Abstracts

The living cell as a sloppy dynamical system

Erez Braun¹

¹ Department of Physics & Network Biology Research Laboratories Technion, Haifa 32000, Israel

The emergence of stable cell states (morphology, metabolism and function) reflects the organization of regulatory modes determining the temporal spectrum of intracellular proteins. In the last decade, our study of cell populations exposed some intriguing characteristics of their behavior, showing that cell-state organization reflects exploratory dynamics in a degenerate, high-dimensional phase-space. I’ll discuss these dynamics, arguing that the living cell belongs to a broad class of systems exhibiting sloppy dynamics, characterized by their insensitivity to the underlying parameters yet efficient convergence to a viable state. Understanding of the physics of such dynamical systems remains elusive.

Molecular Dynamics Simulations of Protein Interactions Leading to Synaptic Vesicle Fusion

Maria Bykhovskaia¹

¹Department of Neurology, Wayne State University , Detroit, Michigan, USA

Neurotransmitters are released via the fusion of synaptic vesicles with the neuronal membrane. Vesicles dock to the membrane via a specialized protein complex termed SNARE. The fusion occurs in response to Ca2+ inflow, and the vesicle protein Synaptotagmin (Syt) serves as a Ca2+ sensor. Syt includes C2A and C2B domains connected by a flexible linker. A cytosolic protein Complexin (Cpx) interacts with the SNARE complex, regulating the fusion time-course. Although molecular interactions of these proteins have been extensively studied, it is still debated how the fusion proteins interact with each other and with lipid bilayers to trigger lipid merging and pore opening. To elucidate this mechanism, we performed molecular dynamics simulations of Syt interacting with the SNARE complex, Cpx and lipid bilayers. Our simulations demonstrated that C2A domain has a strong affinity to lipids, while C2B domain interacts weakly with neutral lipids but deeply immerges into PIP2 containing anionic lipids upon Ca2+ binding. Our simulations also suggest that C2B domain tightly interacts with the SNARE-Cpx complex. Altogether, our computations support a model whereby Ca2+ binding pocket of Syt C2A domain is attached to the vesicle membrane, while C2B domain interacts with the SNARE complex and Cpx, bridges bilayers, and upon Ca2+ binding immerges into the plasma membrane, thus triggering fusion.

New results on collective chemotaxis in colonies

Ramin Golestanian¹

¹Rudolf Peierls Centre for Theoretical Physics, University of Oxford, Oxford OX1 3NP, United Kingdom

I discuss two different problems in which a crude phenomenological description of chemotaxis leads to interesting new perspectives. The first question concerns the competition between chemotaxis and cell division, which might at first sight seem completely unrelated. We have developed a simple model to explore any possible interplay between the two processes, and studied it via dynamical Renormalization Groups methods [1]. We find that whereas details of the microscopic behavior of cells do not impact the collective behavior on a large scale, the interplay between the two general processes of growth and chemotaxis leads to a variety of collective phenomena, which includes a sharp transition from a phase that has moderate controlled growth and death, and regulated chemical interactions, to a phase with strong uncontrolled growth/death and no chemical interactions. Remarkably, for a range of parameters, the transition point shows nontrivial collective motion, which can even be described analytically. The second problem concerns the role of slowly diffusing chemical residues on the behavior of bacteria with twitching motility. We find evidence that a non-trivial perpendicular alignment mechanism tends to modulate the orientation of bacteria [2], and that this new coupling allows us to build a complete quantitative description of the observed collective behavior of such bacteria [3].

