Joshua Zimmerberg, MD, PhD, Head, Section on Cellular and Membrane Biophysics
Paul S. Blank, PhD, Staff Scientist
Svetlana Glushakova, PhD, Staff Scientist
Vadim A. Frolov, PhD, Senior Research Fellow
Pavel Bashkirov, MS, Guest Researcher
Alexander Chanturiya, PhD, Guest Researcher
Jane E. Farrington, MS, Guest Researcher
Glen Humphrey, PhD, Guest Researcher
Dimitry Karpunin, MS, Guest Researcher
Shu-Rong Yin, PhD, Guest Researcher
Andrea Fera, PhD, Postdoctoral Fellow
Vladimir A. Lizunov, MS, Visiting Fellow
Julia Mazar, PhD, Visiting Fellow
Veena Pata, PhD, Visiting Fellow
Gulcin Pekkurnaz, MS, Predoctoral Fellow
Anna Shnyrova, MS, Predoctoral Fellow
Nicole M. Gartner, BA, Postbaccalaureate Fellow
Molly Fisher Thomas, MS, Postbaccalaureate Fellow
Ludmila Bezrukov, MS, Contractor
Elena M. Kapnik, MS, Biologist

We study membranes, membrane mechanics, intracellular molecules, viruses, organelles, and cells in order to understand viral and parasite infection, exocytosis, and apoptosis. We have organized an interdisciplinary attack on the mechanisms of membrane remodeling with varied techniques and approaches, using the physics of continuum bilayers and direct observations of biological fusion, analytical and numerical calculations of membrane energetics, and experiments on phospholipid bilayers, purified proteins, cell expression systems, purified organelles, cell surface complexes, and the physiological and pathogenic event of fertilization, viral infection, malaria, and diabetes. During the past year we (1) discovered that, unlike other bacterial toxins, the toxin causing Escherichia coli disease is highly unusual in its lipid dependence; (2) proposed a structure of gel-phase lipids in cell membranes; (3) developed a general formalism for how proteins can form shape on biological membranes; (4) proposed a new physical model for the exocytosis of synaptic vesicles at the synapse; and (5) performed a quantitative analysis of the nucleation and growth of membrane microdomains.

Paradoxical lipid dependence of pores formed by the Escherichia coli alpha-hemolysin

Chanturiya, Glushakova, Gartner, Mazar, Thomas, Yin; in collaboration with Bakas

The recent outbreak of E. coli toxicosis due to poorly packaged spinach has underscored the need to understand the mechanism of the toxicosis. A strain of E. coli secretes the extracellular protein toxin (117 KDa) alpha-hemolysin (HlyA), which targets the plasma membranes of eukaryotic cells. Using planar phospholipid bilayers, we studied the interaction of this toxin with membranes. For all lipid mixtures tested, the addition of nanomolar concentrations of toxin resulted in an increase in membrane conductance and a decrease in membrane stability. HlyA decreased membrane life time by up to three orders of magnitude in a voltage-dependent manner. Using a theory for lipidic pore formation, we analyzed the data in order to quantify the reduction in line tension of the membrane by HlyA (i.e., the energy required to form the edge of a new pore). However, in contrast to the expectation that adding the positive curvature agent lysophosphatidylcholine would synergistically lower line tension, we discovered that its addition significantly stabilized HlyA-treated membranes. HlyA also appeared to thicken bilayers to which it was added. The results led to new considerations for existing models for proteo-lipidic pores and prompted us to propose a new model for such pores.

A synthesis of membrane biophysics with a new proposal for gel phase lipids in cellular membranes

Blank, Bezrukov, Farrington, Fera, Kapnik, Pata; in collaboration with Hess

In physics, we usually strive to simplify the behavior of a system to fit it to a formula. However, in biology, all details are important. Both organization and non-repeating polymers are essential for life. Thus, the organization of proteins in the plasma membrane must be an essential part of the success of life. Consistent with fact that about a third of the dry weight of a cell is membrane, roughly half of all proteins in a eukaryotic genome are membrane proteins. Thus, roughly half of biological processes occur on membranes, and aspects of each of these processes fall into the realm of physics. It is not surprising that membrane biophysics comprises roughly half the topics for posters and talks at meetings dedicated to all biophysics. Just as water is the solvent for soluble proteins, the phospholipid bilayer membrane is the solvent for membrane proteins and the basis of the biological membrane. As a semi-crystalline array that is ordered in some aspects and disordered in other aspects, a membrane has the characteristics of both a fluid and a solid. It is only two molecules in thickness but can be square millimeters in area (e.g., eggs) or yield cylinders meters in length (e.g., axons in giraffes). Our closest everyday experience with thin films comes during play with soap bubbles, which are made of thin films of water lined by detergents. Fortunately, the phospholipid bilayer itself is stable for many lipid compositions, and bilayers self-assemble upon sufficient hydration of these lipids. Therefore, the bilayer's properties and self-interactions can be extensively studied without proteins in vitro. Our understanding of the physical nature of the membrane backbone comes mostly from spectroscopic, microscopic, and electrophysiological studies of bilayers. In a study of the viral hemagglutinin (HA) of membrane microdomains, we discovered non-circular domains, strongly suggesting that the proteins in these domains are not fluid and that the domains represent areas of gel-phase lipids.

