knot for certain types of surgeries.
  • It has been proposed that there are subatomic particles, called glueballs, which form tight knots.

As for Pi, it is not clear cut whether Rawdon uses it in his work.  He says, “Yes and no.  I think a lot about knots made out of tubes and any time you are you dealing with anything round, Pi is lurking in the shadows. Plus, I teach math, so I probably say Pi and mean the number, as opposed to the dessert, more than the average person.  But I do not think about Pi every day.  Still, each March 14, I take a moment to recognize Pi.

Tomorrow, to celebrate, the Math and Actuarial Science Club at the University of St. Thomas will buy some pies, as they do every year on Pi Day.  Since Pi Day is on the weekend this year, they are celebrating on Friday, March 13.  There are t-shirts and a pie-eating contest.  It promises to be a special event.

This image comes from a paper that was just published and is freely available for downloading: Eric Rawdon, Ken Millett, and Andrzej Stasiak, Subknots in ideal knots, random knots, and knotted proteins Scientific Reports 5:8928 (2015) http://www.nature.com/srep/2015/150310/srep08928/full/srep08928.html In the paper, we are trying to understand what sort of simpler knots lie inside more complicated knots.  These disks are our way of encoding the information.  But in and of themselves, I find them very beautiful.

This image comes from a paper that was just published and is freely available for downloading:
Eric Rawdon, Ken Millett, and Andrzej Stasiak, Subknots in ideal knots, random knots, and knotted proteins Scientific Reports 5:8928 (2015)
http://www.nature.com/srep/2015/150310/srep08928/full/srep08928.html
In the paper, we are trying to understand what sort of simpler knots lie inside more complicated knots. These disks are our way of encoding the information. But in and of themselves, I find them very beautiful.

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Coarse-Grained Model of RNA Provides Insight into RNA Strand Displacement Reaction

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The cover image of Biophysical Journal (March 10, 2015 [Volume 108, Issue 5]) shows an RNA toehold mediated strand displacement reaction, as represented by the coarse-grained oxRNA model. The invading strand (shown in blue) attaches to a single stranded overhang (the “toehold”) of the substrate strand (shown in red) and will eventually replace the incumbent strand (shown in green) because it forms the more stable complex. This process “catalyzes” the detachment of the incumbent strand, which is then available for other reactions.

RNA shows great promise as a material for nanotechnology. Like DNA, it has a four-letter alphabet that facilitates the design of stable, three-dimensional structures with near-atomic precision. Moreover, in vivo, it not only stores genetic material, as DNA does, but also acts as a structural element and can exhibit catalytic activity, much like proteins do. This versatility makes the prospect of using RNA nanotechnology for sophisticated biomedical applications, both in vitro and in vivo, particularly appealing.

Toehold-mediated strand displacement has long been an essential component for designing active DNA machines, because it allows kinetic control over the rates and ordering of key reactions. By combining multiple strand displacement reactions, complex logic operations and computation have been realized. Such success naturally raises the question of how this process could be used to create new applications in RNA nanotechnology. It also suggests that nature may exploit this relatively simple reaction inside the cell.

To unravel the underlying biophysics of strand displacement for RNA, we employed a recently derived nucleotide level coarse-grained model of RNA called oxRNA. Coarse-grained models are necessary to describe such processes that rely on rare events since the relevant time and length-scales are typically not accessible to more detailed atomistic models. OxRNA shares many features with the previously derived oxDNA model, which has successfully been used to describe many processes that are fundamental to DNA nanotechnology. In particular, these processes have reproduced experimentally measured relative rates for DNA displacement reactions with near quantitative accuracy.