• [1] A. Gelimson and R. Golestanian, Phys. Rev. Lett. 114, 028101 (2015)
• [2] W.T. Kranz, A. Gelimson, and R. Golestanian, arXiv:1504.06814
• [3] A. Gelimson, K. Zhao, C. Lee, W.T. Kranz, G.C.L. Wong, and R. Golestanian, unpublished (2016)

Features of Ca²⁺ release and uptake channels mediating ER-to-mitochondrial communication

J. Kevin Foskett¹

¹Departments of Physiology and Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Low-level constitutive inositol trisphosphate receptor (InsP₃R)-mediated Ca²⁺ release from the endoplasmic reticulum (ER) and its uptake by mitochondria through the uniporter Ca²⁺ channel complex (MCU) is essential for maintaining cellular bioenergetics. The kinetic features of the channels and their spatial relationships are crucial to mediate this communication. Kinetic responses of single InsP₃R channels in native ER membrane to rapid ligand concentration changes with msec resolution revealed channel activation and deactivation with novel Ca²⁺ regulation and unexpected ligand cooperativity. We measured i_Ca through an InsP₃R channel in its native membrane environment under physiological ionic conditions to be 0.15 ± 0.01 pA for a ER store with 500 μM [Ca²⁺]ER. The iCa–[Ca²⁺]ER relation suggests that Ca²⁺ released by an InsP₃R channel raises [Ca²⁺]i near the open channel to ~13–70 μM, depending on [Ca²⁺]ER. These kinetic and conductance measurements have implications for Ca²⁺ uptake by MCU. Recent patch clamp recordings of MCU in the mitochondrial inner membrane have provided insights into novel regulation, but high resolution kinetic features of MCU channel gating in response to Ca²⁺ released through an InsP₃R remain to be determined. Modeling will be helpful to determine the required spatial proximity of release and uptake sites and the number of channels involved.

Bacterial cell wall peptidoglycan architecture and dynamics

Simon J. Foster¹

¹The Krebs Institute, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK

Bacterial cell wall peptidoglycan is essential for the life of most bacteria. It determines cell shape, and its biosynthesis is the target for many important antibiotics. The fundamental chemical building blocks of peptidoglycan are conserved: repeating disaccharides cross-linked by peptides. However, despite this relatively simple chemistry, how this is manifested into the myriad bacterial shapes and how this single macromolecule remains dynamic permitting cell growth and division has largely remained elusive. The advent of new microscopy approaches is beginning to revolutionize our understanding of the architecture of this polymer and to reveal novel insights into its biosynthesis and hydrolysis. Atomic force microscopy has demonstrated a complex, nanoscale peptidoglycan architecture in diverse species, which meets the challenges of maintaining viability and growth within their environmental niches by exploiting the bioengineering versatility of the polymer. The application of super-resolution fluorescence microscopy, coupled with new chemical probes has begun to reveal how this essential polymer is synthesized during growth and division.

• [1] Wheeler, R. et al. (2015) mBio 6:e00660
• [2] Bailey, R.G. et al. (2014) Biophysical Journal 107, 2538
• [3] Turner,R.D. et al. (2014) Molecular Microbiology 91, 862
• [4] Turner, R.D. et al. (2013) Nature Communications 4, 1496
• [5] Wheeler, R. et al. (2011) Molecular Microbiology, 82, 1096
• [6] Turner, R.D. et al. (2010) Nature Communications 1, 26

The physical control of CNS development and pathology

Kristian Franze¹

¹Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK

During the development of the nervous system, neurons migrate and grow over great distances. Currently, our understanding of neuronal development and function is, in large part, based on studies of biochemical signaling. Despite the fact that forces must be involved in cell motion, mechanical aspects have so far rarely been considered. Here we investigate how Xenopus neurons respond to their mechanical environment. Axonal growth velocities, directionality, fasciculation, i.e., their tendency to grow in bundles, and maturation all significantly depended on substrate stiffness. Moreover, when grown on substrates incorporating linear stiffness gradients, axon bundles were repelled by stiff substrates. In vivo atomic force microscopy measurements revealed stiffness gradients in developing brain tissue, which axons followed as well towards soft. Interfering with brain stiffness and mechanosensitive ion channels in vivo both led to similar aberrant neuronal growth patterns with reduced fasciculation and pathfinding errors, strongly suggesting that neuronal growth is not only controlled by chemical signals – as it is currently assumed – but also by the tissue’s local mechanical properties.

