Opportunity to Engage: Biophysics Week 2017

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You are scientist. It is your job to be an expert in what you study, to know and understand the tiniest details of your subject matter. You work with others in your field, and teach/mentor students and postdocs with some background related to your work.  You publish your work in specialized publications so that scientists with similar backgrounds and knowledge of your specialized vocabulary understand what you do. But when was the last time you explained your work to someone outside of your very specific field?  Or talked about biophysics and all it encompasses in general?

The second annual Biophysics Week, March 6-10, 2017, is an opportunity to do just that. The Biophysical Society will be hosting a series of events, including webinars on topics ranging from mentoring to getting a biophysics paper published,  and a Congressional briefing.  Lesson plans and profiles of women in biophysics will be released.  Cell Press will create a picture show, illustrating the beauty unveiled by biophysics research.

But to really reach people, the Society needs you to get involved.

We encourage you to plan an educational outreach event , such as a seminar, webinar, information session, lab tour, open house, or other activity that allows you to share what you and your colleagues do with others. The Biophysical Society will advertise your event on its website, in member communications, and through  social media.

And you will have taken your science out of the lab and engaged.  Maybe the effort will result in a student deciding to take a biophysics class, or find a biophysics lab to work in.  Maybe it will introduce a high school student to the term biophysics and teach him to not be intimidated by it. Or maybe your efforts will result in a non scientist developing an appreciation of basic research.  All are important outcomes.  And they will only happen when we all  engage.

Plan your event and register it here.

Stay up-to-date on Biophysics Week 2017 here.

 

Computational Microscopy: Using Simulations to Decode Infrared Vibrations

BPJ_112_1.c1.inddHeterotrimeric G-proteins are molecular switches that are omnipresent in animal and plant cells. They maintain central physiological processes such as vision, scent, or blood pressure regulation. The signal is determined by a small molecule, Guanosine triphosphate (GTP).  GTP binds to the heterotrimeric protein and thereby switches the signal “on.” The off-switch is maintained by hydrolysis of GTP to GDP and a phosphate moiety.

This central molecular reaction has beenthe focus of research for dozens of years, as the mechanism is highly conserved and leads to a plethora of diseases when disturbed, including cancer.

The cover image for the January 10 issue of the Biophysical Journal shows the GTP molecule bound to the active site of Gi1, one of three subunits of heterotrimeric G-proteins. The image was created using the program Blender. The configuration was obtained from coupled quantum mechanics and molecular dynamics simulations of the protein crystal structure. We used these simulations to obtain a structural interpretation of infrared spectroscopy measurements of the protein. Although infrared spectroscopy yields millisecond temporal and sub-Ångstrom spatial resolution, this information is encoded into infrared spectra that can be hard to interpret. However, a figure like the cover image can help this become easier to understand. By combining simulations with experiments one can use the computer as a microscope with subatomic resolution and directly observe structural and electronic changes. We benchmarked this setup by introducing point mutations at the active site (indicated as sticks in the picture) and comparing experimental and computational spectral changes that were found to be in agreement.

Further simulations elucidated details about the Mg2+ cofactor in the active site (green sphere) and about catalysis of GTP hydrolysis by heterotrimeric G-proteins that can be found in our article.

-Daniel Mann, Udo Höweler, Carsten Kötting and Klaus Gerwert

Get to Know: Bert Tanner, BPS Early Careers Committee Chair

We recently spoke with BPS Early Careers Committee Chair Bert Tanner, Washington State University, about his research, his time on the committee, and the years he spent as a gymnast.

tanner-bertWhat is your current position & area of research?

Assistant Professor, Department of Integrative Physiology and Neuroscience, Washington State University

I study muscle biology and teach physiology to undergraduate, graduate, and veterinary students. Research studies within my laboratory focus on normal, mutated, and diseased proteins that influence muscle contraction. We often integrate mathematical modeling, computational simulations, biochemical assays, and biophysical system-analysis to investigate complex network behavior among muscle proteins. We use these findings to describe and illustrate molecular mechanisms of contraction that underlie muscle function at the cellular and tissue levels.

What drew you to a career as a biophysicist?

I studied Physics as an undergraduate student at University of Utah. The last couple years of my undergraduate studies I got the opportunity to further explore bioengineering and computer science, and I participated in a summer research experiences learning about computational biology, remote sensing, and environmental biophysics. Through these experiences, I became increasingly interested at using mathematics, physics, and computation to better understand and describe biological processes. Through a series of injuries, I started learning more about physiology and became increasingly curious about different applications where mathematical modeling could help illustrate complicated, dynamic processes at the molecular, cellular, and organismal levels.  This led me back to graduate school, where I ultimately began studying muscle biophysics.

