The Function of Lung Surfactant and the Lateral Organization of Lipids in Biological Membranes

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The cover image of the September 2 issue of Biophysical Journal shows a lipid monolayer in equilibrium with bilayer vesicles. Both bilayers and monolayers are separated into liquid-ordered and liquid-disordered phases. The bilayers were formed in water (not shown in the image) as a result of monolayer collapse below the equilibrium surface tension. This structure was obtained from molecular dynamics (MD) simulations with the Gromacs software package and the coarse-grained Martini model.

The image was generated from the three-dimensional particle densities using the visualization software Paraview [D. Rozmanov et al., Faraday Discuss., in press]. The densities were sampled on a high-resolution grid (0.2 nm) using a short MD trajectory (1 ns) at the monolayer–bilayer equilibrium. The simulation time to achieve the equilibrium was 25 µs. The monolayer lateral size is 50×50 nm2. The image shows part of the system, corresponding to a patch of ca. 30×22 nm2.

This image is an example of the scale and complexity of systems accessible by the state-of-the-art computer simulations. The coexistence of liquid-ordered and liquid-disordered phases as well as liquid-expanded and liquid-condensed phases was reproduced in a monolayer at an air-water interface, and large-scale collective lipid transformations upon monolayer collapse from the interface were simulated. This work was inspired by questions related to the function of lung surfactant and the lateral organization of lipids in biological membranes. More details on our research can be found on the group webpage at http://www.ucalgary.ca/tieleman/.

- Svetlana Baoukina, Dmitri Rozmanov, Eduardo Mendez-Villuendas, Peter Tieleman

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Everything You Need to Know about BPS Travel Awards for the 2015 Annual Meeting

Only a month remains before the abstract and travel award deadlines for the Biophysical Society’s 59th Annual Meeting, being held February 7-11, 2015 in Baltimore, Maryland. If you are a student, postdoc, early or mid-career scientist looking for a little extra funding to attend the Annual Meeting, be sure to apply for a BPS Travel Award. Check out the FAQ below to learn more about the application process.

What is the Travel Award application deadline?

October 3. Remember: You MUST submit an abstract by October 1 in order to be eligible for a Travel Award.

Can I submit any part of my application late?

No. ALL parts of your application are due by the October 3 deadline – including your letters of recommendation! Start asking your advisers for references now, and be sure to read each award’s description so you know exactly what is required.

I think I’m qualified for both the CPOW and Education Travel Awards. Can I apply for both?

Yes, you can apply for multiple travel awards, as many as you are eligible for. However, you can only be selected to WIN one award.

 Oops! I forgot to submit my abstract by October 1. But I am going to submit a late abstract! Can I still apply for a Travel Award?

No. Only abstracts submitted by the regular deadline (October 1) will be eligible for a Travel Award.

I am a co-author on an abstract, but not a presenting author. Can I apply for a Travel Award?

In most cases, no. For all Education, MAC, and International awards, you MUST be a presenting author on the abstract. If you are not a presenting author, your abstract will be marked as ineligible. This also applies to CPOW awards for postdocs. The only exception is the mid-career CPOW award, for which you must be a co-author or presenting author on a submitted abstract.

When will I find out if I won?

You will be notified on the outcome of your application via email by November 21. Be sure to check your spam folder if you don’t see the email.

My adviser would rather send the letter of recommendation directly to you. Where exactly should he/she send it?

Letters of recommendation can be emailed to Laura Phelan, lphelan@biophysics.org. If your adviser prefers ‘snail mail,’ please have them send it to the attention of Laura Phelan at the Society Office. We are located at 11400 Rockville Pike Suite 800, Rockville, MD 20852. Whether emailed or mailed, all letters must be received (not postmarked!) by the October 3 deadline.

 I am not a US citizen, but I am still a minority researching in the US. Why can’t I apply for the MAC Travel Award?

Because the MAC Travel Awards are funded by an NIH grant, only US citizens or permanent US residents are eligible. Please be sure to check out the Education or CPOW awards to see if you qualify.