Active Brownian particles — spheres, filaments, and mixtures

G. Gompper^1,2,A. Wysocki^1,2,R.G. Winkler^1,2, R.E. Isele-Holder^1,2, and J. Elgeti^1,2

¹Institute of Complex Systems, and ²Institute of Advanced Simulation, Forschungszentrum Jülich, Jülich, Germany

Ensembles of active Brownian particles are highly simplified model systems for a large variety of self-propelled synthetic microswimmers, microorganisms, cells, and biological filaments. The simplicity of the model allows for (i) a description which emphasizes generic behaviors, and (ii) the investigation the collective motion of a large number of particles. The modeling of an active system by Brownian particles emphasizes the roles of volume exclusion, particle shape, and thermal or active noise [1].

Two types of active Brownian particles will be considered. The first is a system of active spheres [2]. This system shows activity-induced phase separation, very similar to a system of passive attractive spheres. However, the collective dynamics is very different, with a spontaneous formation of swirls and jets – despite of the absence of any alignment mechanism. The dynamics becomes even more interesting for mixtures of passive and active particles. Now the interfaces between the dense and the dilute phases become mobile, with a spontaneous symmetry breaking between advancing and receding interfaces [3]. The second is a system of self-propelled filaments, both without [4] and with a load [5]. This is a model, for example, for actin filaments in motility assays. Depending on the size and shape of the load, the bending rigidity of the filament, and the propulsion strength, a large variety of conformations and dynamics is observed, ranging from spiraling and circle-swimming to beating [5].

• [1] J. Elgeti, R.G. Winkler, and G. Gompper, Rep. Prog. Phys. 78, 056601 (2015).
• [2] A. Wysocki, R.G. Winkler, and G. Gompper, EPL 105, 48004 (2014).
• [3] A. Wysocki, R.G. Winkler, and G. Gompper, arXiv 1601.00850 (2016).
• [4] R.E. Isele-Holder, J. Elgeti, and G. Gompper, Soft Matter 11, 7181 (2015).
• [5] R.E. Isele-Holder, J. Jäger, G. Saggiorato, J. Elgeti, and G. Gompper, submitted (2016).

Controlling contractile instabilities in the actomyosin cortex

Stephan W. Grill ¹

¹BIOTEC, Technische Universität Dresden, Germany

Cells and tissues represent active materials that generate stresses for driving morphogenesis. A fundamental challenge is to understand how spatiotemporal patterns arise in such active biological materials, driven by the interplay of active mechanical processes and regulation by signaling pathways. I will discuss the mechanism of spatiotemporal pattern formation in the highly contractile actomyosin cortical layer, where transient accumulations of myosin motor proteins tend to form pulsatile networks to drive morphogenetic events. Using a novel image analysis technique (COmoving Mass Balance Imaging, COMBI) we have determined the kinetic diagram of myosin activation by RhoA in the cell cortex of the polarizing one-cell stage Caenorhabditis elegans embryo. We found that the complete system of myosin activation by RhoA, active stress generation by myosin, and RhoA advection by actomyosin gel flow is unstable. Notably, the dynamic pattern in the unstable regime appears to be under separate regulatory control, and I will discuss general means of how introducing regulatory processes to active materials gives rise to novel pattern forming states.

Molecular mechano-sensors: translating force into biochemical signals

Frauke Gräter^1,2 and Camilo Aponte-Santamaria^1,2

¹Interdisciplinary Center for Scientific Computing, Heidelberg University and ²Heidelberg Institute for Theoretical Studies, Heidelberg

How can a cell sense force and translate it into downstream signaling cascades leading to changes in cellular behavior such as differentiation, proliferation, or motility? It is increasingly recognized that mechanical force can reversibly change protein conformation, thereby allosterically switching proteins on and off, reminiscent of protein regulation by biochemical signals such as co-factor binding. I will present recent results of two proteins, the von Willebrand factor (VWF) and focal adhesion kinase (FAK), for which we have successfully revealed how they work as mechano-sensors [1-5]. To this end, we used molecular modeling, Molecular Dynamics (MD) simulations and a novel Force Distribution Analysis to reveal the inner working and change of function of these proteins under the influence of mechanical load. Our results can be used to extrapolate to the more complex cellular environment of these molecules, and have implications for the underlying processes, namely blood coagulation (VWF) and stem cell differentiation pathways (FAK).

