What Makes Neurons Contract to Generate Tension?

BPJ_111_7.c1.inddWhen preparing for the cover image for the October 4 issue of Biophysical Journal, we started with an image that resembled an art painting, probably something between Monet and Pollock (not claiming that it’s up to their standards). It was pretty, at least to us, but we thought that it didn’t tell a story. So we thought to ourselves, “What is the message that we want to tell if we have the chance?”

This is how the current version of our cover image came about. On the left, it has a bunch of curved green and red lines, while on the right there is the same set of lines but straightened. These are actually real experimental images achieved by genetically encoding the neuronal membrane to fluoresce in green—thanks to the technology enabled by the late Roger Tsien, while staining the cytoskeleton, in this case microtubules, in red. The implication is that buckled (curved) neurons would always contract and become straight again in less than 5 minutes with the contraction vs. time profile following an exponential decay. We used genetic and pharmaceutical tools to study this phenomenon and found that the acto-myosin machinery was the active driver, while microtubules contributed in a resistive/dissipative role.

The neurons contracted because the machineries were trying to build up a mechanical tension, which was shown to be critical for vesicle clustering, a process central to signal propagation across neurons. Yet, we do not know how tension leads to clustering. It’s like we know that we can drive from New York to Los Angeles, but we do not know the path. Unfortunately, there isn’t an app for that in our case. Knowing the players (tension generators) involved is the first step to answer this question.

There are quite a few implications, mostly to our understanding of mechanics in neuroscience. A better understanding always leads to new ideas and approaches to solve problems, which, in the context of nervous system health, are usually costly and sometimes a matter of life and death.

– Alireza Tofangchi, Anthony Fan, Taher Saif

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Feeling for Filaments

BPJ_109_4.c1.inddThe cover image on the Biophysical Journal issue released August 18, 2015 features a live cultured endothelial cell that was imaged using two different techniques: The green fluorescence part was recorded with an optical confocal microscope and displays actin filaments inside the cell in a maximum intensity projection.  The greyscale square shows a shaded visualization of the sample surface as probed by atomic force microscopy (AFM).

The AFM is capable of ‘feeling’ the rigid structures of the cortical cytoskeleton through the cell membrane, hence the rough appearance of the cell’s surface. We developed a technique to image this cortical cytoskeleton web with high resolution and quantify its density. This enables us to observe dynamic behavior and study the effects of pharmacological treatments. Simultaneous confocal fluorescence microscopy enhances the method in terms of molecular labeling; it both allows correlation with AFM data and completes the three-dimensional view of the cell towards the basal side.

Our aim is to elucidate the link between the structure of the cortical actin cytoskeleton, cell mechanics, and physiology. The research in vascular endothelium is inspired by findings on how cell mechanics determine endothelial function. Thus it deals with the pathophysiological background of hypertension and other cardiovascular diseases. Another application can be found in oncogenesis and metastasis, where cell mechanics are also altered.

Our paper is also, hopefully, an interesting read for anyone using AFM in other cell imaging applications because of the methodological issues it addresses: Force stability with uncoated cantilevers, contact-mode versus newer, fast force mapping modes, image processing to extract and segment features on large scale objects, and more.

More information on the endothelial physiology research, AFM mechanobiology and microscopy applications can be found on our website.

–Cornelius Kronlage, Marco Schäfer-Herte, Daniel Böning, Hans Oberleithner, and Johannes Fels

The Science Behind the Image Contest Winners: MinD, Spirals, and Turing Patterns

Each year the Biophysical Society hosts an image contest in conjunction with the Annual Meeting. And each year we are blown away by the beauty, the variety, and the scientific advancements encaptured by the submitted images.

Annual meeting attendees vote for their favorite images and the top three vote getters are recognized with prizes, as well as in the Society newsletter. To follow-up this year, we will be featuring the winners here on the BPS blog, so that readers can learn more about the images and the research behind them.  Today, we start off with the third place winner, Anthony G. Vecchiarelli, a research fellow in the laboratory of Kiyoshi Mizuuchi at the National Institutes of Health, where he uses microfluidics and single-molecule microscopy to reconstitute and visualize self-organizing systems involved in intracellular organization. Vecchiarelli notes that “Collectively, these studies have fundamentally changed our understanding of intracellular spatial organization and unveiled a new mode of transport that uses protein patterns on biological surfaces for the positioning of DNA, organelles, and the cell-division apparatus.”