We find that RNA displacement reaction rates are dominated by a complex interplay of enthalpic and entropic effects at the junction between the invading and incumbent strands. These processes cannot be captured by simple secondary structure models, and so a fully three-dimensional model such as oxRNA is necessary. We predict up to six orders of magnitude speedup between the rate for a toehold of length 1 and the saturated maximum speed for a toehold of length 5 and more. However, in contrast with DNA systems, we find that the displacement reaction is faster (by about a factor of two to nine) depending on which end of the substrate (3′ or 5′) the toehold is placed, with the 5′ toehold being faster. This difference arises from the asymmetry of the A-form helix adopted by RNA duplexes, which results in bigger stabilization of an invading strand at the 5′ end of the incumbent-substrate duplex. We also study the displacement rate at different temperatures, and find that for longer toeholds, the displacement slows down with increasing temperature.

Our results provide new insight into the fundamental biophysics of the RNA strand displacement reaction, which can be exploited to modulate reaction rates. Thus, our results improve the accuracy and flexibility of RNA nanotechnology design.

Further information about oxRNA, including a publicly accessible code and instructions for its use, can be found at http://dna.physics.ox.ac.uk.

– Petr Sulc, Thomas Ouldridge, Flavio Romano, Jonathan Doye, and Ard Louis

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Heart Beats and Biophysics

February has been designated Heart Month by the American Heart Association, the CDC, and several other organizations concerned with heart disease and ailments.  The goal of Heart Month is to raise awareness of heart diseases and steps individuals can take to prevent them.  It is also a good time for the Biophysical Society to highlight how advances in basic research contribute to our understanding of these diseases.  BPS member David Eisner, the BHF Professor of Cardiac Physiology at the University of Manchester in the United Kingdom has taken the time to share his lab’s research on heart functioning with us.

eisner picWhat is the connection between your research and heart disease, heart attacks, or heart functioning?

We study calcium signaling in the heart and, specifically, what controls the intracellular concentration of calcium ([Ca2+]i ).  Each heartbeat is initiated by a rise of [Ca2+]i; the greater the rise, the stronger the heart beats.  During exercise, the stronger contraction of the heart results from an increase in the size of the rise of  [Ca2+]i. As well as studying the normal regulation of [Ca2+]i, we are also interested in what happens in disease situations such as  heart failure and cardiac arrhythmias. One of the reasons that the hearts beats more weakly in heart failure is because the rise of [Ca2+]i is smaller than in healthy conditions.  The mechanisms responsible for this are still being unraveled. Many cardiac arrhythmias result from abnormalities of calcium signaling, in particular involving a rise of [Ca2+]i which occurs at the wrong part of the cardiac cycle.  Again, our research aims to understand the origins of these changes.

Why is your research important to those concerned about these diseases?

Understanding cardiac disease requires a much better understanding of the basic physiology of the heart. The fundamental unit of the heart is the cardiac muscle cell (myocyte) and it is at this level that most of our work is focused. We use  patch clamp to measure the movements of calcium across the membranes surrounding these cells and combine this with the use of fluorescent indicators to measure changes of [Ca2+]i. As well as providing information about the normal working of the heart, these sort of  studies will reveal the changes in disease. Furthermore, cellular studies are essential for developing therapies against these conditions. In this context it is important to note that, although enormous progress has been made, the progonosis for someone diagnosed with heart failure is still bleak.

How did you get into this area of research?

When I was at school I wanted to study physics and had never heard of physiology. At university I was taught physiology as the application of physics to the body.  Following undergraduate studies, I did my PhD with Denis Noble in Oxford where I worked with Jon Lederer (now at the University of Maryland) studying the control of contraction in the heart.  I can still remember the sense of immediate gratification when one pushed  a sharp microelectrode into a piece of cardiac muscle  and heard the change of pitch of the audio amplifier.  At that time it was impossible to do electrophysiological recordings on single cells and methods were not available to measure intracellular calcium.  However advances in these areas meant that it became possible to study calcium signaling in the heart.

How long have you been working on it?

Since the early 1980s!   My own research interests began very much at the basic science end of the subject but, over time, together with my long term collaborator Andrew Trafford, we have investigated disease models.

Do you receive public funding for this work? If So, from what agency?