• [1] Posch S, Aponte-Santamaría C, Schwarzl R, Karner A, Radtke M, Gräter F, Obser T, König G, Brehm MA, Gruber HJ, Netz RR, Baldauf C, Schneppenheim R, Tampé R, Hinterdorfer P. J Struct Biol. 2016 Apr 23. pii: S1047-8477(16)30081-8.
• [2] Zhou J, Aponte-Santamaría C, Sturm S, Bullerjahn JT, Bronowska A, Gräter F. PLoS Comput Biol. 2015 Nov 6;11(11):e1004593.
• [3] Aponte-Santamaría C, Huck V, Posch S, Bronowska AK, Grässle S, Brehm MA, Obser T, Schneppenheim R, Hinterdorfer P, Schneider SW, Baldauf C, Gräter F. Biophys J. 2015 May 5;108(9):2312-21.
• [4]Zhou J, Bronowska A, Le Coq J, Lietha D, Gräter F. Biophys J. 2015 Feb 3;108(3):698-705.
• [[5] Goñi GM, Epifano C, Boskovic J, Camacho-Artacho M, Zhou J, Bronowska A, Martín MT, Eck MJ, Kremer L, Gräter F, Gervasio FL, Perez-Moreno M, Lietha D. Proc Natl Acad Sci U S A. 2014 Aug 5;111(31):E3177-86.

Unexpected ordered phases in active matter systems

Michael F. Hagan¹ , Gabriel S. Redner¹, Caleb G. Wagner¹, and Aparna Baskaran¹

¹ Martin Fisher School of Physics, Brandeis University, Waltham, MA, USA

Active matter describes systems whose constituent elements consume energy to generate motion. I will describe computer simulations of two recently developed active matter systems, and unexpected ordered phases that arise as a consequence of activity in these systems. (1) Self-propelled colloids with repulsive interactions and no aligning interactions are a minimal model active matter system. We and others have shown that this system undergoes athermal phase separation. Despite the intrinsically nonequilibrium nature of the phase transition, I will show that the kinetics can be described using a framework analogous to equilibrium classical nucleation theory, governed by an effective free energy of cluster formation, with identifiable bulk and surface terms. I will also show that when these particles are confined they undergo another transition, in which the particles become confined to the boundary, with a density that depends on the local curvature radius of the boundary. (2) Active nematics are liquid crystals which are driven out of equilibrium by energy-dissipating active stresses. The ordered nematic state is unstable to the proliferation of topological defects, which undergo birth, streaming dynamics, and annihilation to yield a seemingly chaotic dynamical steady-state. In this talk, I will show that order emerges from this chaos, in the form of heretofore unknown broken-symmetry phases in which the topological defects themselves undergo orientational ordering.

Cell Penetration and Membrane Fusion: Molecular Dynamics and Fluorescence Spectroscopy

Pavel Jungwirth¹

¹ Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2, 16610 Prague 6, Czech Republic

First, molecular dynamics simulations, together with fluorescence spectroscopy and biomimetic colorimetric assays, have been performed in search of explanations why arginine rich peptides with intermediate lengths of about ten amino acids translocate well through cellular membranes, while analogous lysine rich peptides do not. We observe a strong tendency of adsorbed arginine (but not lysine) containing peptides to aggregate at the bilayer surface. We suggest that this aggregation of oligoarginines leads to partial disruption of the bilayer integrity due to the accumulated large positive charge at its surface which increases membrane-surface interactions due to the increased effective charge of the aggregates. As a result, membrane penetration and translocation of medium length oligoarginines becomes facilitated in comparison to single arginine and very long polyarginines, as well as to lysine containing peptides.