What do you find unique or special about BPS? What have you enjoyed about serving on the Early Careers Committee?

I love the rigor, diversity, and plasticity of the Biophysical Society, as well as the annual Biophysical Society meeting.  I’ve been attending and presenting at the national meeting since 2004, and I am really impressed by the high-quality science and constructive engagement of many society members—many of whom have become great friends and colleagues over the years. I also really appreciate the strong commitment to training young scientists in a rigorous, difficult field that is demonstrated by the BPS and its engaged membership. I enjoy being a member of the Early Careers Committee because it is a platform that enables education and programming for early career biophysicists via the newsletters, society webpage and blog posts, and annual meeting events.  These early career biophysicists are among the best and the brightest minds in the world, and our committee feels it is critical to help them learn about the myriad career paths where their skills will make an impact: academia, industry, small business, national laboratories, science writing and education, public policy, etc.

Who do you admire and why?

I admire many people from many different walks of life, but I often think most of the people that have impacted my education in a positive way. This includes a handful of teachers from elementary, middle school, and high school, all of whom made a really big impact on my thinking and career choices. Just like the impact these teachers made on me, other teachers work tirelessly to educate students each day; the well-being of our society greatly benefits from their efforts.  A second tier of people that I really admire are the approachable, engaging, unselfish, and constructively-critical mentors or colleagues that I get to interact with each year.  These people inspire me to try and do my best each day, and to treat people kindly.

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What do you like to do, aside from science?

I love the outdoors and to exercise. When I can pair these two up, it is even better.  My favorite hobby is skiing, just being out in the snow and gliding down the mountain, trail, or path is fantastic.  The past few years I’ve spent all my spare skiing-time on the ‘magic carpet’ teaching my son how to ski.  He is 5 now, and getting pretty good at the ‘blue squares’.  On our last ski day in Spring of 2016, my daughter (then about 18 months old) even skied by herself for about 60-100 feet.  She loves skiing and spent most of her first couple seasons skiing in a backpack on my back. I cannot wait to watch her keeping up with her big brother soon.

What is your favorite thing about living in Washington?

The diversity of the outdoor activities.  My family and I get to live in a small town and I get to work at a Pac-12 university with wonderful colleagues and great resources to pursue my research.  However, we are only 30 minutes to 2.5 hours away from world-class white water rivers, camping, hiking, backpacking, and pretty good skiing.  This accessibility to nature, and the diversity of options is really special to me and my family.

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

I was a gymnast until age 18.  I loved it, but it took a lot of time and I decided not to pursue it as a collegiate athlete.  However, it was pretty fun watching some of the fellow gymnasts that I’d trained with, and competed against as I grew up, perform in the Olympics over the past 15-16 years.

Do you have a non-science-related recommendation you’d like to share (book, movie, TV show, etc.)?

The recent Zootopia movie has a classic and wonderfully painful scene with sloths running the DMV.  For a quick laugh (2-3 min segment) you should check it out on YouTube.

What drives immune cells to engulf pathogens?

BPJ_111_12.c1.inddMacrophages and neutrophils (phagocytes) are the front-line defenders in your body’s immune system. They seek out, ingest, and destroy pathogens and other debris through a process called phagocytosis.  Typically, phagocytosis is initiated when receptors on the immune cell surface bind to ligands which have coated a pathogen particle. Once the cell’s receptors have found their target ligands, they initiate a chemical cascade within the cell which recruits the biochemical machinery necessary to drive the cell to envelop its target, forming a vacuole in which the pathogen can be degraded.

On a small scale, nearly every cell type in your body internalizes nutrients and various signals through a similar engulfment process called endocytosis What makes immune cells and phagocytosis unique is the relative size of the internalized particle. During endocytosis, cells internalize small objects, typically no larger than 100 nanometers, a fraction of the cell’s size (usually 10-30 micrometers). However, during phagocytosis, immune cells need to be able to internalize very large particles such as bacteria, which could be several microns long, and debris like dead cells, which could be larger than the immune cell itself.

Phagocytes accomplish this seemingly heroic feat by leveraging the biomechanical machinery typically involved with cellular migration, specifically the actin cytoskeleton and myosin molecular motors. Once phagocytosis has been initiated, actin monomers within the cell begin to polymerize near the location of the bound particle. As the polymerized network forms, it pushes the cell’s membrane around the particle, forming what is called a phagocytic cup. Interestingly, as a particle is internalized, the leading edge of the phagocytic cup constricts, pinching down on the particle.