I am applying for an Education Travel Award as a postdoc. Why is the application asking me to answer all the questions for undergrads and grad students?

Postdoc Education Travel Awards only require a CV and a copy of your abstract. Please fill out all of the extra questions with ‘n/a’. When the site asks you to upload a copy of a faculty recommendation letter, simply upload a copy of your CV instead. Let us know if you have questions.

I’m international but I live/research/study in the US. Aren’t I still eligible for an International Travel Award?

No, you are not eligible. You must be living and conducting research OUTSIDE of the US in order to qualify for an International Travel Award. If you live/work/study in the US, no matter your origins, you are not eligible for this award.

 But I’m an international postdoc living/researching in the US. Does this mean there are no Travel Awards available to me?

All postdocs are eligible for the Education Travel Award. If you are a female postdoc, you may also be eligible for the CPOW Travel Award. Be sure to review eligibility requirements online.

I am currently a graduate student. However, by the time of the Annual Meeting I will be a postdoc. What award should I apply for?

You should apply for the awards that fits your career level as of October 3. In your case, you must apply as a graduate student.

I am no longer a student or a postdoc. Am I eligible for a Travel Award?

MAC, CPOW, and the International Relations Committee all offer travel awards for junior, senior, and/or mid-career scientists. Please check eligibility requirements online to see if you qualify for any of these awards.

Still confused? Please contact the Society Office at (240)-290-5600 or lphelan@biophysics.org.

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Biophysical Tools Key to Understanding How Ebola Spreads

On most days, biophysicists go about their work with little public interest.  The things they work on are complex, very specific, and impossible to see with the naked eye. Every once in a while though, biophysicists find their work in the spotlight due to public events such as a disease outbreak. With the ebola outbreak in west Africa in the news, the research of Biophysical Society members Michelle Digman, University of California Irvine, and Robert Stahelin, University of Notre Dame has attracted increased attention. Digman was kind enough to answer some questions about her work for us.

Actin cytoskeleton (red) and Ebola VP40 virus budding (green).

Actin cytoskeleton (red) and Ebola VP40 virus budding (green).

What is the connection between your research and Ebola?

My research aims are to develop enabling technologies in optical microscopy to be able to measure protein dynamics in living cells. These fluorescent fluctuation measurements measure spatial-temporal interaction at the molecular scale in real time. My colleague, Professor Robert Stahelin at the Indiana University School of Medicine and the University of Notre Dame, who is studying protein-lipid interactions in the life cycle of the Ebola virus, wanted us to use these biophysical approaches to measure the membrane binding behavior of the Ebola virus matrix protein, VP40. Given that we both are interested in understanding protein regulation and function we teamed up to understand how VP40 from the Ebola virus assembles into virus like particles (VLPs) in live cells.

Why is your research important to those concerned about finding a cure/stopping the spread of Ebola?

Our research involves studying the egress of the virus particles and understanding the mechanism of spreading. There is not a large body of literature that addresses how the ebola virus assembles and how it interacts with endogenous proteins, lipid membranes, or cytoskeletal structures in living cells. I think this information can be useful in developing small molecule targets for therapy.

How did you get into this area of research?

This collaboration, with Stahelin, started mutual interests to learn more about virus protein interactions in living cells. Our microscopic tools allowed us to measure protein assembly, aggregation and interactions. We realized once the VP40 protein was expressed in live cells with a fluorescent tag that the best way to follow protein assembly was to use dual color particle tracking.

How long have you been working on it?

We have been collaborating for 3 years.

Do you receive federal funding for this work? 

Yes, I am the Co-PI of the Laboratory for Fluorescence Dynamics (LFD) which is a P41 Biomedical Technology Resource Center (BTRC) funded by NIGMS at NIH. We have 5 core technological research projects, over 10 driving biological problems, collaborative research projects as well as training and dissemination of the technologies developed at the LFD. As such, our work with Stahelin also included training one of his graduate students, Emmanuel Adu Gyamfi, who spearheaded this project for his Ph.D. thesis.

Have you had any surprise findings thus far? 