Second, we aim at understanding of interactions of calcium with lipid membranes at the molecular level, which is of great importance in light of the involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. Time-resolved fluorescent spectroscopy of lipid vesicles and second harmonic generation spectroscopy of lipid monolayers are used to characterize local binding sites of Ca2+ in zwitterionic and anionic model lipid assemblies while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environment. To gain an atomistic-level information about calcium binding, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively including electronic polarization effects. We demonstrate that the membranes have very high calcium-binding capacity, with several types of binding sites present, with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.

Single vesicle recordings in hippocampal ‘xenapses’ reveal diffusional dispersion of synaptic vesicle proteins after fusion

Jürgen Klingauf¹

¹Institute of Medical Physics and Biophysics, Westfälische Wilhelms-Universität Münster, Münster, Germany

In order to maintain neuronal transmission after exocytosis of synaptic vesicles (SVs), the vesicular proteins have to be cleared away from the active zone. Until now it remained controversial whether SV components remain clustered during translocation from sites of exocytosis or disperse by free diffusion. To address this question, we developed a novel purely presynaptic neuronal preparation which enables single vesicle recording by TIRFM.

Using click-chemistry we functionalized micropatterned coverglasses with protein domains of synaptic cell adhesion molecules, serving as artificial postsynapses. On these host substrates purely presynaptic boutons form ‘en face’ directly onto the coverslip, termed ‘xenapses’. Serial section TEM as well as focused-ion-beam SEM showed that xenapses contain a few hundred SVs, many of them docked in several clusters at the bottom membrane. 4Pi and TIRF-STORM confirmed the existence of several active zones. Thus, xenapses offer the unique opportunity to record exocytosis of single vesicles by TIRFM. Using fusion constructs of the pH-sensitive pHluorin, single fusion events were visible as diffraction-limited spots on stimulation with single action potentials. We could localize fusion events synchronous to action potentials with ~20 nm precision and follow the fate of released SV proteins. We observed diffusional dispersion of vesicular proteins post fusion with diffusion constants in the range of 0.1 µm²/s. Thus, our results point to free diffusion as mechanism for fast clearance.

Snapshot of sequential SNARE assembling states between membranes shows that N-terminal transient assembly initializes fusion

Frédéric Pincet¹

¹Ecole Normale Supérieure, Laboratoire de Physique Statistique, 24 rue Lhomond, 75005 Paris, France

Many prominent biological processes are driven by protein assembling between membranes. Understanding the mechanisms then entails determining the assembling pathway of the involved proteins. Because the intermediates are by nature transient and located in the intermembrane space, this determination is generally a very difficult, not to say intractable, problem. In this presentation, I will present a new setup with sphere/plane geometry. Using this setup, we have been able to freeze one transient state in which the N-terminal domains of SNARE proteins are assembled [1]. A single camera frame is sufficient to obtain the complete probability of this state with the transmembrane distance. I will show that it forms when membranes are 20 nm apart and stabilizes by further assembling of the SNAREs at 8 nm. This setup that fixes the intermembrane distance, and thereby the transient states, while optically probing the level of molecular assembly by Förster resonance energy transfer (FRET) can be used to characterize any other transient transmembrane complexes.

• [1] Y. J. Wang, F. Li, N. Rodriguez, X. Lafosse, C. Gourier, E. Perez, F. Pinceta, PNAS 113, 3533-3538 (2016).

Buckling the cell membrane

Aurélien Roux¹

¹Department of Biochemistry, University of Geneva, CH-1211 Switzerland

Cells and organelles are delimited by lipid bilayers. Since these membranes are impermeable to most solutes, in order to exchange material with their environment, organelles and cells have developed a large protein family involved in budding membranes to form membrane carriers,. These carriers transport material between organelles. Proteins involved in intracellular membrane traffic can remodel the membrane by several ways. Clathrin, for example, polymerizes into a spherical cage onto the membrane, forcing it to curve. Here we describe a recently discovered protein complex called ESCRT-III, which has the property of forming spirals at the surface of the lipid bilayer. This unique structural feature did not suggest any known mechanism by which it could deform the membrane. It was theoretically proposed that, while growing into a spiral, it accumulates stress energy which can be released by buckling of the central part of the spiral 1. By using high-speed AFM and biophysical tools to measure membrane elasticity we show how the elastic and polymerization properties of the ESCRT-III filament are compatible with such model 2. We further investigate the dynamics of the complex.

