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

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

Mechanical Interplay in Clot Contraction

BPJ_112_4.c1.inddBlood clotting, thrombosis, and blood cells all have great biological and clinical significance. Clotting is necessary to stop bleeding yet thrombi can obstruct blood flow, which can cause heart attacks, strokes, venous thrombosis, and pulmonary embolism. Although much is known about various aspects of clotting, much less is known about clot contraction or retraction. Clot contraction is thought to play a role in hemostasis, wound healing and the restoration of flow past otherwise obstructive thrombi.

The cover image for the February 28 issue of the Biophysical Journal shows a colorized scanning electron microscope image of a coronary artery thrombus extracted from a heart attack patient. We chose this image because contraction occurs in such thrombi and all of the elements described in our paper are visualized here: platelets (gray), fibrin (brown) and red blood cells (red). Thus, this image represents a real-world example of the practical significance of our research. Furthermore, we have found that clot contraction is altered in patients with certain thrombotic disorders, such as acute ischemic stroke. Our model provides the fundamental mechanical basis for understanding the contraction of blood clots.

The contraction of blood clots and thrombi is an interdisciplinary problem related to fundamental aspects of cell biology, including cell motility and interaction of cells with an extracellular matrix. The biophysical mechanisms of clot contraction have been poorly understood, although it has been shown that it results from the interaction of actively contracting platelets with the fibrin network, the structural matrix of the clot that has unique mechanical properties. Though many of the same basic principles of motility of other cells are employed in this system, the specialized mechanisms of cellular contractility represent a novel biological application. The consequences of cell-matrix interactions in blood clots are unique and result in massive compaction of the network, rather than motility or alignment of fibers that occur in other cellular contractile environments.

Blood clot contraction is driven by platelet-generated contractile forces that are propagated by the fibrin network and result in clot shrinkage and deformation of red blood cells. We developed a model that combines an active contractile motor element with passive viscoelastic elements consisting of fibrin and red blood cells. This model predicts how clot contraction occurs due to active contractile platelets interacting with a viscoelastic material, and explains the observed dynamics of clot size, ultrastructure, and measured forces.

– Andre E.X. Brown, Chandrasekaran Nagaswami, Valerie Tutwiler, Hailong Wang, Rustem Litvinov, Vivek Shenoy, and John Weisel

Note: This image originally appeared in a different form in Science 325:651, 2009.

Unraveling the genome biophysics puzzle

BPJ_112_3.c1.inddWhile digitizing the human genome is one of the recent major scientific achievements, unraveling genome structure and, more importantly, structure–function relationships has been a major preoccupation for many scientific teams worldwide. Indeed, the understanding of structure from the DNA level to nucleosomes, chromatin fibers, genes, and chromosomes holds the key to interpreting many of the associated genome functions from DNA repair and duplication to gene transcription.

To celebrate the anniversary of Biophysical Journal‘s newest section Nucleic Acids and Genome Biophysics, we present this Special Issue devoted to Genome Biophysics.

The cover image for this issue conveys the excitement in the field, as many techniques are being developed and applied by both experimentalists and modelers to decipher aspects of the genome puzzle. Specifically, the cover image highlights the genome puzzle from multiple scales and viewpoints. The illustration, created by G. Bascom and T. Schlick, features images from the contributions by A. Onufriev and colleagues on partially assembled nucleosomes (left top puzzle piece), B. Zhang and P. Wolynes on chromosomal domains (right bottom puzzle piece), and G. Bascom and T. Schlick on looping networks in fibers and genes (central image and background fibers).

We hope BJ readers will enjoy the excellent contributions in this issue that reflect an exciting range of topics, as well as the breadth and depth of this fascinating subject.

– Tamar Schlick

Three Coexisting Phases of Lipid Membranes

BPJ_112_2.c1.inddWhile there has been a large amount of research into the phase behavior of lipid membranes in the past decade driven by the biological relevance of ordered micro-domains (often termed “lipid-rafts”), images of three coexisting phases are very rare. Researchers have difficulties preparing samples with a high proportion of ordered phase, and finding reliable methods to sufficiently discriminate the phases as fluorescent dyes often prefer one phase and are excluded from the remaining two. The cover image of the January 24th issue of the Biophysical Journal shows a collection of atomic force micrographs (AFM) exhibiting three-phase coexistence in a model cell membrane under water.

Three phases are found in lipid compositions. They lie in a narrow triangle below the more commonly studied two-phase micro-domain region, which contains the liquid disordered (Ld – yellow/red) and liquid ordered (Lo – magenta) phases, together with a gel phase (Lb – blue/green). Each phase has a different degree of chain packing order leading to varying depths. A surprising and counterintuitive finding of this study is that the gel phase, while definitely solid, is more disordered and slightly lower in height than Lo. It is also structurally very weak. This is explained by the small but significant quantity of cholesterol that disrupts the ordered solid phase, while being insufficient to form the Lo phase. Relative proportions of the three phases are governed by their position in the three-phase triangle. Domain morphology is controlled by the mechanism of phase separation. We observed examples of both spinodal and nucleated domains of each phase, and in some cases both mechanisms in the same image An example of this can be seen in the cover image where a nucleated gel phase (green) is surrounded by a percolated Lo/Ld  structure (yellow/magenta). Another interesting finding was the signature of a radially varying composition across the nucleated gel domains, reflecting the kinetically trapped solid state in the process of separating from a compositionally varying melt. This effect has been commonly observed in metallurgy, but not in lipids.

Each image was produced within the standard AFM analysis software, Bruker Nanoscope v1.5. Manual coloring in Photoshop has not been used, rather a standard color look-up table (No 9) is mapped directly to the topography data. The contrast was adjusted so that the lowest phase is on the yellow/red transition, and the highest phase is magenta, resulting in a blue/green middle phase. A problem with this approach is the tiny 0.6 nm difference in height between the highest and lowest phases, which calls for accurate levelling to remove image bowing artefacts common in AFM, provide defined peaks in the depth histogram, and  a uniform color across each phase. This task was made even more painstaking by the presence of three phases interfering with the thresholding and masking approach normally used. The final composite was created simply in Powerpoint.

Our work is part of an EPSRC (Engineering and Physical Sciences Research Council) Program entitled CAPiTALS, which is based around the fundamental understanding of the physics governing lipid membrane curvature, asymmetry, and patterning, and the technological uses arising from this new knowledge.

–  Simon Connell, Anders Aufderhorst-Roberts, Udayan Chandra.

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