Earlier studies have shown that actin tends to accumulate in a dense ring at the point of constriction. Unfortunately, due to limitations of the microscopy techniques available at the time, the precise structural organization of actin filaments within this ring could not be resolved. Consequently, exactly how the actin ring facilitates constriction remained elusive.

Using super-resolution fluorescent imaging (Structured Illumination Microscopy) we sought to illuminate how actin is reorganized during phagocytosis, with the goal of providing insight as to how phagocytes constrict around their targets.  One of the challenges in using microscopy to study phagocytosis is that particle internalization is three-dimensional, yet nearly all microscopy techniques are inherently two-dimensional. To side-step this issue, we turned to a planar technique called Frustrated Phagocytosis. Instead of presenting immune cells with pathogen particles, we deposited cells onto glass coverslips functionalized with antibodies. When the immune cells contact the surface, they perceive it as a giant pathogen and begin to flatten and spread as if trying to phagocytose the entire plane, yielding an unfolded view of what’s happening at the cell-target interface.

The cover image in the December 20th issue of the Biophysical Journal shows several macrophage cells at various stages of the frustrated phagocytic process. In the image, each cell’s actin cytoskeleton is shown in green (using Atto488-phalloidin) and the nuclei are shown in blue (using DAPI). During the early stages of phagocytosis (top left), actin polymerizes at the leading edge of the cell, forming a dense zone. This is similar to the structure formed at the leading edge of migrating cells. As actin polymerizes at the edge, it pushes the membrane outward causing the cell to spread. As the cell nears its maximum contact area, the actin zone begins to dissociate (top center) and actin-filaments throughout the body of the cell bundle to form fibers (middle right). As the cell enters the later stages of phagocytosis those bundles reorient, surrounding the perimeter of the cell (bottom center and middle left). With actin bundles surrounding the cell, myosin motor proteins exert tension between adjacent bundles. This tension causes the network to contract, forcing the cell to pinch down on the substrate. For frustrated phagocytosis, this constriction drives the cell to retract from the surface, leaving fragments of actin and tethered membrane in its trail (spinney protrusions around bottom center and middle left cells).

The mechanism that triggers the bundles to form and reorganize around the cell perimeter remains a mystery; although, there is mounting evidence that mechanical factors such as the cell’s membrane tension are involved in signaling transitions to late-stage phagocytic behavior.  These images, along with other studies of phagocyte mechanics, illustrate the robust and dynamical processes that unfold when immune cells carry out their essential task of clearing debris and eliminating pathogens.

– Wenbin Wei, Patrick Chang, Jan-Simon Toro, Ruth Fogg Beach, Dwight Chambers, Karen Porter, Doyeon Koo, Jennifer Curtis, Daniel Kovari

BPS Members Making a Difference Beyond the Lab: Karen Fleming

Society members make a difference in their communities in many ways.  BPS member Karen Fleming a faculty member and undergraduate program director in biophysics at Johns Hopkins University, has taken on the barriers facing women in science, and decided to do something about it on her campus. 

Know a member making a difference in their community that should be featured here?  Let us know. We would like this to be the first of a series!

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Pictured, (Left to Right), all of Johns Hopkins University: Dominic Scalise, graduate student in chemical and biomolecular engineering and Women’s of Hopkins Exhibit team member; Erin Gleeson,  Project and Events Specialist, Office of Instituional Equity; Gail Kelly, one of the Women of Hopkins; Ron Daniels, President; Karen Fleming; and Jeannine Heynes, Director, Office of Gender Equity.*  Photo Credit: Will Kirk, homewoodphoto.jhu.edu

Role Models.

We hear it over and over again—the need for diverse role models so that diverse students can see themselves succeeding in science. This includes gender.  Women often are underrepresented on panels, at conferences, and as recipients of prestigious awards.  BPS member Karen Fleming decided to do something about it.

Fleming, along with a handful of other JHU staff and graduate students, formed a committee, successfully sought institutional funding and support, and put together an exhibit entitled “Women of Hopkins.” The purpose of the exhibit was to highlight the many successful women that have graduated from the university and been pioneers in their fields, especially for current students.   Nominations were accepted and vetted by the committee, resulting in a photographic exhibit of 23 Hopkins graduates, displayed on the walls of Hopkins buildings. The women highlighted represent many different fields-not just science—and include Bonnie Basler, Bernadine Healy, Carol Greider, Mary Guinan, Nitza Margarita Cintrón, and Florence Sabin.  The project also includes a Women of Hopkins website, which has a biography for each woman included in the exhibition, as well as a presence on social media platforms Facebook and Twitter, giving the project greater visibility.