• [1] M. Lenz, D. Crow, and J.-F. Joanny, Physical Review Letters 103 (2009)
• [2] N. Chiaruttini, L. Redondo-Morata, A. Colom, F. Humbert, M. Lenz, S. Scheuring, and A. Roux, Cell 163, 866 (2015)

Overlapping roles of Synaptotagmins 1 and 2 in triggering transmitter release at fast CNS synapses

Ralf Schneggenburger ¹Brice Bouhours, Enida Gjoni, Norbert Babai, Olexiy Kochubey

¹Brain Mind Institute, School of Life Sciences, EPFL.

At a synapse, an action potential (AP) invading a presynaptic nerve terminal causes the Ca2+ - dependent release of neurotransmitter, by opening voltage-gated Ca2+ channels in the presynaptic nerve terminal. In this process, the rate of transmitter release evoked by a presynaptic AP ("evoked release") is increased by several orders of magnitude (up to ~ 1-millionfold) as compared to the background rate of release. To enable this high dynamic range, Ca2+ must act on a highly non-linear Ca2+ sensor, which are represented by the double C2 domain containing proteins Synaptotagmin (Syt)-1, or Syt2. Combined patch-clamp and Ca2+ uncaging studies at the large calyx of Held synapse, have shown that Syt2 KO mice have a shallow Ca2+ dose-response curve of transmitter release (slope ~ 1 in double logarithmic coordinates) as compared to wild-type synapses (slope ~ 4; ref. [1]). Thus, the Syt2 protein confers highly-non-linear Ca2+ sensing during the triggering of evoked release at the calyx synapse. Interestingly, Syt1 and Syt2 are closely related genes with a high sequence homology. Syt1 is expressed in forebrain, whereas Syt2, an isoform found only in vertebrates, is highly expressed in hindbrain. We now find that in various excitatory and inhibitory hindbrain synapses, Syt1 and Syt2 have overlapping roles. This could either indicate a developmental expression switch from Syt1 towards Syt2 as we showed for the nascent calyx synapse recently [2], or else, some synapses, especially inhibitory synapses, might use both Syt1 and Syt2 to achieve high rates of evoked release.

• [1] Kochubey and Schneggenburger (2011). Neuron 69, 736-748.
• [2] Kochubey et al. (2016) Neuron, in press.

Direct observations of transition dynamics from macro- to micro-phase separation in asymmetric lipid bilayers induced by externally added glycolipids

S. F. Shimobayashi, ¹ M. Ichikawa, ² and T. Taniguchi ³

¹Department of Chemistry, Ecole Normale Supériure, Paris, France, ²Department of Physics, Kyoto University, Kyoto, Japan and ³Department of Chemical Engineering, Kyoto University, Kyoto, Japan

We present the first direct observations of morphological transitions from macro- to micro-phase separation using micrometer-sized asymmetric lipid vesicles exposed to externally added glycolipids (GM1:monosialotetrahexosylganglioside). The transition occurs via an intermediate stripe morphology state. During the transition, monodisperse micro domains emerge through repeated scission events of the stripe domains. Moreover, we numerically confirmed such transitions using a time-dependent Ginzburg-Landau model, which describes both the intramembrane phase separation and the bending elastic membrane. Our findings could provide important mechanistic clues for understanding the dynamics of the heterogeneities exiting in cell membranes.

• [1] S. F. Shimobayashi, M. Ichikawa, and T. Taniguchi, EPL 113, 56005 (2016).

Macrophage dorsal ruffles as dynamic platforms for signaling and endocytosis

Jennifer L. Stow ¹ Adam Wall, Lin Luo, Nicholas Condon, Jeremy Yeo

¹Institute for Molecular Bioscience and IMB Centre for Inflammation and Disease Research, The University of Queensland, Brisbane Australia.