Most of my funding comes from the British Heart Foundation (BHF).   This is a charity supported by the general public.  It is chastening to know that our research is supported by countless volunteers.  The funding environment in the UK  is very different from that in the US with a much smaller fraction of research supported by government funds.

Have you had any surprise findings thus far?

We obtained one very surprising result when we did experiments to increase the opening of the sarcoplasmic reticulum (SR) release channel  (the ryanodine receptor, RyR).  We had confidently expected that this would increase the size of the Ca signal and contraction.  However,  we found that the calcium signal was only increased for a couple of couple of beats before returning to normal levels.   The explanation of this result turned out to be that the increased release of calcium from the SR decreased SR Ca content.   This was the first hint we had of what has turned out to be a much more general phenomenon; the interaction between the various Ca handling pathways results in complicated, emergent behavior which is difficult to predict in advance.  At the simplest level, these results arise from the fact that the cardiac cell is in calcium flux balance.  On each beat the amount of calcium that enters the cell must exactly balance that which leaves.  This highlights the need to study calcium signaling in an integrated way.

What is particularly interesting about the work from the perspective of other researchers?

As a result of our work, others now appreciate that, on each beat, the cell is in calcium flux balance.   This point has to be borne in mind when trying to explain changes in cardiac contractility.

What is particularly interesting about the work from the perspective of the public?

The general public always seem fascinated when they are shown electrical and calcium signals from cardiac cells.  They are amazed by the fact that the heart beats repetitively even when outside the body.  It is a privilege to lecture to the public.  I always think that it is a pity that most people know much more about outer space than about their own bodies.

Do you have a cool image you want to share with the blog post related to this research?

The image at the top of this blog post (work by Jessica Caldwell, Andrew Trafford and colleagues) shows ventricular cells connected together.  The horizontal bands are the transverse tubules which invaginate the cell.  We are currently studying the cellular mechanisms that ensure that the transverse tubule network is laid down in this precise arrangement and why it disappears in heart failure.

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Visualizing Chromosomes, Cell Cycles, and Entropy

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David Goodsell is a legendary figure whose scientific illustrations have inspired many. When I first entered the bacterial chromosome field in 2004 as a theoretical physicist, his illustration was a definite guide to the inner space of Escherichia coli. It conveyed the right sense of scale and the numbers of different types of proteins and biomolecules inside the cell. Goodsell regularly updates his illustrations to incorporate up-to-date information from the scientific literature. A detailed explanation behind his work process can be found in Miniseries: Illustrating the Machinery of Life, Escherichia coli (Biochem Mol Biol Educ. 2009 [37]: 325-332).

In recent years, my lab has been taking multidisciplinary approaches to understand the physical principles that drive organization and segregation of the chromosomes in bacteria. For example, we have revealed the fundamentally “soft” nature of the bacterial chromosome and the entropic forces that can compact it in a crowded intracellular environment (Pelletier et al. Physical manipulation of the Escherichia coli chromosome reveals its soft nature. PNAS Plus. 2012; 109 [40]: E2649–2656), which motivated the work by Shendruk et al. in this issue of Biophysical Journal (108 [4]: February 17, 2015).

In the meantime, Stuart Austin’s group at the National Cancer Institute was tackling what had been considered impossible. They started the measurements and analysis of intracellular positions of dozens of dual genomic loci markers under overlapping cell-cycle conditions. This is a daunting task because, for any given moment, every cell contains several homologous copies of each genomic locus, and deciphering the organization and dynamics of the whole chromosome based on their positional information was something that had never been done before. Nevertheless, Stuart’s group relentlessly pushed their efforts without publishing anything for several years. When the task was done, the end result was simple, elegant, and surprising. The organization of the chromosome during multifork replication was that of a simple branched donut, with the two arms of the chromosome occupying each cell half along the radial axis of the cell. This result could not have been predicted based on our knowledge of the slowest-growing cells, but it encompasses all the previously known results and moves beyond them into something new and general (Youngren et al. The multifork Escherichia coli chromosome is a self-duplicating and self-segregating thermodynamic ring polymer. Genes Dev. 2014 [28]: 71-84).

