The Ribosome Running at (Half-)Full Tilt

BPJ_113_11.c1.inddThe ribosome, Nature’s ubiquitous nano-assembler for proteins, is not only very old, but complex, sophisticated, and evolving. In bacterial cells, it adds 20 amino acids per second to a polypeptide chain during the elongation phase of synthesis. It only incorporates the wrong amino acid or erroneously shifts the reading frame on the messenger RNA (mRNA) less than 1 in 1,000 elongation cycles. Even the best typist will make more mistakes. Enough free energy is liberated on formation of the peptide bonds to power synthesis, but two extra units of energy are spent in the splitting of GTP by elongation factors (EFs) Tu and G to ensure the fidelity of amino acid selection and the maintenance of the reading frame. How these accessory factors lower the error rate is incompletely understood.

X-ray, cryo-EM, and single-molecule fluorescence assays have suggested that the two main subunits of the ribosome and transfer RNAs (tRNAs) spontaneously fluctuate between so-called classic and hybrid states before EF-G binds to catalyze translocation 3 bases along the mRNA. Most of these studies analyzed ribosomes that had been stalled by antibiotics or lack of EF-G, but we had a hint from earlier work that the situation might be different with ongoing synthesis. Our study, shows that during ongoing synthesis there is indeed insufficient time for the ribosome and tRNAs to settle into classic-hybrid fluctuations. Rather, the pre-translocation tRNAs reside in intermediate locations similar to some of the “chimeric” positions that structural studies have detected. Our results demonstrate that fully operational molecules may be imperfectly represented by those which are artificially inhibited and highlight the synergy between high resolution snapshots and real-time structural dynamics.

The cover drawing evokes the power, intricate complexity, and early origins of this machinery in the “Steampunk” visual style influenced by Victorian industrial technology and the scientific romances of Jules Verne, HG Wells, Mary Shelley, and Arthur Conan Doyle. The integrated metal, wood and glass materials are a reminder of the hybrid RNA-protein composition of the ribosome. The 2014 Oscar-winning, charming animated short Mr. Hublot by Laurent Witz and Alexandre Espigares, and the darker, full-length movie City of Lost Children by Marc Caro and Jean-Pierre Jeunet inspired us to adopt this style. The artwork was drawn by  Patrick Lane at Sceyence Studios (www.sceyencestudios.com), who also produced our previous Biophysical Journal cover, myosin V as Robert Crumb’s Mr. Natural, March 19, 2013.

More about our research on molecular motors and protein synthesis, emphasizing single molecule, optical trapping, and fluorescence microcopy can be found on the Goldman Laboratory website.

– Ryan Jamiolkowski, Chunlai Chen, Barry Cooperman, Yale Goldman

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Biophysics on World AIDS Day

December 1 is World AIDS Day. Globally, there are an estimated 36.7 million people who have the virus. Despite the virus only being identified in 1984, more than 35 million people have died of HIV or AIDS, making it one of the most destructive pandemics in history.

In recognition of World AIDS Day, we spoke with Biophysical Society member Leor Weinberger, University of California, San Francisco, whose research focuses on how HIV makes a fate decision between active replication and latency.

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What is the connection between your research and HIV/AIDS?

Our lab focuses on fundamental aspects of how HIV makes a fate decision between active replication (turning on) and a long-lived dormant state called latency (turning off).  Latency is the chief barrier to curing a patient of HIV.  We use quantitative single-cell approaches and mathematical models to map the transcriptional circuitry that controls this fate decision.

Many years ago we found that HIV harnesses and amplifies stochastic fluctuations (noise) in gene expression to control its active vs. latent fate decision (back in the early 2000’s HIV, in fact, provided the first experimental evidence of a noise-driven fate decisions).  Recently, we discovered that noise can be manipulated with small molecules and we are developing therapies that disrupt HIV circuitry by manipulating noise.

Why is your research important to those concerned about HIV/AIDS?

