Two phases on two faces

109-11 Cover ImageThis image of domains on a lipid vesicle was captured by Matthew Blosser, who is now an NIH NRSA fellow at the University of Oxford. In the image, the lipids within the membrane of a ~70 micrometer giant unilamellar vesicle have demixed into coexisting liquid phases. The bright domains are the liquid-disordered phase, which are diffusing in a background of dark, liquid-ordered phase.  The image was obtained with an epifluorescence microscope using a 40x air objective. The spherical vesicle was imaged at several different focal planes, and these images were then projected onto one 2-dimensional image.

One striking aspect of our image is how cleanly the vesicle divides into regions of light and dark. All of the have nearly the same brightness (accounting for geometric effects), and each individual domain has a single, uniform brightness. This means that, although this vesicle is made of several different lipid components, only two phases are present within the membrane, each with well-defined physical properties. It also means that the domains on each face of the membrane are perfectly aligned with each other, at least on length scales observable by fluorescence microscopy. As a result, the two monolayer leaflets of the membrane must be coupled.  This result is somewhat non-intuitive because interactions between the two leaflets are at least partially mediated by the lipid acyl chains, which are floppy.

We were inspired by our collaborator Aurelia Honerkamp-Smith and by other researchers to measure this effect. Specifically, we measured the energy penalty a vesicle would have to pay in order to misalign the domains on each face of the membrane. Because we knew that domains did not misalign on micron length scales of their own accord, we decided to give a push to the domains on only one side of the membrane.  Because that push had to be a very strong one, we built a microfluidic chamber into which we flowed vesicles, which burst on the solid support. Flowing water through the chamber moved the upper monolayer of the membrane over the lower monolayer, which was firmly stuck to the substrate. By measuring how hard we needed to push an individual domain to make it move (and by collaborating with Mikko Haataja and Tao Han, who produced some beautiful theoretical work), we extracted a value for the interleaflet coupling. The value we found agrees with a previous theoretical prediction (there were no previous measurements to our knowledge), and it could help explain how changes in lipid organization are communicated across cell membranes. More information about our research is found in our article in the new issue of Biophysical Journal and at

-Matthew Blosser, Aurelia Honerkamp-Smith, Tao Han, Mikko Haataja and Sarah Keller

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.


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

3D Cellular Membrane Systems Highlighted on BiophysJ Cover

bpj_104_11_coverMasa Hoshijima and Christian Soeller, authors on the latest paper to be highlighted on the cover of the Biophysical Journal, detail the process of creating the image below. The paper, Nanoscale distribution of ryanodine receptors and caveolin-3 in mouse ventricular myocytes: dilation of t-tubules near junctions, was co-authored by Joseph Wong, David Baddeley, Eric A. Bushong, Zeyun Yu, Mark H. Ellisman, Hoshijima, and Soeller.

The cover image shows a 3D snapshot of cellular membrane systems that are essential to the rapid electrical activation of cardiac muscle cells. We acquired hundreds of serial high-resolution scanning electron microscopy (SEM) images from a heart tissue-embedded plastic block, determined membrane profiles, and created the rendering of the transverse tubular system (green), which is a complex network of membrane invaginations continuous with the surface membrane (blue-black). We are also showing junctional contacts (red) between the transverse tubular system with the sarcoplasmic reticulum, an internal membrane-bound compartment that is an intracellular reservoir of calcium, which acts as a chemical activator of the contractile proteins. The signaling between these two membrane systems occurs at the junctional contacts, where they come to within ~15 nm of each other.

The serial imaging method is termed serial block-face SEM, one of the newer volume electron microscopic technologies, which are gaining in popularity. In this method, the surface of tissue blocks is sectioned in situ in specially designed SEM vacuum chambers. The original interest, which drove the development of volume electron microscopy methods, was largely in the visualization of neuronal circuits. However, the method is generally useful to visualize a whole cell or a large fraction of a whole cell, with extremely fine details of their structural complexity. We were struck by the dramatic and beautiful features of the rendered structures and thought it would make a suitable cover of Biophysical Journal.

The nanoscale detail of the membrane network architecture is biophysically very important as it determines propagation of electrical signals, diffusion of signaling and messenger compounds (such as calcium) and – via junctional contacts – strongly affects signaling between channels in the surface membrane and the sarcoplasmic reticulum. All of these interactions are critical for the forceful and rapid contraction of heart muscle. Our manuscript reveals details and structural specializations of the transverse tubular system that were not shown before in such clarity and identified local swellings of the tubules that often occurred at junctions with the sarcoplasmic reticulum.

Our accompanying publication was the result of a collaboration between the University of Auckland (now University of Exeter, Soeller) and University of California San Diego (UCSD) (Hoshijima) using a range of imaging techniques: (1) the SEM imaging highlighted on the cover, (2) EM tomography for the highest resolution in small volumes and (3) optical super-resolution imaging which we used for quantification and protein specific imaging of key molecules associated with these membranes. We believe our paper demonstrates the power of correlative imaging between modern EM techniques and optical super-resolution with fluorescent markers. We are continuing to pursue this theme in a new collaboration between our laboratories in San Diego (Hoshijima), Exeter (Soeller), and in addition colleagues in Kyoto (Dr. Hiroshi Takeshima), supported by a recently funded Human Frontier Science Program research project to reveal the makeup and assembly of nano-signaling structures.

Imaging is the key technology in both of our laboratories and as a result our work is generally very visual. In addition to the scientific information the images often have a great aesthetic appeal to us. The fact that one of our images has been chosen as cover art of the current issue of biophysical journal is greatly encouraging since it means that others can see the aesthetic value, too. Most important to us, however, is the scientific value of our images – they contain important information and we see it as one of our key tasks to reduce the data in our images to biophysical quantities that can be related to hypotheses and quantitative models. This brings us to perhaps the most significant reason we are very happy that our art is on the cover of the Biophysical Journal: it implies that one of our manuscripts has been accepted and that our image data and the information it conveys has passed the peer review of BJ.

Links to further work of Dr. Soeller’s laboratory and imaging activities are available at To learn more about the active development of various advanced microscopic imaging technologies at UCSD, please visit