Min-Spiral---3rd-place_2My submission to the 2015 BPS image contest shows one of the many stunning and dynamic patterns that this minimal system can achieve when reconstituted in our flowcell. At a wave front, MinD binds the membrane. Towards the rear of the wave, MinE accumulates up to a threshold density that results in the precipitous release of both proteins. Once MinE also releases the bilayer, MinD initiates another wave. As shown in our image, this interplay can form spirals, which are consistent with Turing patterns based on reaction-diffusion principles.

Similar spirals were first reconstituted on flat bilayers by Martin Loose while in the Schwille group (Loose et al., 2008). Together, the Schwille and Mizuuchi labs have used this technique to provide significant advances in understanding the patterning mechanism (Loose et al., 2008; Ivanov et al., 2010; Vecchiarelli et al., 2014). It is an exciting time to study this unique positioning mechanism as a growing diversity of intracellular cargos have been shown to use related systems for their subcellular organization (Vecchiarelli et al., 2012). We anticipate that surface-mediated bio-molecular patterning will become an emerging theme throughout all kingdoms of life.

I submitted this image to the BPS Image contest because I love how it emphasizes what can be achieved with proteins when released from the confines of the cell. In vivo, MinD and MinE form a pole-to-pole oscillator. But on an expansive flat bilayer, their biochemical potential for pattern formation is unleashed. MinD and MinE can form a variety of patterns including the spirals shown in my image. Dissecting how these patterns form outside of the cell is unraveling the oscillatory mechanism used inside the cell.

“What I cannot create, I do not understand.”  This quote by Richard Feynman is the essence of synthetic biology. To say that we fully understand a cell, we must first build one from the bottom up. We are approaching a time where it is feasible to experimentally challenge the concept of a protocell. The MinCDE system, and related positioning systems, will be key to such an endeavor because they are composed of a minimal number of components that self-organize to position essential cargos, such as the cell division apparatus. But first we must define the systems and develop a full biochemical understanding of the components, which can be achieved via cell-free reconstitution experiments presented in the image. I hope this image gets others just as excited and motivated as I am about studying this fascinating pattern-mediated positioning system, which may turn out to be the norm as opposed to the exception in all cells.

This image shown is a Turing pattern. Alan Turing was first to describe a mathematical model called “reaction-diffusion” that explains how the concentration of one or more substances distributed in space changes after a local reaction and subsequent diffusion. Alan is mainly known for conceiving of a machine that we now call a computer, and for breaking the German Enigma code, which played a pivotal role in ending WWII. But he always had a fascination with patterns in nature and his one and only Biology paper that describes reaction-diffusion is also his most cited. Turing was not applauded for these extraordinary efforts. Rather, because Alan Turing was gay at a time it was illegal, he was sentenced to chemical castration, labeled a security risk, and lost his job as perhaps one of the best code breakers that ever lived. The resultant depression led to suicide. I hope this image reminds viewers of Alan’s ongoing contributions to science.

Supporting Scientific Information

The intricate subcellular organization of a eukaryotic cell is mainly communicated by actin filaments, microtubules, and motor proteins that drive along these cables. Recent improvements in microscopy have recently shown that bacteria also display complex intracellular organization. The view of bacterial cells as simply sacks of enzymes is obsolete. But instead of using cables and motors for moving the ‘innards’ of a bacteria cell, dynamic protein patterns on biological surfaces, such as the inner membrane, are emerging as the critical driving forces for positioning a wide variety of cargos such as the bacterial chromosome, plasmids, organelles, and even the cell division apparatus.