*Women of Hopkins team members not pictured are:  Anna Coughlin, graduate student in chemical and biomolecular Engineering; Jeff Gray, Professor of chemical and biomolecular engineering; and Valerie Hartman, instructional designer at JHMI.

Probing Water and DMSO near Lipid Membrane Surfaces

BPJ_111_11.c1.inddDimethyl sulfoxide (DMSO) is a powerful anti-freezing agent and has been used in biology as a cryoprotectant of cells. Thanks to a series of experiments and computer simulations  bulk properties of DMSO solution are reasonably well understood, yet the effects of DMSO on water molecules near lipid membrane surfaces, which are more relevant for elucidating the underlying physical chemistry of DMSO as a cryoprotectant, still remain elusive.

The consensus from a number of different experiments is that DMSO dehydrates phospholipid bilayer surfaces, which our study confirms. However, the DMSO-enhanced water diffusivity at solvent-bilayer interfaces, was not confirmed in our simulations. In order to resolve this discrepancy, we explicitly modeled Tempo-PC by appending Tempos to a few choline groups and conducted simulations and analyses.

Our cover image for the December 6th Issue of the Biophysical Journal depicts a snapshot from the molecular dynamics simulation of POPC phospholipid bilayer in 7.5 mol% DMSO solution. The lipid tails are rendered in grey, and the regions corresponding to phosphatidylcholine head groups are depicted in pale blue. Four Tempo-PCs, in the upper and lower leaflets are highlighted with the tail domain in yellow and the Tempo appended to the choline group in blue. Of particular note is that in contrast to the original intent of Overhauser Dynamic Nuclear Polarization (ODNP) measurements using Tempo-PC lipids to probe the surface water dynamics, the Tempo moieties are predominantly equilibrated at 8 − 10 Å below the solvent-bilayer interface, probing the water dynamics in the interior of bilayer. The water and DMSO molecules around the Tempo moieties are depicted in stick and surface representations, respectively. The inset magnifies the snapshot of water and DMSO molecules around Tempo. The image was produced using the molecular visualization system, PyMOL.

DMSO deposited beneath the PC head group, where Tempo moieties are equilibrated, increases the area per lipid slightly, and hence water diffusion probed by Tempo is detected to increase with increasing DMSO. Our study suggests that the experimentally detected signal of water using Tempo, stems from the interior of lipid bilayers, not from the interface. The only viable tool for the direct probe of water dynamics on biological surfaces at present is ODNP measurements using a Tempo spin label. Given its significance, the equilibrated location of Tempo moiety in lipid bilayers revealed here calls for adequate interpretation of data and careful re-evaluation of the technique.

—Yuno Lee, Philip A. Pincus, Changbong Hyeon

Biophysics on World AIDS Day 2016

December 1 is World AIDS Day, a global awareness day to bring attention to the disease, the research being conducted in relation to it, and the many people living with HIV/AIDS. In honor of World AIDS Day this year, we spoke with Gildas Loussouarn, University of Nantes, about his research on cardiac channels dysfunction in Long QT Syndrome, a disorder seen much more frequently in HIV patients as compared to the general population.

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

With the worldwide development of antiretroviral therapies, HIV patients now live longer. As a result, they encounter additional pathologies and these pathologies are over-represented as compared to the general population. Among these pathologies, the Long QT syndrome, a heart rhythm condition associated with arrhythmias. In the general population, this syndrome is a rare disorder, characterized by a delayed ventricular repolarization leading to tachycardia and/or sudden cardiac death. HIV patients present with a higher prevalence of Long QT syndrome, as compared to the general population suggesting a higher risk of sudden cardiac death. Here at the institute du thorax, the global objective of our team is to decipher molecular mechanisms of cardiac ion channels and their dysfunction in cardiac arrhythmias, in order to identify new therapeutic targets. We are thus interested in cardiac channels dysfunction in the context of HIV.

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

More than 10 studies lead to the conclusion HIV patients have a higher prevalence of Long QT syndrome, as compared to the general population. Despite that, it is still difficult to address whether the LQT syndrome is due to the virus itself or to drugs that are proposed to HIV patients, which are known to prolong cardiac repolarization. Sorting this out is essential, in order to limit arrhythmias and the potential sudden cardiac death in HIV patients. By directly looking at the effect of HIV proteins on cardiac repolarization, we aim at addressing if the virus itself participate to cardiac repolarization prolongation. Importantly, we have already observed that one of the viral protein, Tat, can delay repolarization in human cardiomyocytes generated from induced pluripotent stem cells.