Macrophages engage in surveillance, detection and phagocytosis of pathogens using receptors displayed transiently on the cell surface. We have shown that Toll-like receptors (TLRs), along with regulatory molecules and adaptors and kinases, are localized in surface dorsal ruffles which give rise to macropinosomes and phagosomes, creating sequential signaling environments for differential macrophage outputs. Newly identified signaling adaptors for TLRs further bias signaling outputs. Lattice light sheet imaging reveals the extremely dynamic nature and unique structural features of F-actin-rich dorsal ruffles on activated cells. I will present a sequence of ruffle-associated Rab GTPases and their effectors that control aspects of ruffle behavior and TLR-induced PI3K-Akt-mTOR signaling. 4D live cell imaging and 3D electron microscopy are helping to characterize these signaling membrane domains and the endosomal network responsible for receptor trafficking. The highly dynamic properties of these membrane niches and compartments sheds new light on the spatiotemporal regulation of innate immune responses.

Multi-level effects of cholesterol on the formation of exocytotic fusion pores

Lukas K. Tamm^1,2 Alex Kreutzberger^1,2and Volker Kiessling^1,2

¹Center for Membrane and Cell Physiology and ²Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA

Cholesterol modulates the structure and dynamics of biological membranes in multiple ways. It changes the fluidity, thickness, mechanical properties and intrinsic curvature of lipid bilayers. Cholesterol also induces phase separations in multicomponent lipid mixtures, partitions selectively between different coexisting lipid phases, and causes integral membrane proteins to respond by changing conformation or redistribution in the membrane. In this contribution, we discuss which of these often overlapping properties are important for the formation exocytotic fusion pores and how they affect the distribution of the relevant SNARE and accessory proteins in the plasma and vesicle membrane. We also discuss how cholesterol affects the balance between hemi- and full fusion as measured by single vesicle fusion with millisecond time resolution [1-4].

• [1] Domanska, M.K., Kiessling, V., Stein, A., Fasshauer, D., and Tamm, L.K. (2009) Single vesicle millisecond fusion kinetics reveals number of SNARE complexes optimal for fast SNARE-mediated membrane fusion. J. Biol. Chem. 284:32158-32166.
• [2] Murray, D. and Tamm, L.K. (2011) Molecular mechanism of cholesterol- and phosphoinositide-mediated syntaxin clustering. Biochemistry 50:9014-9022.
• [3] Lukas K. Tamm,1,2 Alex Kreutzberger1,2 and Volker Kiessling1,2.
• [4]Kreutzberger AJB, Kiessling V, and Tamm LK (2015) High cholesterol obviates a prolonged hemifusion intermediate in fast SNARE-mediated membrane fusion. Biophys J 109:1-11.

β-arrestin1 as a pivotal regulator in pediatric leukemia

Lin Zou¹ , Yi Shu¹, Shan Liu¹, Haiyan Li¹, Juan Long¹, Kang Li¹, Xiaoyan Zhou¹, Xinkun Qi¹, and Hongxu Wang¹

¹Center for Clinical Molecular Medicine, Children’s Hospital of Chongqing Medical University, Chongqing 400014, China

Leukemia is the most common childhood malignancy. Pediatric acute leukemia (AL) mainly includes acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) subtypes, which occupy approximately 76% and 20% of childhood leukemia respectively. Although the long-term disease-free survival (DFS) and overall survival (OS) rate of pediatric leukemia has been improved recently, there is still about 25% relapse rate. Novel molecular mechnism for therapetic target are still more concerned. β-arrestin1, the multifunctional scaffold protein, is found to mediate many intracellular signaling network, and to be involved in many tumors. However, little is known in leukemia. Here we present the aberrant β-arrestin1 expression and regulation in different kinds of pediatric leukemia subtypes, by binding with several proteins and mediating their corresponding signalings, to be potential therapeutic targets for childhood leukemia.

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