In E. coli, the MinCDE system self-organizes into a cell-pole-to-cell-pole oscillator that positions the divisome at mid-cell so that daughter cells are equal in size. The MinD protein is an ATPase that binds the membrane in its ATP-bound form and recruits the cell division inhibitor MinC. MinE stimulates the ATP-hydrolysis activity of MinD, which releases MinD from the membrane. The perpetual chase of MinD by MinE creates the pole-to-pole oscillator, which maintains a low level of the division inhibitor at mid-cell where divisome assembly and cell division is allowed to take place. How Min proteins interact with the membrane surface to generate the in vivo oscillations is a subject of intense study.

The number of factors involved in subcellular organization makes it difficult to study individual systems under controlled conditions in vivo. We in Dr. Kiyoshi Mizuuchi’s group developed a cell-free technique to visualize and study spatial organization mechanisms.  The MinD protein was fused to Green Fluorescent Protein and the MinE protein was labeled with an Alexa dye. The two proteins were mixed in a buffer containing ATP and infused into a flowcell that had its surfaces coated with a flat lipid bilayer, which acts as a biomimetic of the inner membrane. The dynamics of MinD and MinE were then visualized by total internal reflection fluorescence microscopy (TIRFM), a technique that specifically resolves surface-associated processes.

References

Loose M, Fischer-Friedrich E, Ries J, Kruse K, Schwille P. 2008. Spatial Regulators for Bacterial Cell Division Self-Organize into Surface Waves in vitro. Science 320: 789
Ivanov V, Mizuuchi K. Multiple modes of interconverting dynamic pattern formation by bacterial cell division proteins. 2010. Proc Natl Acad Sci USA 107: 8071
Vecchiarelli AG, Li M, Mizuuchi M, Mizuuchi K. 2014. Differential affinities of MinD and MinE to anionic phospholipid influence Min patterning dynamics in vitro. Mol Microbiol 93: 453
Vecchiarelli AG, Mizuuchi K, Funnell BE. 2012. Surfing biological surfaces: exploiting the nucleoid for partition and transport in bacteria. Mol Microbiol 86:513

Patterned EGF Reveals Differential Distributions of Activated Kinase and Integrin Activities

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This confocal fluorescence image on the cover of Biophysical Journal volume 107, issue 11, shows a single cell attached to micron-sized patterns labeled with red fluorescence (immobilized streptavidin). These patterns present irreversibly attached epidermal growth factor (EGF), which engages EGF receptors expressed at the cell surface. A consequence of this engagement is stimulated phosphorylation of tyrosine residues at the patterned features, detected as blue fluorescence with a monoclonal anti-phosphotyrosine antibody. Concentration of phosphotyrosine labeling at the patterned sites can be seen more clearly in the single color images of this cell (not overlayed) in Figure 4 of our paper in this issue of the Journal.

Most interesting in this image is the concentration of the beta1 subunit of an integrin in green. The striated labeling around the more peripheral EGF features that are engaging the cell suggests that focal adhesion complexes are formed at these sites. As quantified in our paper using a novel radial analysis method, we find that this integrin is more concentrated at patterned features in contact with the cell periphery, rather than at the cell center, where the tyrosine phosphorylation is just as abundant as it is in the periphery. Together, these results suggest that the earliest signaling steps mediated by EGF receptors are activated similarly wherever the patterned EGF engages the cells, while more downstream signaling that recruits integrin-containing focal adhesion complexes is preferentially activated at the cell periphery. Possibly tension forces at these peripheral sights are greater, and inside-out activation of integrin signaling complexes may result.

The experiment represented by this cover image was carried out by Devin Wakefield, a graduate student in our laboratory. We were prompted to submit a cover image by Dr. Kirsten Bryant, a former graduate student in our laboratory and a co-author in this study. The image represents the multidisciplinary efforts and interests in our laboratory, that range from efforts to understand molecular mechanisms of store-operated calcium entry in mast cells to super resolution imaging of receptor clustering dynamics. These efforts provide new tools and methodologies for application to a broad range of cell signaling questions. Our laboratory website summarizes our research activities: http://baird.chem.cornell.edu

– Amit Singhai, Devin L. Wakefield, Kirsten L. Bryant, Stephen R. Hammes,
David Holowka, and Barbara Baird