HIV latency is the chief barrier to curing a patient of HIV.  There is no vaccine for HIV and current anti-retroviral therapies only halt active viral replication; if a patient discontinues therapy, HIV will reactivate from latently infected cells and viral levels in the blood rebound to pre-treatment levels within a few weeks.  We now know the most problematic (i.e., longest-lived) latent reservoir exists in a type of white blood cell called a CD4+ T cell, the same cell type that HIV actively infects.  HIV remains ‘silent’ and integrated as a “provirus” in the genome of these cells.  So, the field is actively pursuing approaches to better understand and attack the proviral latent reservoir.

Our work showed that a major mechanism HIV uses to promote its silencing is harnessing stochastic fluctuations in a transcriptional feedback circuit and gene-expression noise is now acknowledged as a primary clinical barrier to reversing HIV latency and curing HIV. Recently, we identified molecules that modulate stochastic fluctuations and could be a new class of therapeutic candidates for reversing latency.

How did you get into this area of research and how long have you been working on it?

I’d always hoped to make a contribution to medicine but as a student I became enchanted with the beauty of nonlinear dynamics theory and the intellectual approach of physics.  This eventually led me to train with a group of physicists who were developing mathematical models to explain HIV infection dynamics in patients.  At the time many physicists were modeling biology but very few models were grounded by experiment (HIV was one of the very few).  A problem, however, was convincing experimental collaborators to test the most interesting predictions.  So, in grad school I decided to learn wet-lab molecular biology and eventually started my own experimental lab.

This combination of modeling and experiment helps the science move faster.  Plus, I greatly enjoy training students and postdocs at the interface of these disciplines and translating basic discoveries into potential therapies is both fun and rewarding.  So, my lesson to aspiring biophysicists is: try to find training opportunities that cross disciplinary interfaces (theory and experiment, basic and applied, etc…) it can be challenging at times but is also very rewarding.

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

We received generous funding to develop a radical new class of ‘autonomous’ antiviral therapies from the Defense Advanced Research Projects Agency (DARPA) as well as the NIH, especially the NIH Director’s Common Fund.  DARPA actually started a program called INTERfering and Co-Evolving Prevention and Therapy (INTERCEPT) to fund this work.  The NIH Common Fund was what allowed us to start out with this research; it is a unique and important program that funds high-risk, high-reward research through the Director’s Pioneer and New Innovator Awards and has mechanisms to support young researchers setting off in bold directions.

Have you had any surprise findings thus far?

Probably our most surprising finding was discovering noise-enhancer molecules (Dar et al. Science 2014).  These are small molecules (some are approved drugs) that alter transcriptional fluctuations without changing the mean level of gene expression and act like Bunsen burners for cells: they appear to potentiate cell-fate decisions in diverse systems.

The analogy can be made to physical and chemical systems, where many processes are enhanced by catalysts but also by increasing the thermal fluctuations (e.g., kT in the Arrhenius equation).  Catalysts deterministically lower activation-energy barriers on potential-energy landscapes but when deterministic drivers are insufficient to cross the barrier, amplifying thermal fluctuations (e.g., with a Bunsen burner) provides an added perturbation for crossing activation-energy barriers.

Remarkably, we found that this concept applies to gene regulation during cell-fate decisions; the novel class of noise-enhancer molecules act like biological Bunsen burners.  As a model system, we focused on HIV where the leading HIV-cure strategy requires latent virus be reactivated and then purged—but where current reactivation schemes are ineffective.  Strikingly, these Bunsen burner-like noise-enhancer compounds potentiated transcriptional activators to greatly enhance HIV reactivation in patient cells.  We also also identified noise-suppressor compounds (effectively ‘ice packs’) that inhibit reactivation.

It is important to note that noise modulators starkly contrast with generic stress responses (e.g., starvation), which can increase noise but necessarily attenuate transcriptional activators.  Thus, noise modulation is a fundamentally departure from generic stress responses.  Preliminary evidence indicates that noise-modulating molecules could provide a general tool to manipulate diverse cell-fate decisions from antibiotic persistence to cellular reprogramming and cancer.