Everything we learned with the Ebola VP40 protein was a surprise. It was never studied before in living cells and in real time. Using the number and molecular brightness (N&B) and with the 3D particle tracking methods, we were able to calculate the aggregation process on the actin filaments.

What is particularly interesting about your work on the Ebola VP40 protein from the perspective of other researchers?

The Ebola VP40 protein is sufficient to make virus like particles from human cells.  Thus, we study the assembly of one single virus particle at a time directly inside the cell to understand the spatial and temporal distribution of VP40 within the plasma membrane before a new virus particle leaves the cell membrane.

What is particularly interesting about your research from the perspective of the public?

With the recently identified structural information on VP40 that came available from Dr. Saphire’s lab at The Scripps Research Institute, we are starting to unravel the mechanism of how new virus particles are formed to sustain and spread Ebola infections.

With Ebola a top story in the news, are you receiving more inquiries about your work?  Does the attention to the disease affect you in other ways?

Yes, mostly from family and friends. Stahelin, on the other hand, has appeared on a local news broadcast, been interviewed on a radio show, and interviewed by the local newspaper.   has received the most inquiries.

 

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Turning to Micropattering As a Way to Control Cell Shape

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While significant progress has been made in understanding the biochemical and biophysical interactions of a wide variety of individual molecules, we know surprisingly little about how the cell coordinates hundreds and thousands of simultaneously active molecules across both time and space. For instance, we know in great detail the mechanochemical cycle of a non-muscle myosin II motor, but that doesn’t tell us how a cell determines where to put down an adhesion or how hard to pull on it as it polarizes and migrates. These types of questions are further complicated by the fact that many suspected regulatory factors are tightly coupled, making it difficult to establish distinct regulatory roles.

In this case, we wanted to know how the cell determines the magnitude and distribution of traction stresses when adhered to a surface. When it became apparent that cell geometry was important we turned to micropatterning as a way to tightly control cell shape. In this way we could decouple the roles of the suspected regulatory parameters: spread area, cell shape, number of adhesions and the stiffness of the substrate. By combining our traction force experiments with micropatterning, we could isolate single parameters to vary. The image selected for the cover demonstrates the beauty and precision of this approach. It depicts the actin cytoskeleton, stained by a fluorescent phalloidin, in an experiment where we held the spread area constant while altering the cell shape.

Using these techniques we were able to establish that the amount of work done by the cell was regulated by the spread area alone, and was independent of the substrate stiffness or the number of focal adhesions. Changes in cell shape served to regulate the distribution of stresses on the substrate, but did not change the overall contractility of the cell. These results enabled us to build a simple yet accurate physical model of the cell that worked for all cell shapes, not just the micropatterened ones. We can now use this model to investigate the molecular mechanisms driving these physical processes. Our aim is to highlight the interesting mechanical means that a cell can use to regulate molecular interactions at the cellular scale.

For more information please visit our websites: http://squishycell.uchicago.edu/ and https://mcmarche.expressions.syr.edu/

- Patrick Oakes, Shiladitya Banerjee, Cristina Marchetti & Margaret Gardel

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Canine Cell Scattering

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The image on the August 5 issue of the Biophysical Journal captures the moment that canine kidney cells on cover glass begin to break free of each other in response to stimulation with soluble growth factor. The image was obtained by chemically fixing the cells, labeling two specific proteins (the cytoskeletal protein actin and the cell-cell adhesion molecule E-cadherin) with fluorophore-conjugated chemical or antibody, and imaging the cells on a fluorescence microscope. Two fluorescence channels were color-combined to produce the final image. Even though several biochemical changes are known to accompany this process called cell scattering, the image qualitatively illustrates that the cell adhesion plaques and the actin cytoskeleton are involved in a fundamentally mechanical process. Our work attempts to quantify such mechanical changes in order to gain a physical understanding of this process.

