Lipid vesicles on the beat

BPJ_112_8.c1.inddThe cell plasma membrane serves not only as a protective barrier but as the first responder to a changing environment. One environmental challenge these cells face is osmotic stress — where an imbalance of, for instance, ions or sugars, across the plasma membrane exerts osmotic pressure on the membrane. In order to deal with osmotic stress, mammalian cells have evolved complex protein machineries. But how do simpler cells respond to an osmotic onslaught? We answer this question by studying cell-sized lipid vesicles in osmotic stress. Surprisingly, they display a pulsatile behavior, swelling, bursting, and resealing their membrane cycle after cycle!

The cover image for the April 25 issue of the Biophysical Journal is an artistic rendering of the pulsatile dynamics of cell-sized vesicles in osmotic stress. The red fluorescent vesicles represent experimental observations of giant unilamellar vesicles at three stages of the osmotic swell-burst cycle: relaxed (left), swollen (middle), and ruptured with a burst of fluorescent green sucrose (right). On the piece of paper, the vesicles are projected onto corresponding schematics depicting the different stages of the cycle. The leading processes driving the pulsatile behavior, osmotic pressure, surface tension, and leak-out velocity, are defined in mathematical terms. On the top-right of the paper, is one of the key equations we proposed, representing the dynamics of a pore in the membrane taking into account stochastic pore nucleation induced by thermal fluctuations.

This cover was inspired by the combined experimental and theoretical approaches we embraced in this study. It shows how, from experimental observations (the red vesicles), a conceptual model can be built and formalized into mathematical terms (the schematics) to understand the underlying mechanisms of pulsatile vesicles. By mirroring “realistic” vesicles with “hand-drawn” schematics, the cover illustrates how the complexity of a real world process can be reduced to its essential features through modeling. This fundamental scientific approach allows us to build a testable hypothesis to unravel the biophysical principles behind experimental observations.

Through our study and this cover, we highlight how combining experimental and theoretical approaches can be a powerful way to tackle complex challenges, especially for fundamental problems at the frontier between biology, chemistry, and physics.

—Morgan Chabanon, James CS Ho, Bo Liedberg, Atul Parikh, Padmini Rangamani

Meet a Biophysicist Marching for Science

As an official partner of the March for Science, the Biophysical Society encourages members to participate in the event, in person or virtually, and speak up for science. Prior to taking to the streets on Saturday, April 22, in over 525 cities worldwide, meet a BPS member planning to March:  Connie Jeffery.  Connie is an
associate professor in the Department of Biological Sciences at the University of Illinois at Chicago.  Her lab works on protein structure and function using biochemistry, biophysics, and bioinformatics methods.  The lab has projects in basic science and also focused on diseases – cancer, tuberculosis, and inflammatory bowel disease (Crohn’s and Ulcerative Colitis).  She will be marching in Chicago, Illinois on April 22.

 

Dr. Constance Jeffery poses in front of a ribosome sculpture at a Cold Spring Harbor meeting.

 

Why did you sign up to march?

I’m concerned about the huge cuts in the proposed federal budget for NIH, NSF, and other parts of the government that fund scientific research.  I am also concerned about potential cuts to agencies that protect the public like the EPA and the FDA.  I am also concerned about so much “pseudoscience” that is misinforming the public, especially things like incorrect information about what to eat or not to eat, quack cures, anti-GMO activists, and anti-vaccination drives that can harm people.  On the more positive side, I would like to share information about the importance of science and what scientists do.

What do you hope to get out of the day personally?

I’d like to share my love of science and encourage young people to consider a job in science, help inform the public about the importance of science and what scientists do, and also learn from others interested in science, including other scientists, but also environmentalists and people with family members who are suffering from diseases that can be potentially cured in the near future (as long as funding is not cut).

What do you hope it will accomplish?

I hope we can better inform the public and our representatives at the local, state and federal level about the importance of science and that there are many American voters who care about science.  There have been such amazing breakthroughs in the past 15 years that we have the potential to find better treatments and ease a lot of suffering soon, but the opportunity will be missed and many people will continue to suffer needlessly if funding is cut.

What will your sign say?

I’m planning to make multiple signs, and to have messages on both sides – things like “Prevent suffering in children:  Fund Research on Childhood Arthritis”, “Fund Cancer Research”, “Fund Autism Research” and from growing up in Cleveland “Before the EPA the Cuyahoga River was so polluted it BURNED {picture of one of the fires}.  Not just once – THE RIVER BURNED MULTIPLE TIMES. Today with the EPA: {and then a picture of how clean and beautiful it looks today}”, “Vaccinations Save Lives”, etc.