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

I often hear from colleagues that they most appreciate that our work shows a biological role for stochastic ‘noise’.  Before the HIV example, there had been beautiful and elegant work from colleagues measuring and identifying the sources of stochastic gene-expression noise in laboratory organisms (E coli and yeast).  However, it was not clear if cells ‘cared’ about the noise or simply ignored it.  The HIV example (Weinberger et al. Cell, 2005) was the first to experimental evidence that stochastic noise in gene-expression could flip a genetic switch and drive a biological fate decision (i.e., developmental bet-hedging).  Developmental bet-hedging–the concept that organisms harness intrinsic variability to enable bet-hedging decisions between alternate developmental fates, similar to how financial houses diversify assets to minimize risk in volatile markets—had in fact been hypothesized since the 1960s.  Subsequent work from some of my scientific heroes—and now friends—showed similar results in B subtillis, stem cells, and cancer cells.  Recently, we have gotten a lot of requests from colleagues for our noise enhancer molecules and we send out samples every few weeks.  I suspect that these molecules may end up being the more helpful contribution to the field.

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

Typically, it is a different thread of research in our lab that catches the public’s attenuation.  Many years ago we hypothesized the idea of treating infectious diseases by engineering therapies that co-evolve and co-evolve TIPs mentioned above (Metzger et al. PLoS Comp. Biol. 2011) has garnered the most public interest.  Existing measures for infectious disease control face three ‘universal’ barriers: (i) Deployment (e.g. reaching the highest-risk, infectious ‘superspreaders’ who drive disease circulation); (ii) Pathogen persistence & behavioral barriers (e.g. adherence); (iii) Evolution (e.g. resistance and escape). To surmount these barriers, we have proposed a radical shift in therapeutic paradigm toward developing adaptive, dynamic therapies (Metzger et al. 2011). Building off data-driven epidemiological models, we show that engineered molecular parasites, designed to piggyback on HIV-1, could circumvent each barrier and dramatically lower HIV/AIDS in sub-Saharan Africa as compared to established interventions. These molecular parasites essentially steal replication and packaging resources from HIV within infected cells thereby generating Therapeutic Interfering Particles (TIPs), which deprive HIV of critical replication machinery thereby reducing viremia. The fundamental departure from conventional therapies is that TIPs are under strong evolutionary selection to maintain parasitism with HIV and will thus co-evolve with HIV, establishing a co-evolutionary ‘arms race’ (Rouzine and Weinberger, 2013).  Like Oral Polio Vaccine, (OPV)—currently used for the W.H.O. worldwide polio-eradication effort—TIPs could also transmit between individuals, a recognized benefit for OPV.  TIP transmission would occur along HIV-transmission routes (via identical risk factors), thereby overcoming behavioral issues and automatically reaching high-risk populations to limit HIV transmission even in resource-poor settings.

Analysis of a Brain Plasma Membrane

BPJ_113_10.c1.inddCellular plasma membranes (PM) contain hundreds of lipid species that are heterogeneously distributed in the membrane plane, forming local domains of altered composition. Cells tightly regulate their lipid composition because lipids influence membrane protein function both directly through lipid-protein interactions and indirectly through changes in bilayer properties. Yet larger differences in lipid composition can be observed between different cell types. By constructing a realistically complex lipid model of a neuronal PM, we can determine the membrane’s properties and compare to that of an average PM. Even though the neuronal lipid mixture is significantly different from the average mixture, the main changes in mixture are carefully counter balanced, resulting in a range of surprisingly similar membrane properties while domain behavior is unique.

The cover image for the November 21 issue of the Biophysical Journal illustrates the vast possibilities for analysis of a neuron’s realistically complex lipid membrane. A composition of many of our analysis features is shown stacked on top of a computer-rendered neuron-like cell. The features show a range of system characteristics from the distribution of the lipids and their tail types to analysis of membrane thickness and the lateral flow of the lipids to the fitted curvature surface of the membrane and distribution of cholesterol domains. The figure captures the depths of analyses that are required to properly characterize our simulations. The layers of analysis are complementary to each other, and all are required to fully appreciate and understand the sophistication and subtlety of complex, dynamic membranes.

The trajectory images of the membrane were rendered using Tachyon in VMD. The membrane thickness and lipid flows were calculated from the simulation and rendered with Python tools. The membrane curvature surface and the cholesterol domains were rendered using Paraview. The neuron-like cell was made and rendered using Blender 3D.

