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

Temperature-Adapted Zebrafish Membranes Show Their Stripes

BPJ_113_6.c1.inddOrganisms that do not maintain a constant body temperature must have some mechanism to adapt their physiological functions in order to survive at a range of temperatures.  For individual cells within the organism, the cellular membrane serves as a platform for cellular signaling and cell-cell interaction. The organization and physical properties of plasma membrane lipids are sensitive to changes in temperature. When giant plasma membrane vesicles (GPMVs)— derived from cells grown in culture— are cooled slightly below growth temperature, they separate into distinct ordered and disordered liquid phases. These phases can be observed through fluorescence imaging of a phase-selective dye. ZF4 cells, derived from zebrafish, can be adapted to grow at a range of temperatures and GPMVs derived from these cells were used in our study to examine how these cells change the makeup of their plasma membranes to adapt to changes in temperature.

The cover image of the September 19th issue of Biophysical Journal focuses on an imagined “zebrafish,” which was composed of a fluorescence image of a ZF4-derived GPMV with stripe-like phase separated domains, combined with photographs of a zebrafish and a zebra.  These three images were blended together in Photoshop to create our chimeric zebrafish. In this cover image, we wanted to highlight the idea that lipid organization of the plasma membrane could be central to the physiology and function of the organism as a whole. For this reason, we used the fluorescence image of the phase-separated GPMV as the focal point of our “zebrafish,” and used the common visual motif of stripes to draw a comparison between the organization at the sub-cellular level of plasma membrane lipids to the organization at the level of the whole organism. This zebrafish swims through a sea of phase-separated GPMVs, shown in the background, again highlighting the theme of lipid organization.

Projects exploring how the physical properties of the plasma membrane impact membrane organization and function are ongoing in the Veatch Laboratory. This interest applies to a variety of biological processes, from immunoreceptor function to general anesthesia.

– Margaret Burns, Kathleen C. Wisser, Jing Wu, Ilya Levental, Sarah Veatch

For Microtubule Sliding, One Arm Is Better Than Two

BPJ_113_5.c1.inddThe versatile and dynamic network of the cytoskeleton scaffold would be stagnant and lifeless if not for the tiny nanoscopic machines called molecular motors. Kinesin motors, in particular, have captured the imagination of biologists and physicists because of their ability to transform ATP into anthropomorphic walking patterns on polar microtubule filaments, which make up a significant portion of the cytoskeleton. Recent experiments have shown that kinesin motors can crosslink adjacent microtubules and facilitate sliding between them resulting in cytoplasmic streaming in Drosophila cells. This facilitates faster distribution of molecules and organelles, and determines cell-shape.

But how do motors bring about microtubule sliding? How does the collective motion of microtubules depend on the movement of motor arms? In our work, we answer these questions by studying the effect of dimeric (one active arm, one anchored arm) and tetrameric (two active arms) kinesin motors on the dynamics of confined microtubules. Through our computer simulations we find that single-armed kinesins bring about much faster dynamics in specific regions of the confinement, compared to their two-armed counterpart. This goes against the intuitive idea that more arms pull more.

The cover image for the September 5th issue of the Biophysical Journal is our rendering of filament organization for two different motor types and the effects of these differences in the large-scale structure and dynamics of confined microtubules. The green shapes on the left represent the active motor heads that walk on polar microtubules. These are depicted as a linear array of dark-blue and yellow circles. The red blob depicts the anchor belonging to the single-armed, dimeric motor. Motor arms walk in specific directions on microtubules, and stretch, producing a sliding stress between microtubules.

The structures shown in the circular confinement on top consist of sluggish filament packages formed by tetrameric motors. The arrows at the bottom represent the highly dynamic microtubule arrangement formed by dimeric motors. Here, we also depicted the trajectories that three selected microtubules have taken. The cover image was crafted to highlight the large-scale biophysical implications of seemingly trivial and counterintuitive details in biology. Through this work we emphasize the vastly different cytoskeletal dynamics due to dimeric and tetrameric motors. By way of the trajectories, we capture the active layer of microtubules close to the circular confinement we observed for the single-armed motor systems.

– Arvind Ravichandran, Gerard Vliegenthart, Guglielmo Saggiorato, Gerhard Gompper, Thorsten Auth

Exploring the Interstitial Space

BPJ_113_4.c1.inddThe cover art for the August 22nd issue of Biophysical Journal was composed with the intention of incorporating elements from both the theoretical and experimental sides of modern biology. It depicts the interstitial space present between cells that adhere to one another with varying degrees of adhesiveness. The top right shows an electron micrograph of a tissue from a frog embryo, in which the interstitial space appears as pale, approximately triangular gaps. One such triangular gap is inset by a 3D representation of a gap derived from our theoretical model, acting as a visual link between theory and experiment. The size and interconnectedness of the modelled interstitial space increases as the eye moves towards the lower left, reflecting the different gap shapes and sizes predicted for different conditions of cell adhesion. The interstitial space is filled with fluid (in fact just over a quarter of the water in the human body is contained in the interstitial space!), and this is reflected in the watery, liquid-like appearance of the model. The electron micrograph and 3D model are reminiscent of the moon and stars – this is meant to invoke a feeling of a new frontier being explored, as biologists move more and more towards mathematical and computational modeling as a means to describe the living world around us. The cover art was produced using Blender, an open-source 3D modeling program, which has helped us to visualize and internalize the geometry of the interstitial space as the model became more complex with more interacting elements.