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This figure shows that HIV-Tat is detected intracellularly in human cardiomyocytes but not simian fibroblasts (COS-7), after a 24h external application (200 ng/ml). Tat immunostaining is shown in red, plasma membrane is identified by hERG channel immunostaining (green), nucleus is in blue (DAPI). Tat remains in the extracellular compartment of COS-7 cells (red arrow) while in human cardiomyocytes, Tat is located inside the cytoplasm (asterisks) and colocalizes with hERG at the plasma membrane (yellow arrows).* (Adapted from Es-Salah-Lamoureux et al, 2016, JMCC 99:1-13, with permission.)

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

I was first contacted by a colleague, Dr. Bruno Beaumelle (CNRS Research Director, Montpellier). Dr. Beaumelle had previously observed that due to its high affinity to the membrane phospholipid PIP2 (phosphatitylinositol 4,5-bisphosphate), HIV-Tat could interfere with some PIP2-dependent mechanisms of neurosecretion. Bruno Beaumelle had read our previous works showing the impact of a decrease in available PIP2 on cardiac repolarization through specific cardiac channels KCNQ1 (Kv7.1) and hERG (KV11.1). Our previous works indeed suggested that a decrease in PIP2 or a decrease in KCNQ1 / hERG channels affinity for PIP2 leads to a decrease in the activity of the repolarizing channels and hence leads to Long QT syndrome. Bruno Beaumelle anticipated that we will be interested in looking at a potential effect of Tat on these channels. The hypothesis was that Tat may be a link between HIV and LQT syndrome. Our results seems to confirm this hypothesis since expression or extracellular application of Tat leads to a decrease in the activity of the repolarizing channels KCNQ1 and hERG. In human cardiomyocytes, HIV-Tat leads to a delayed repolarization and other Action Potential alterations, which are common triggers of cardiac arrhythmias.

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

Our work that was just published in the Journal of Molecular and Cellular Cardiology was partially funded by the Fédération Française de Cardiologie, the Fondation Genavie, the Marie Curie European Actions, the French Regional Council of Pays-de-la-Loire, the National Research Agency, the Fondation Lefoulon Delalande, the Fondation pour la Recherche Médicale, the Association of Scientific Orientation and Specialization and Campus France. We hope to get specific funding from AIDS focused agencies allowing us to study the effects of Tat in further details.

Have you had any surprise findings thus far?

Yes! Our first surprise was that external application of Tat, which is known to penetrate cells, had an effect on hERG channels depending only in specific cell type! Tat did not have any effect on hERG channels expressed in COS-7 cells. Paradoxically, the same application halved the activity of the same channels in cardiomyocytes! The absence of Tat effect in COS-7 cells is surprising since Tat is a promiscuous ligand that binds a plethora of receptors (such as Heparan Sulfate Proteoglycans), internalization of which supposedly allowing Tat entry. To identify the reason of this cell-specific effect, we looked for intracellular Tat in both models, after its extracellular application. We observed that intracellular Tat could be detected by immunofluorescence, in cardiomyocytes but not in COS-7 cells (cf. also figure). We suppose that Tat requires specific cellular components to be internalized in sufficient amount to have an effect on the potassium channels. It has been shown that Tat interacts tightly with some receptors such as LRP and CXCR4 receptors, which represent a cell-specific way for internalization. Such receptors may lack in COS-7 cells. In addition, these observations illustrate the great value of induced pluripotent stem (iPS) cells derived cardiomyocytes, a model closer to mature human cardiomyocytes as compared to COS-7 cells. Human iPS cells are quite easy to get: they were obtained by reprogramming renal cells contained in the urine of a patient. This is clearly a non-invasive (but long!) way of getting human cardiomyocytes.

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

Tat is present in the patient’s serum and seems to target different organs and interfere with various PIP2-dependent processes in these organs: Bruno Beaumelle showed that Tat can interfere with the secretory activity of neuroendocrine cells, by sequestering PIP2. We then showed that Tat can interfere with cardiac repolarization, also by sequestering PIP2. Now, we could test if Tat is also a link between HIV and epilepsy, which is also overrepresented in HIV patients.  This hypothesis is founded on the observation that PIP2 activates neuronal channels (KCNQ2/KCNQ3), alteration of which leads to epilepsy.  We can hence speculate that AIDS may be seen as a “PIP2-pathie” with multiple organs targeted by Tat.

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

I think it is important for the public to bear in mind that despite huge progresses made in treatments against HIV, living with AIDS is associated to many other severe pathologies, including cardiac diseases. It is thus important to remain aware of risk- behaviors. Regarding potentially new therapies, an anti-Tat vaccine may represent a new therapeutic strategy, as tested currently by another French laboratory (ETRAV laboratory, Aix Marseille University/CNRS).

 

*Image scale= 5μm.