– Helgi Ingólfsson, Timothy Carpenter, Harsh Bhatia, Timo Bremer, Siewert Marrink, Felice Lightstone

American Education Week

American Education Week (1)

November 13-17 is American Education Week, highlighting the importance of K-12 education. We asked Biophysical Society Education Committee members to tell us about teachers who made an impact on them.


stoked01-hero“In high school there was a fierce science teacher named Mr. Housek. I was unimpressed with the butterflies that my fellow students were collecting in Biology and, against all advice, took his Electronics class instead, where I learned to use a slide rule and to build circuits. Somehow, we got along and his Physics class provoked my curiosity in understanding how things worked. Little did I know how very complicated it was going to be to apply this understanding to biology.”

– David Stokes, New York University

 

“I had a remarkable science teacher, Mr. Griffith, at Wy’east High School, a small high downloadschool in rural Oregon – he was known for his pointed and sarcastic remarks, as well as his ability to teach science. He taught me Chemistry, Physics and Semi-Micro Quantitative Analysis, and oversaw my senior research project, which won a semi-finalist place in the Westinghouse Science Talent Search. He also graciously disposed of the batch of chemicals that I once mixed and realized after making it, that it might not be entirely safe. He taught science to many students, including a colleague at UNC Chapel Hill, Richard Cheney, who grew up in a small town up the road from my parents. Sadly, Mr. Griffith is no longer with us.”

– Sharyn Endow, Duke University

 

Me in Office“The biggest impact on me was not from any one teacher but from a family of teachers at St. Stephen’s Elementary School in Milwaukee.  They watched over me and my brother during difficult times in a turbulent family.  My debt to them is immeasurable.”

– Alex Small, Cal Poly Pomona

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“I would like to honor Mrs. Mader from Quarton Elementary School in Birmingham, MI. She taught us about self-esteem and confidence. She told me that you could go a long way in life if you believed in yourself, and she was right!”

-Ashley Carter, Amherst College

 

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“I did have a 9th grade math teacher who I have always remembered.  He recognized that I had some talent and let me work on my own in the back of the room during class.  I was a bit introverted at that time, and working on my own at my own pace really motivated me.”

-Allen Price, Emmanuel College

 

Linda Columbus Investigates Cell Membranes With Large New Grants

“Many teachers come to mind when asked about K-12 teachers that made an impact on me. I couldn’t read when I started first grade and my teacher took the time and effort to get me up to speed and performing well by the end of the year. I am confident without her attention to the way I learn and realizing it wasn’t for the lack of ability that I would not be a scientist. Another was my seventh grade science teacher that had very visual exams, which stimulated me. We were learning anatomy and doing dissections so on the day of the exam there were about 25 dissected animals or products from our labs that were tagged with numbers and we had to go around the classroom to identify or answer specific questions about the visual product. This was so aligned with my learning style and curiosity that I loved the exams. My third grade teacher let us self-pace in math if we wanted. So we could just keep going in the math book and several of us chose to do math instead of free time some days. Fourth grade was a shocker when we weren’t allowed to do that anymore. Another was a teacher in high school that I had for two classes, Calculus and Computing. She just got how I learned and most lessons were open ended or if we finished we were asked to help others in the class. Looking back, it seems the teachers that were inclusive of different learning styles and instruction were the ones I remember.”

– Linda Columbus, University of Virginia

 

 

Get to Know: Joanna Swain, BPS Council Member

We recently spoke to Biophysical Society council member Joanna Swain, Bristol-Myers Squibb, about her research, meeting her heroes, and what she loves about living in New England.


Joanna Swain_pictureWhat is your current position & area of research?

I am currently a Senior Principal Scientist in Molecular Discovery Technologies at Bristol-Myers Squibb, where I work to discover transformative medicines for patients whose medical needs are not being met by currently available treatments. My team uses in vitro selections to discover cyclic non-natural peptides that bind to pharmacologically important targets with high specificity, but that are small enough to hold the promise for intracellular delivery and oral bioavailability.

What drew you to a career as a biophysicist?