Knowledge of the context-dependent physical forces exerted by cells is necessary to gain a complete understanding of various physiologically relevant phenomena. For example, how cell-generated forces shape the embryo and whether cell-generated forces play a role in cancer metastasis are broad, open questions in biology. Uncovering the relationship between the mechanical nature of cells or cellular processes and their biochemical underpinnings can be expected to continue to yield new insights into cellular and multi-cellular function. More details on our research can be found at our lab website at http://squishycell.uchicago.edu/

- Venkat Maruthamuthu and Margaret Gardel

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Using Biophysics to Understand the Basis of Allosteric Inhibition in the Hepatitis C Virus Polymerase

July 28 is World Hepatitis Day, drawing attention to the 240 million people chronically infected with the hepatitis B virus and 150 million people with the hepatitis C virus. To recognize this worldwide awareness day, BPS asked member Ian Thorpe, Assistant Professor of Chemistry and Biochemistry at the University of Maryland Baltimore County, to answer a few questions about his research on hepatitis C.

HCV blog

What is the connection between your research and hepatitis?

We study the RNA polymerase responsible for replicating the Hepatitis C virus (HCV) genome. There is no vaccine for HCV infection and this enzyme has been extensively targeted with the goal of developing HCV therapeutics. One reason for this is the crucial role the enzyme plays in generating new viral particles. In addition, because it is an RNA-dependent RNA polymerase, there is no homologous human version of this enzyme. Thus, if this enzyme is targeted there should be a decreased likelihood of generating side effects from impacting human proteins. The search for therapeutics has yielded many small molecule inhibitors of the polymerase, including allosteric inhibitors that bind distal to the active site. We employ molecular modeling and simulation to understand the physical processes that underlie this allosteric regulation.

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

Understanding how allosteric inhibitors modulate the function of the HCV polymerase provides insight into what intrinsic properties of the enzyme allow it to effectively replicate viral RNA. In addition, understanding the underlying molecular mechanisms involved may allow for the development of novel and more effective inhibitors. While there are treatments currently available to treat this disease, these are generally expensive, can require significant time investments and often have serious side effects. In addition, the HCV RNA polymerase does not contain proofreading ability. The resulting high error rate during replication induces an elevated incidence of mutations in the virus and facilitates the development of viral resistance to treatment regimens. Thus, there is a continuing need to identify new molecules that could serve as HCV therapeutics.

How did you get into this area of research?

During my postdoctoral position at the University of Utah with Professor Gregory Voth (now at the University of Chicago), I began to consider research projects that I could pursue during my independent career. My wife worked in the field of liver transplantation at the time. She was familiar with HCV because it is one of the leading reasons for liver transplantation in the United States. She also recognized the need for additional studies of HCV due to the limited treatment options and extensive gaps in our knowledge of how the virus functions. HCV infection is a burgeoning health crisis because the virus was only conclusively identified within the last thirty years and many people were likely infected (for example via blood transfusions) before a test became available to screen for HCV infection. Those who are infected often go decades without displaying symptoms, only to be diagnosed later with serious complications including cirrhosis and liver cancer. Thus, a large fraction of the population who were infected in the past may be asymptomatic and are only now starting to display symptoms (or will do so in the near future).

How long have you been working on it?

I began working in this field after becoming a faculty member in the Department of Chemistry and Biochemistry at the University of Maryland, Baltimore County in 2009.

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

I do not currently receive federal funding for this work. However, I do receive federal support in the form of compute time on supercomputing resources supplied by the National Science Foundation via the Extreme Science and Engineering Discovery Environment (XSEDE).

Have you had any surprise findings thus far?