Thanks to Connie and everyone else planning to celebrate science at the March!

How to Prepare for a Non-Bench Career

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Professor Molly Cule is delighted to receive comments on her answers and (anonymized) questions at mollycule@biophysics.org, or visit her on the BPS Blog.

There is an increasing interest for science PhD students to pursue an “alternate” career beyond the traditional bench research followed by a tenure-track faculty position. The options include marketing, sales, intellectual property, policy, and writing, among others. This article highlights four important steps you can take to prepare for any of these non-bench careers.

  • Do your research: Do not go into another non-bench career just for the sake of it. The career sections of most societies, as well as top journals like Science and Nature have a treasure trove of information on various alternative careers. Reach out to alumni from your school or your lab, as well as to friends and family members, or use social media (Twitter/LinkedIN) to directly speak with people who have made the transition.
  • Along the same lines, make a list of your transferrable skills. These skills could have been built up either as part of your graduate research (e.g., data mining and analysis), or at home or through community work (e.g., did you demonstrate leadership skills through some sort of volunteer work?).  Then note how they align with the careers you are considering.
  • Work on your communication skills: Most non-bench careers involve effective communication, whether it is written or verbal. Two particular skills that will be useful to master include (a) the ”elevator pitch” — a quick summary of who you are and/or what you do and why it’s valuable, and (b) communicating technical information to a lay audience.
  • Gain experience outside of your work: It can be difficult to break into a new industry without prior experience. However, it is possible to gain experience in other ways. If you are interested in science writing, think of maintaining an active blog, or contribute to your school or society newsletters; see if you can volunteer at your institute’s technology commercialization office if you are interested in patent law. Employers also tend to look favorably upon those who have demonstrated a willingness to broaden their horizons beyond bench research.
  • Network: It’s gotten to be a cliché now, but the value of the mantra ”Network, network, network” cannot be overstated. Apart from helping you land that next job, networking will help all of the above — researching alternate careers, communicating, and broadening your horizons!

Modeling Unravels the Upper Limit of Mitotic Spindle Size

BPJ_112_7.c1.inddWhen talking about organelle size, most people believe the bigger the cell, the larger the organelle should be. In fact, this is not true, at least for mitotic spindles. Recent studies showed that the mitotic spindle size scales with the cell size in small cells, but approaches an upper limit in large cells. However, how the spindle size is sensed and regulated still eludes scientists.

The cover image for the April 11 issue of the Biophysical Journal shows a configuration sampled by a three-dimensional computational simulation guided by a general model for mitotic spindles. The model explicitly shows microtubules (colorful rods), centrosomes (green sphere), and chromosomes (pink bulks). Microtubules can be nucleated from the centrosomes, grow outward, and show the dynamic instability. When microtubules encounter the cortex or chromosome arms, they can generate pushing forces (red rods) due to the polymerization of microtubules. Various molecular motors on cortex and chromosomes, including dyneins (yellow dots) and kinesins (green dots), can bind to microtubules and generate pushing forces (green rods) or pulling forces (blue rods). Therefore, the centrosomes and chromosomes can move under these forces. In this way, the mitotic spindle can be self-assembled to form a bipolar structure with certain size, positioned to the cell center, and orientated to the long axis. Meanwhile, the chromosomes can be attached correctly and aligned on the equatorial plate.

This computational model is very useful for studying the size regulation of mitotic spindles. The spindle size is usually defined as the pole-to-pole distance, so that the problem of spindle size regulation is dependent on the positioning of two poles. The position of each pole is determined by the mechanical equilibrium between the cortical force and the chromosome force on the spindle pole.  For each pole, the chromosomes and the cortex are geometrically asymmetric. In small cells, the geometric asymmetry is small and the pole is nearly positioned to at the center of each half cell so that the spindle size scales with the cell size. However, in large cells, because few microtubules can reach the cell boundary, the geometric asymmetry is large and the spindle size is only determined by the chromosomes; that is, the spindle size approaches the upper limit. Therefore, this work revealed a novel and essential physical mechanism of the spindle size regulation.

There are certainly many other factors that influence spindle size but only quantitatively; they cannot explain the existence of the size limit of spindles.