I was initially drawn to the field of structural biology by the idea of seeing the unseeable, with NMR as my first tool to illuminate protein structure and dynamics. I was captivated by the theoretical models of protein allostery, and wanted to understand allostery at a mechanistic level – how are protein structure & dynamics impacted by ligand binding, and how can information about binding site occupancy be transmitted to distal sites? It has been immensely rewarding to channel my interest in modulating protein activity toward drug discovery in an industry setting.

What do you find unique or special about BPS? What have you enjoyed about serving on Council?

In my early career, BPS was the meeting for finding other people who shared my interests and for learning about new technologies and applications that could be relevant to my own research. Since joining Council, I have been particularly impressed by the commitment of the Society to maintaining diversity at the podium in all of its meetings. Serving on Council has also given me the opportunity to meet many academic peers that I would not otherwise have gotten to know. I have to admit to being a little starstruck in a recent Council meeting sharing breakfast with my heroes Angela Gronenborn and Jane Dyson!

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Swain modeling her mother’s 1970s era ski gear. “It’s still going strong! Wooden waxless skis with real mohair strips, and leather boots with no insulation whatsoever! Perfect for New England winters,” she jokes.

Who do you admire and why?

I admire women like Margaret Oakley Dayhoff, who pushed their way past boundaries and first claimed access to male-dominated scientific fields, allowing me to follow my interests and gather opportunities that were never so easily offered to them. I admire local and global citizens, faith leaders, and activists, who build me up with messages of hope for a more just world, and give me both strength and mechanisms to help make it happen.

What do you like to do, aside from science?

Raising a family alongside an active career has not left much time for other pursuits, but now that my children have grown to teenagers, I look forward to a future that involves a whole lot more bicycling and travel, hopefully at the same time!

What is your favorite thing about living in New England?

I love the change of the seasons, and long cold snowy winters. It’s not everyone’s cup of tea.

What is something BPS members would be surprised to learn about you?

Sometimes the news on NPR’s Morning Edition becomes too much for me on my drive to work, so I start the day with Red Hot Chili Peppers or Ani DiFranco at volume 11 instead.  With enthusiastic tuneless singing.

Do you have a non-science-related recommendation you’d like to share?

This might be a better answer for the last question, but my guilty pleasure is the TV show “Shameless.” I think William H. Macy’s portrayal of deadbeat dad/addict Frank Gallagher is just brilliant. Again, it’s not everyone’s cup of tea.

Do leaf hairs swing to a caterpillar beat?

BPJ_113_9.c1.inddIn 2014, when Heidi Appel and Rex Cocroft demonstrated the ability of Arabidopsis leaves to respond to the sound of Pieris caterpillars feeding, people reacted with either disbelief or a sense of playfulness. For this cover image for the November 7 issue of Biophysical Journal, we have picked up the fanciful idea that plants can appreciate music.

The cover image conjures up the ghosts of science fairs past: playing music to plants, a favorite high school student project for well over half a century. Whether or not responses were measured, some responses should in theory occur if plant and parameters are selected appropriately. Many plants are extremely sensitive to small mechanical stimuli, and with a well-chosen plant it should be necessary to only include the right frequencies and a (perhaps unreasonably) high volume. Nevertheless, although plants have not evolved to appreciate Chopin and the Beatles, some certainly may have evolved to listen in on chompin’ and the beetles.

The cover illustrates the possibility that trichomes (hairs) of the weed called Arabidopsis thaliana are acoustic sensors. The trichomes are well known to have many other functions–for example protecting against the overly bright sunshine of the cover image, creating a layer of surface moisture to lessen dehydration, and greeting herbivores with a shield of distasteful toxins. But their evolution may have also been driven by the mechanical inputs shown. For example, they are the first cell type that insects touch when settling on an Arabidopsis leaf. And, as illustrated in the archetypal simulations on the cover, they have the form of miniature mechanical antennae.

The cover reflects how the percussion section contributes as well. Our recent study of trichome mechanoresponses showed that touching or brushing led to diverse complex patterns of acidification and cytosolic Ca2+ oscillations in the stalk, branches, and subsidiary cells (Zhou et al., Plant, Cell & Environment, 40:611-621. 2017).   Morphological observations suggest that information propagates to the leaf as a whole, where it was already shown that caterpillar feeding elicits rapid production of deterrent toxins.

