What’s the Buzz? – Membrane Leaflet Crosstalk

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The cover image for the January 9 issue of Biophysical Journal is an artistic rendering of vesicles made up of lipids with shapes resembling either an inverted cone (cyan heads), or a right cylinder (yellow heads). In one type of vesicle, the cyan and yellow lipids populate the inner and outer leaflets, respectively, while in the other type of vesicle, they are reversed. A closer look reveals that the vesicle with an inner leaflet of cyan lipids has acyl chains that are “straight” in both leaflets, implying an ordered state or gel phase. However, when the yellow lipids are placed in the inner leaflet, their acyl chains go from being straight to being “wavy,” implying hydrocarbons in a disordered state or fluid phase. The difference between the two types of vesicles is related to interleaflet coupling. In other words, when the inverted cone (cyan) lipids are located in the inner leaflet, they are able to “communicate” information about their ordered state to the right cylinder with yellow lipids in the outer leaflet. Interestingly, when their places are inverted, this communication is lost.

We studied ~100 nm diameter asymmetric lipid vesicles composed of palmitoyl oleoyl phosphatidylethanolamine (POPE) and palmitoyl oleoyl phosphatidylcholine (POPC). Cyan lipids in the cover image represent POPE and yellow lipids represent POPC. Combining elastic X-ray/neutron scattering techniques with differential scanning calorimetry, dynamic light scattering, nuclear magnetic resonance spectroscopy, and cryo-electron microscopy allowed us to compare leaflet- specific structural properties of vesicles with POPE-rich inner leaflets and POPC-rich outer leaflets, to vesicles with reversed lipid asymmetry. Our data reveal when the inner leaflet is predominantly made up of POPE, it is able to induce a gel phase in the POPC-enriched outer leaflet. When reversed, this communication between the leaflets is lost and the POPC-enriched inner leaflet remains fluid. This can be understood in terms of an energetic benefit of locating inverted cone-shaped lipids, such as POPE, in the inner monolayer, which is better able to match the overall vesicle curvature, compared to when it is located in the outer lipid monolayer.

Signal transduction and intercellular communication are essential for membranes, and are mostly due to integral proteins. However, physiological processes that require communication between, for example, receptors secreted to the exoplasm and components of signal transduction pathways in the cytoplasm, may rely on a bilayer leaflet coupling mechanism such as the one described  in our manuscript.

-Barbara Eicher, Drew Marquardt, Frederick Heberle, Ilse Letofsky-Papst, Gerald N. Rechberger, Marie-Sousai Appavou, John Katsaras, Georg Pabst

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A Second in the Life of Blood Cells

BPJ_113_12.c1.inddMicrovascular networks are highly complex structures comprised of the smallest blood vessels. Some of their major utilities include gas and nutrient exchange to surrounding tissues, and regulation of blood flow in individual organs. Blood cells squeeze and deform as they flow through the vessels that comprise them, an ability that is critical to the healthy functioning of the circulatory system. While much is known about the average behavior of blood cells flowing within these networks, little is known about the individual cellular-scale events giving rise to this average behavior. What is going on at the cellular-scale as the actual blood cells flow through such torturous geometries? To better understand this, we use a recently developed state-of-the-art simulation tool to model nearly one second in the life of red blood cells as they squeeze, twist, and tumble through physiologically realistic microvascular networks. The results are indeed surprising!

An image based on one such simulation is presented on the cover of the December 19 issue of the Biophysical Journal. Specifically shown is a snapshot of red blood cells flowing and deforming through a microvascular network. This network was designed following in vivo images and data, and was constructed by digitally rendering the geometry using a standard CAD software.  The 3D vascular surfaces were then imported into our simulation tool, and using this we captured the 3D flow field as well as the large deformation and dynamics of each individual blood cell. The blood cells shown are suspended in plasma, which conveys them through the network. The cells are modeled using the finite element method, with each cell surface discretized by about 5000 elements. During the nearly one second simulated, thousands of cellular-scale events occur. The statistics associated with the resulting hemodynamic quantities in vessels are utilized to better understand microvascular network blood flow.

The inspiration behind the simulation shown on the cover is our desire to mimic what actually occurs in physiology as closely as possible. That is, we wanted to capture the realistic features of the geometries through which cells flow without sacrificing the particulate nature of blood, and vice versa. In doing so, we uncover new and interesting features associated with the cellular-scale dynamics. Furthermore, we reveal some surprising counter-intuitive behavior that can only be captured by considering the time-dependent 3D deformation and dynamics of each individual cell.