This computational model provides a very powerful and robust tool. It can be combined with existing biochemical techniques to explore many important and interesting phenomena, including the positioning and orientation of mitotic spindles, the spontaneous oscillation of chromosomes, the mechanical response of spindles under various forces, and many other relevant questions.

– Jingchen Li and Hongyuan Jiang

Understand the Regulation of Learning and Memory Formation from a Molecular Prospective

BPJ_112_6.c1.inddWhen most people talk about calcium (Ca2+), they think about building bones and muscle contraction. In fact, calcium is also essential for learning and memory formation. Molecular basis for learning and memory formation has aroused attention since 1980s.  So what does calcium do with learning and memory formation? The calcium-modulating protein calmodulin (CaM) coordinates the activation of a family of Ca2+-regulated proteins, which are crucial for synaptic plasticity associated with learning and memory in neurons. These proteins include neurogranin (Ng) and CaM-dependent kinase II (CaMKII). In a resting cell, CaM is mostly reserved by Ng and free of Ca2+, whereas in a stimulated cell, CaM is able to bind Ca2+ and activate CaMII, which plays a pivotal role in learning and memory formation for both long-term potentiation and mechanisms for the modulation of synaptic efficacy.

The cover image for the March 28 issue of the Biophysical Journal shows the crystal structure of CaM-CaMKII peptide and the structure of CaM-Ng from coarse-grained molecular simulations. CaM molecules are ribbons in silver, calcium ions are represented by yellow beads, CaMKII peptide is in green surface representation, and Ng peptide is in red surface representation. One CaM-Ng peptide complex is near, where the Ng is aligned with a “pry” (pink); the other is far, indicating rich level of Ng. The images were rendered using the software Visual Molecular Dynamics developed by University of Illinois at Urbana-Champaign with the built-in Tachyon ray tracer. The illustration of a neuron in hippocampus is taken from Shelley Halpain, UC San Diego. Dendrites are green, dendritic spines red, and DNA (in cell nucleus) blue. The illustration of a human brain contains red dots to indicate active parts of the cerebral cortex.

Our computational study provides the very first detailed description at atomistic level ofhow binding of CaM with two distinct targets, Ng and CaMKII, influences the release of Ca2+ from CaM, as a molecular underpinning of CaM-dependent Ca2+ signaling in neurons. We believe this study bridges the molecular regulations in atomistic detail and the understanding of cellular process cascade of learning and memory formation.

– Pengzhi Zhang, Swarnendu Tripathi, Hoa Trinh, Margaret S. Cheung

Biophysics and Bleeding Disorders

March is Bleeding Disorders Awareness Month in the US. More than three million Americans who have hemophilia, von Willebrand disease, and other rare bleeding disorders. These conditions prevent blood from clotting the way it should, which can lead to prolonged bleeding after injury, surgery, or physical trauma. We spoke with Biophysical Society member Valerie Tutwiler, an American Heart Association graduate research fellow in the lab of John Weisel at the University of Pennsylvania, about her hemostasis and thrombosis research.

What is the connection between your research and bleeding disorders?

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This recent cover of Biophysical Journal shows Tutwiler, Wang, Litvinov, Weisel, and Shenoy’s image of a colorized scanning electron microscope image of a coronary artery thrombus extracted from a heart attack patient.

Blood clotting or hemostasis is the process that stems bleeding. On one hand if you have insufficient clotting this can result in prolonged bleeding, on the other hand a hypercoagulable state can result in thrombosis. Thrombi can result in the obstruction of blood flow, which can cause heart attacks and strokes. My thesis research pertains largely to studying one portion of the coagulation process blood clot contraction, or the volume shrinkage of the clot, which has been implicated to play a role in hemostasis and the restoration of blood flow past otherwise obstructive thrombi.

Why is your research important to those concerned about bleeding disorders?

While there is much known about the various aspects of blood clotting relatively little is known about the process of clot contraction despite the clinical implications of its importance in the formation of a strong hemostatic seal and the restoration of blood flow past otherwise obstructive thrombi. The study of clot contraction is a highly interdisciplinary problem and as a result can be of interest to researchers from many different fields. Platelets are active contractile cells, which interact with an extracellular matrix of fibrin, a naturally occurring polymer with unique mechanical properties. The fibrin matrix can be imbedded with other blood cells, such as red blood cells, as well. From a biophysical standpoint the mechanisms of clot contraction have not been well understood. To better elucidate this process, we performed a systematic study on how the molecular and cellular composition of the blood influences the rate and extent of clot contraction along with the mechanical properties of the contracting clot using a novel application of an optical tracking system.