The idyllic scene on the cover of Biophysical Journal highlights how the mechanoresponsive trichome is an idyllic system for studying plant signaling. The guard cells of the stomata that control gas exchange have been considered the premier system for such study, and it may be significant that the trichomes derive from the same kind of stem cells.  Evidences such as presented in our study play up the importance of the historical transition from the belief that walls are dead, to the concept that they play active and vital regulatory roles of mechanical, electrical, and biochemical character, especially when jamming out like the walls on the cover.

For more on our work in this area, please visit these websites: www.cemb.org, and http://bebc.xjtu.edu.cn/English/

– Shaobao Liu, Jiaojiao Jiao, Tianjian Lu, Feng Xu, Barbara Pickard, Guy Genin

On the State of Professional Opportunities for Women in Biophysics: Marina Ramirez-Alvarado

To investigate perceptions about the state of women in science, the BPS Committee for Professional Opportunities for Women is hosting a blog series where members can express their views on the subject by briefly answering these four questions: In your opinion,

  1. What is the current state of gender equality in science and biophysics?
  2. What is the value of having equality and true inclusiveness?
  3. What is one area that needs attention; and
  4. What is the one thing that can be done right away?

You are encouraged to read and comment on these blog posts, and to volunteer your own answers by emailing them to Laura Phelan at lphelan@biophysics.org.


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Marina Ramirez-Alvarado, Mayo Clinic

What is the current state of gender equality in science and specifically in biophysics?

MRA: Gender equality has definitely improved over the past 25 years since I finished college and embarked on a scientific career, but unfortunately, things are still not great for women in science and biophysics.  We are all struggling with implicit biases that diminish and discourage the work of female scientists and biophysicists. Also, most often, when couples are faced with the two-body problem, the man’s career takes precedence. Many measures of progress, such as for example, the percentage of women professors in STEM fields, show that the pipeline is still leaky, especially at the top of the ladder, for full professors and leadership positions. In my view, a current problem is patchy support: some institutions are supportive, while others are less so; some colleagues are sensitive, supportive, and inclusive, while others are not. Further, even within biophysics, some sub-fields include more female speakers than others, and some journals (including Biophysical Journal) have more female representation in their editorial board than others. The same colleague/interaction/experience may be positive for one female scientist and negative for another. It can be confusing!

What is the value of striving towards equality and true inclusiveness?

MRA: More and more data show that diverse teams are more productive, more creative, and more successful. Female CEOs and female leaders are more effective in making their companies/institutions financially solvent. Female leaders inspire more loyalty and create welcoming and productive atmosphere where everyone feels valued. These seem to me compelling advantages for inclusive environments.

What is one area that needs attention?

MRA: Effective mentoring and increased visibility of female role models can help, and we must concentrate on closing this existing gap. Female scientists need mentors who can guide and advise them along fulfilling roles in research and leadership; further, it will be important to help women scientists identify possible sponsors and develop with them supportive and durable relationships. Mentors must recognize and acknowledge the multiple identities and roles that their mentees have; must learn to empower them to overcome barriers whether in the form of implicit bias, administrative burdens, and the sometimes-dangerous political waters of scientific careers.

What is the one thing that can be done right away?

MRA: We (all scientists) have to get involved. To those of us who are aware of the still real problem of gender inequity in science, I ask that that you speak up, voice your concerns and propose solutions; we have to express our concerns anytime we see gender inequality. To my colleagues who think we solved the problem of gender equality in science, I invite you to listen more to female colleagues, friends, and even relatives; every one of them has a story where she had to work harder than a male peer had to, for the same recognition.

Scientific societies, such as BPS, can play an important role by programming sessions at their annual meeting dedicated to addressing this problem.

In addition to programming educational sessions at their annual meetings, scientific societies can encourage all members to fill out the implicit bias assessment https://implicit.harvard.edu/implicit/education.html

One last thing: while we have to do more to include women scientists, we must extend an unbiased and welcoming hand to all colleagues regardless of ethnicity, race, sexual orientation, disabilities, etc. We need a broader view about what a diverse and inclusive environment is and work diligently to achieve it.