Yes, several! One of the key features of the HCV polymerase is that it likely undergoes transitions to distinct conformational states as it replicates RNA. These include a closed state required for the initiation of replication and an open state associated with elongation of the newly synthesized RNA strand. We have discovered that components of the polymerase may have a regulatory function by restricting the conformational sampling of the enzyme. In addition, we have seen that while diverse allosteric inhibitors can induce distinct effects on conformational sampling and enzyme dynamics, shared characteristics exist that may underlie the inhibitory action of these small molecules. Specifically, most inhibitors we have studied disrupt the conformational sampling of the enzyme in ways that can be related to their inhibitory capability. Some discourage conformational transitions by overly stabilizing one or the other conformational state, while others may destabilize both states to the extent that neither can be stably occupied. Finally, we have observed that free enzyme is able to explore both the open and closed states thought to have functional roles in RNA replication. This result is unanticipated given that the free enzyme lacks other components of the replication complex such as RNA template or nucleotides. This observation suggests that the presence of ligands does not engender new enzyme conformational states, but instead shifts the populations of preexisting enzyme conformations. Such a phenomenon is consistent with the conformational selection model of allostery.

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

Our studies highlight mechanisms of allosteric regulation that provide insight into how allosteric inhibitors decrease enzyme activity and suggest experiments that can be carried out to validate our hypotheses. Understanding the molecular mechanisms of allosteric inhibition is important in the development of HCV therapeutics because this knowledge may allow the discovery of new and more effective inhibitors or new ways of using existing inhibitors, such as in novel combination therapies. These studies also illuminate the fundamental processes involved in RNA replication by viral polymerases. HCV is related to several viruses that are serious human or agricultural pathogens, including the viruses that cause Dengue Fever, Yellow Fever, West Nile disease and Bovine viral diarrhea. Thus, knowledge gained from studying the HCV polymerase may be applicable to polymerases from these related viruses as well. More generally, our research helps to elucidate the many ways that allosteric regulation of enzyme function can occur. Allosteric regulation is a key way in which protein function can be modulated in biological systems. Thus, better comprehending the underpinnings of allosteric regulation may be applicable in diverse contexts such as in determining the molecular origin of disease states or in discovering drugs to treat other ailments.

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

While this work is fundamentally basic science research, the knowledge we obtain may ultimately be useful in identifying novel and more effective treatment options for infection by HCV and related viruses.

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Learning from Industrial Expertise: A Change in Perspective  

By Cecilia Read, Summer Research Program in Biophysics 2014 participant

When I think of industry, I think of cubicles and an endless amount of paperwork that consumes any livelihood you might have had at the beginning of the work day.  As an undergraduate in the field of biomedical engineering, industry is a very real possibility for me after college.  However, the mental image I’ve developed of industry since entering my field of interest has pushed me from even considering it as an option after I graduate; I would rather continue in academia.

My view on industry changed with the visit of Dr. Deborah Thompson and Dr. Drew Applefield, two employees of the Biotechnology Center in North Carolina who came as speakers for the Biophysical Society Summer Research Program I am participating in.  They provided great insight into what to expect if entering industry, the benefits of an academic background in industry (basically having a Ph.D.), and how to prepare an application for a job in industry.  They each talked about their personal backgrounds and finding their passion in industry.  While one was involved with organizing internships and industrial research, the other worked in advising new businesses and individuals looking for employment in industry. They spoke about finding a balance when choosing what field of industry to work in and the importance of recognizing “What I want” versus “What I bring” to the work place.  Each had their own perspective that made the presentation informative and useful.

When talking about what stood out from the presentation, my friend, Amanie Power, another student in our summer program, reminded me of the speakers’ most important advice: be aware of your own transferable skills.  What exactly did they mean by this? Each person has skills that they may not initially view as important for other fields of work.  In actuality, many skills are transferable; as Amanie describes, “skills that are good for anything.”  It’s hard to see one’s own skills as good for everything, but it’s important to recognize and highlight transferable skills, because they can be used for every setting.  My other friend and fellow student in the Biophysical Summer program, Olivia Dickens, described the whole experience in the most direct way possible:  “it was the best speaker presentation we’ve had all summer… they came, they presented, and they left.  They were well prepared and said exactly the right information that was useful for us.”

I still want to pursue a degree after my undergraduate experience, but I am no longer as opposed to industry as I was before.  Both Dr. Thompson and Dr. Applefield did a tremendous job in enlightening my misconceived notions about industry.   Industry is now no longer a negative alternative to academia in my mind; it’s just another option that can lead to future adventures, with its own kind of fulfillment.

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