Additionally, to further explore the mechanical nature of the clot contraction process we developed a mathematical model that couples active platelets with a passive viscoelastic matrix made up of fibrin and red blood cells. The model predicts the process of clot contraction and explains some of the experimental observations of clot size, structure and mechanical forces. Interestingly, we found that clot contraction is altered in thrombotic states such as ischemic stroke patients. Collectively, these findings show that the study of clot contraction has the potential to inform the development of diagnostics and therapeutics.

How did you get into this area of research?

Since beginning research I have been interested in applying engineering techniques to answer biological questions. I became interested in hemostasis and thrombosis research while completing my first co-op experience in undergrad.

How long have you been working on it?

I began doing hemotology research during my undergraduate career. However, I started studying clot contraction specifically when I started my PhD research.

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

I am currently funded by the American Heart Association as a pre-doctoral fellow, although we also receive funding from the National Institute of Health and National Science Foundation.

Have you had any surprise findings thus far?

We were surprised to find such a striking decrease in the extent of clot contraction in ischemic stroke patients compared to healthy subjects. Correlations with stroke severity suggest that clot contraction may be a potential pathogenic factor in ischemic stroke. These findings have led us to expand our study to other pathological conditions as well.

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

Due to the conservation of the basic principles of contractile proteins and motility, the information learned from the development of a mathematical model of active contractile cells interacting with a viscoelastic matrix can be applied to a variety of different processes.

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

Bleeding and thrombotic conditions remain leading causes of death and disability worldwide. Gaining a more thorough understanding of the processes involved in hemostasis and thrombosis will lead to the development of more effective diagnostic tools and more targeted therapeutics.

PhosphoHero is in Charge of Neurofilaments’ Order

BPJ_112_5.c1.inddGels are neither solids nor liquids but rather a network of deformable and crosslinked polymers. Therefore, it is not surprising that the mechanical properties of synthetic gels are controlled by the degree of cross-linking, achieved, for example, by photopolymerization or the addition of chemical agents. One of the best examples of mechanically supporting bio-gels is the cytoskeleton, where crosslinked polymers (actin, microtubules and intermediate filaments) form a viscoelastic network. For microtubules and actin networks, analogues biological cross-linkers (associated proteins) have been identified. Nonetheless, in some important cases, the biophysical crosslinking mechanism or the existence of associated crosslinking proteins have not been identified.

Neurofilaments (NF) are neuronal specific intermediate filaments that form spaced filamentous networks in the long axon projections. Each neurofilament resembles a bottlebrush: a semi-flexible filament decorated with protruding floppy (intrinsically disordered) long carboxyl terminal tails. The tails engage in extensive crosslinking interactions, which have been the focus of many studies.

In addition to their lack of secondary rigid structure, NF tails contain many “phosphorylation sites”. These sites are specific amino-acid sequences recognized by enzymes that can add or remove charged phosphate groups, known as phosphorylation and dephosphorylation, respectively.

Our cover image for the March 14 issue of the Biophysical Journal illustrates an NF gel made of well aligned bottlebrushes at the front, and un-oriented ones at the back. The superhero (PhosphoHero) artistically illustrates the roles of NF phosphorylation. On the one hand, PhosphoHero increases the cross-linking between the filaments via the generation of ionic bridging between opposite charged residues. This in turn aligns the red filaments in nematic liquid crystalline order, as depicted by the crossed polarized NF hydrogel microscopy in the background. On the other hand, phosphorylation also increases the tails’ net negative charge, and consequently its compression response. Thus, phosphorylation acts as a regulatory knob to control the structure, orientation and mechanical properties of the cellular scaffold, the cytoskeleton.

Future studies into the role of intrinsically disordered proteins, and in particular their tunable phosphorylation states and their role in long-range alignment should be full of further surprises. Intrinsically disordered proteins were evolutionally selected to hold functional, although sometimes atypical properties, characteristic to superheroes.

The cover was hand-drawn and then digitally colored in Photoshop by Eliran Malka.

– Eti Malka-Gibor, Micha Kornreich, Adi Laser-Azogui, Ofer Doron, Irena Zingerman-Koladko, Jan Harapin, Ohad Medalia, Roy Beck