Because of these radical new findings, I felt that David Goodsell’s famous illustration of E. coli would benefit from revision. Our cover image is the result of 12 revisions. A high-resolution file of Goodsell’s E. coli illustration can be downloaded from my lab web site: http://jun.ucsd.edu.

–Suckjoon Jun

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Bye bye Baltimore

For the first time in days, I woke up in the hotel room with sunlight sifting through the curtain. What a nice day with fresh air and ample daylight! Even for the last day, the schedules are packed with multiple platform talks and poster showing.

I want to feature the Cryo-EM structure talk in the afternoon, as it did cover a great variety of research topics and new progresses. All four speakers are working with very challenging protein systems and getting enough particles for comprehensive structural details is only the last thing on their to-do list. Due to highly dynamic nature of the protein system movements, the inherent heterogeneity of the particle conformations is probably the biggest challenge. To solve the problem involves locking the system in a particular state with different substrates, finding the right conditions for the protein preparation, and also collect particles with different orientations on the surface. I really appreciate that several speakers took the time to explain to the audience the technical difficulties they encounter during the structural solving processes. In a way, the story not only tells about a sophisticated structure, but also shows us countless trial-and-error progress from highly driven scientists.

I really do enjoy the BPS experience, even when I felt rather clueless at some moment.

Bye bye Baltimore, and hopefully see you’all soon in Los Angelos.

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t-loop formation at single molecule level.

The average lifespan of a cell is approximately 50 cycles after which the cells go into senescence, inability to replicate. Early published work clearly suggests that the growing cells have an inherent knowledge of the number of cycles they have divided and this attribute of the cells is very much dependent on the structures on the end of the chromosomes known as the telomeres. These structures at the end of the chromosomes are known to shorten after every cell cycle. Once the telomere reserve has run out, cells stop dividing. Telomeres play important roles in maintaining the stability of linear chromosomes. The telomeric structure allows a cell to distinguish between natural chromosome ends and double-stranded DNA breaks. Telomere dysfunction and associated chromosomal abnormalities have been strongly associated with age-associated degenerative diseases and cancer. Telomere maintenance involves dynamic actions of multiple proteins on a long complex DNA structure. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure-function relationships that govern telomere maintenance. These telomeres form little loops at the end of the chromosomes, which are called the t-loops. These are formed by inserting the ends of the chromosome, which is usually 3’ overhang back into the DNA of the chromosome. Thus, very short telomeres, which is the scenario in old aging cells or sometimes cancer cells, can no longer form t-loops. The exposure of these chromosome ends, 3’ overhangs which cannot be inserted back into the DNA of the chromosome, would alert the cells and thus stop cells from dividing. If we can elucidate the mechanism of this t-loop formation, we can introduce methods to stop shortening of these telomeres and make drugs to stop these processes.
There were two talks this year focusing on the t-loop formation at the single molecule level. While Xi Long talk in the DNA structure session used Magnetic tweezers to understand this, Hong Wang’s talk in the Nanoscale Biophysics session on Saturday talked about these structures , using new technique developed in their lab , DREEM ( Dual Resonance Enhanced Electrostatic force Microscopy). Xi long et al was able to show the melting of telomeric DNA substrates on applying torque and the binding of these substrates with the single stranded oligos but the major drawback in her work being the absence of TRF2 protein from the shelterin complex, which has been shown to be required for the formation of t-loops. Hong et al was able to predict a possible mechanism of the t-loop formation based on the interaction of the telomeric DNA with the shelterin protein, very cool work and technique , actually showing the DNA inside the protein DNA complexes. How cool to be able to see what happens inside the protein DNA complex!!!
Would be looking forward to BPS 2016 for their work to better understand this mechanism…

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