While our study made use of frog early embryos, interstitial spaces are present in all animals, from invertebrates to vertebrates, and in both diseased and healthy tissues. The interstitial space is involved in fluid balance in the body, transport of metabolites and signaling molecules, and determination of the mechanical properties of a tissue, and it is curious that even in tissues where the cells are very tightly packed, this space is maintained.

—Serge E. Parent, Debanjan Barua, and Rudolf Winklbauer.


Mesoscopic Adaptive Resolution Scheme toward understanding of interactions between sickle cell fibers

BPJ_113_1.c1.inddSickle cell disease (SCD) is a molecular disease that affects hemoglobin. To understand the altered morphologies and mechanical properties of sickle red blood cells in SCD, it is important to investigate polymerization of the mutated form of hemoglobin (HbS) and subsequent interaction with the red blood cell (RBC) membrane.

The cover image for the July 11 issue of the Biophysical Journal is an artistic rendering of several RBCs and a white blood cell in the blood flow. Some RBCs are sickle-like shape. The largest RBC in the bottom left corner is shown in the protein level. The red, blue, and green particles represent the lipid particles, spectrin proteins, and actin junctions in the RBC membrane, respectively. Inside the RBC,  a single HbS fiber consists of 14 chains of HbS tetramers arranged in a 7 double-stranded configuration. The 14 chains of HbS tetramers are twisted about a common axis in a rope-like fashion. In the image, one small yellow particle is one HbS molecule, and  56 small particles are coarse-grained as one large yellow particle.

As shown in the image, the proposed hybrid HbS fiber model seamlessly couples these two HbS fiber models at different length scales by applying a mesoscopic adaptive resolution scheme (MARS). The stiff HbS fibers interact with the RBC membrane and distort the RBC to the sickle shape. Sickle RBC morphologies are determined by the number of HbS fiber domains and the structure of each domain inside the cells. In return, the RBC membrane also suppresses the growth of the HbS fiber. In addition to irregular shapes, sickle RBCs are characterized by increased cell rigidity due to intracellular fiber structures, resulting in blood flow impairment and vaso-occlusive crises in the microcirculation.

This cover was inspired by the pressing need to understand the integrated process of HbS nucleation and polymerization, and subsequent alterations of cell morphology, which is a multi-scale process ranging from nanometers to micrometers. In order to accurately describe this process, the hybrid HbS fiber model is employed to capture the dynamic process of polymerization of HbS fibers, while maintaining the mechanical properties of polymerized HbS fibers, thus providing a means of bridging the subcellular and cellular phenomena in sickle cell disease.

—- Lu Lu, He Li, Xin Bian, Xuejin Li, and George Em Karniadakis

Frozen Single Cell under Raman Spectroscopy

BPJ_112_12.c1.inddCryopreservation is the technology used to stabilize cells for a variety of applications, including diagnosis and treatment of disease. Because we don’t completely understand the mechanisms of freezing damage, poor or inadequate methods of preservation have limited our ability to use cells for cell therapy. Also, observations of cell responses could not be correlated to viability on a cell-by-cell basis using conventional low-temperature microscopy techniques. This study establishes our ability to measure the viability of individual frozen cells based on the correlation of cytochrome c distribution, a signal that can be detected using Raman spectroscopy, with trypan blue staining. With Raman spectroscopy, we are able to observe cells during freezing, and identify specific chemical and morphological changes inside the cell that result in life or death.

The cover image for the June 20 issue of the Biophysical Journal is an artistic rendering of frozen cells surrounded by extracellular ice and unfrozen solution. The background image is Lake Michigan in cold winter. Floating ice is separated by unfrozen water. The distribution pattern of the floating ice and unfrozen water is just like a frozen sample: ice crystals are separated by unfrozen concentrated solution. Schematic diagrams of frozen cells were imbedded in the background image to mimic a real frozen cell sample. In the diagram, the blue area represents unfrozen solution, the white area represents extracellular ice, the red line represents a region of cell membrane in close proximity to extracellular ice, and the black line represents a region of cell membrane in unfrozen solution between adjacent extracellular ice crystals. The schematic diagrams were precisely positioned in the background image such that the unfrozen solution in the diagram was co-located with unfrozen water in the background image and the extracellular ice crystals in the diagram were co-located with the floating ice in the background image. Our studies found that interactions between the cell membrane and extracellular ice resulted in intracellular ice formation (IIF), and increasing the distance between extracellular ice and cell membrane decreased the incidence of IIF.

Raman spectroscopy has enhanced our understanding of freezing damage. These studies can enable the development of new and improved cell preservation protocols and eventually improve the growth of cellular therapies and our ability to treat patients.

– Guanglin Yu, Yan Rou Yap, Katie Pollock, Allison Hubel