– Peter Balogh, Prosenjit Bagchi

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

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

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

Architecture of Bacterial Cell Division Protein FtsZ Polymers

BPJ_113_8.c1.inddFtsZ is a self-assembling protein that forms the contractile ring guiding the cell division machinery in most bacteria. FtsZ is structurally homologous to tubulin, the subunit of eukaryotic microtubules. FtsZ monomers associate head-to-tail forming  single-stranded filaments that hydrolyze GTP, in a partially understood process. However, how FtsZ filaments organize in the dynamic division ring is still a challenging problem. Rather than forming a well-defined structure, such as band or tubule, FtsZ filaments laterally associate among them in a relatively disordered fashion.  FtsZ filaments bind partner and regulatory proteins, including those tethering them to the inner face of the plasma membrane.

The cover image for the October 17 issue of Biophysical Journal is an artistic representation of the organization that we propose for FtsZ assemblies.  FtsZ filaments made of FtsZ monomers laterally associate through the disordered C-terminal tails, forming loose bundles. Small-angle X-ray solution scattering results (exemplified by the graph on the left) indicated a characteristic 7 nm center-to-center lateral spacing between FtsZ filaments. By modeling comprehensive building and scattering calculations we saw that multiple associated filaments of variable curvature and length were required to reproduce the X-ray scattering features. These calculations also showed a 2-nm gap was left between core filament structures. We hypothesized that the gap would be bridged by the FtsZ intrinsically disordered C-terminal linker region, as in the model bundle in the center of the image. Cryo-electron microscopy provided views of unstained individual assemblies in vitrified solutions (blue background in the bottom half). Analyzing polymers assembled from FtsZ protein constructs with diverse C-termini supported the model.

Combining several biophysical approaches has provided insight into the self-organizing properties of FtsZ that we think underlie the assembly of the bacterial division ring. It should be noted that bacterial division is still a clinically unexplored target for the discovery of new antibacterials needed to counter the spread of antibiotic resistant pathogens.

-Sonia Huecas, Erney Ramirez-Aportela, Albert Vergoñós, Rafael Nuñez-Ramirez, Oscar Llorca, David Juan-Rodriguez, María A. Oliva, Patricia Castellen, and José M. Andreu

An Optically Induced Electrokinetics Microfluidics Chip

BPJ_113_7.c1.inddUsing florescence molecules for bio-marking of cells is a widely accepted technology. However, the auto-fluorescence on the surface of living cells strongly influences the fluorescence-based detection of labeled molecules and is also harmful to cells. Then again, cell dielectric information can be obtained through non-invasive and label-free techniques, which have been shown as a possible bio-marking method.  For instance, the structure-based microfluidics method is a prevalent technique that can obtain cell membrane capacitance/conductance through use of custom-designed microfluidics structures. However, the measurement efficiency and performance of this scheme depend strongly on the use of microstructures with specific and sophisticated designs tailored to the cell size. The microstructures also cannot be altered after they are fabricated by the conventional micro-matching technique. In our paper, we report a new method to determine cell dielectric properties by using a coupled optical-electrical based microfluidics technique.

The cover image for the October 3 issue of the Biophysical Journal is an illustration of the optically induced electrokinetics approach developed by our team to acquire the cellular electrical conductance and capacitance using a specialized microfluidics chip. In the foreground of the cover is an exploded view of the optically induced electrokinetics (OEK) microfluidics chip which is able to generate localized force to trap each individual cell. The nine red and yellow pillars are the simulation results of the optically projected virtual electrodes showing the electric field distribution in the OEK microfluidic chip. In the back of the illustration is a light projector and a microscope. They are used to direct and shrink the light patterns projected onto the OEK microfluidics chip to trap the cells.

This image was inspired by combing the theoretical simulation and experimental approach we performed in this study. It presents how the incident light can trap individual cells using the optically induced electrokinetics forces exerted on the cells.  Depending on how each cell reacts to the exerted electrokinetics forces, we can determine the dielectric parameters of individual cells using the specialized microfluidics chip shown in the cover image.

—Yuliang Zhao, Lianqing Liu, Yuechao Wang, Wen Jung Li, Gwo-Bin Lee, Wenfeng Liang