A general formalism for shape determination on biological membranes

Frolov, Bashkirov, Pekkurnaz, Karpunin; in collaboration with Kozlov

We are interested in the mechanisms whereby proteins produce biological shape. Biological membranes exhibit various function-related shapes, and the mechanism by which these shapes are created remains largely unclear. We have classified possible curvature-generating mechanisms that are supplied by lipids that constitute the membrane bilayer and by proteins that interact with, or are embedded in, the membrane. We have described membrane elastic properties in order to formulate the structural and energetic requirements of proteins and lipids that would enable them to work together to generate the membrane shapes seen during intracellular trafficking.

A new mechanism for synaptic vesicle release at the synapse: rings of proteins pulling stalks into pores

Frolov, Akimov, Lizunov, Shnyrova, Humphrey; in collaboration with Cushman, Rahamimoff, Reese

One of the abiding mysteries in biology is the great speed of synaptic transmission, where synaptic vesicles laden with neurotransmitter fuse to the presynaptic membrane and release their content to the synaptic cleft. The Ca2+-triggered release of neurotransmitter begins some tens of microseconds after Ca2+ floods the presynaptic intracellular release site. Thus, the mechanism of membrane fusion must account for how Ca2+ triggers the extremely fast formation of a fusion pore linking vesicular and plasma membranes that were hitherto stable and non-leaky. In the current paradigm for exocytotic fusion, the trans-SNARE complex, composed of proteins localized in vesicular and plasma membranes, is the minimal fusion machine. Calcium dependence of fusion is believed to be regulated by proteins such as synaptotagmin 1, which acts as both a "calcium sensor," mediating Ca2+-triggering, and a regulator of fusion pore dynamics during neurotransmitter release. However, synaptotagmin 1 may have a more central role in mediating fast synaptic fusion.

We propose that ring assemblies of SNAREs and synaptotagmin complexes form to concentrate and orient C2b domains of synaptotagmin appropriately. The ordered domains then create an electrostatic tunnel for membrane fusion that is extended by the polybasic linker regions of syntaxin and synaptobrevin. As to the role of calcium, Ca2+ first turns on an "electrostatic switch" initially proposed for synaptotagmin-syntaxin interaction, but better suited for instantaneously stressing the phospholipid bilayers of the presynaptic membrane and synaptic vesicle, resulting in the ultra-rapid exocytosis seen in the nervous system. Second, even without synaptotagmin, Ca2+ considerably speeds up fusion of SNARE-reconstituted membranes. Perhaps Ca2+ plays a direct role, electrostatically complexing phosphatidylserine headgroups to promote fusion between negatively charged phospholipid bilayers.

Ultimately, synaptotagmin, SNAREs, and the other proteins that constitute the exocytotic fusion machine must cajole lipids to move through a pathway that culminates in fusion pore opening. Our view is that exocytotic fusion follows the pathway of phospholipid membrane fusion. The role of proteins along this pathway is to lower the several energy barriers to membrane fusion, just as enzymes lower the energy barriers to their respective reactions. Given that the reaction coordinate for membrane fusion is the radius of the stalk, pore proteins controlling radial forces should regulate forward and backward passage through the pathway towards complete fusion. The SNARE proteins and synaptotagmin are the guides that walk and pull the membrane through a bumpy stalk-pore path, with electrostatic interactions playing a larger role than hitherto realized.

Entropic traps in the kinetics of phase separation in multicomponent membranes stabilize nanodomains

Zimmerberg; in collaboration with Akimov, Chizmadzhev, Cohen, Kuzmin

We describe quantitatively the creation and evolution of phase-separated domains in a multicomponent lipid bilayer membrane. The early stages, termed the nucleation stage and the independent growth stage, occur at an extremely rapid pace (characteristic times are submillisecond and millisecond, respectively), and the system consists of nanodomains of about 5-50 nm average radius. Next, mobility of domains becomes consequential; domain merger and fission become the dominant mechanisms of matter exchange, and line tension is the main determinant of the domain size distribution at any time point. For sufficiently small line tension, the decrease in the entropy term that results from domain merger is larger than the decrease in boundary energy, and only nanodomains are present. For large line tension, the decrease in boundary energy dominates the unfavorable entropy of merger, and merger leads to rapid enlargement of nanodomains to radii of micrometer scale. At intermediate line tensions and within finite times, nanodomains can remain dispersed and coexist with a new global phase. The theoretical critical value of line tension required to form large rafts rapidly is in accord with the experimental estimate from the curvatures of budding domains in giant unilamellar vesicles.

COLLABORATORS

Sergey Akimov, MS, Frumkin Institute of Electrochemistry, Russian Academy of Sciences, Moscow, Russia
Laura Bakas, PhD, Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP), La Plata, Argentina
Yuri Chizmadzhev, PhD, Frumkin Institute of Electrochemistry, Russian Academy of Sciences, Moscow, Russia
Fredric S. Cohen, PhD, Rush University, Medical School, Chicago, IL
Samuel W. Cushman, PhD, _Diabetes Branch, NIDDK, Bethesda, MD
Samuel T. Hess, PhD, University of Maine, Orono, ME_
Michael Kozlov, PhD, School of Medicine, Tel Aviv University, Tel Aviv, Israel
Peter Kuzmin, MS, Frumkin Institute of Electrochemistry, Russian Academy of Sciences, Moscow, Russia
Rami Rahamimoff, MD, Hebrew University of Jerusalem, Jerusalem, Israel
Thomas S. Reese, MD, Laboratory of Neurobiology, NINDS, Bethesda, MD

For further information, contact joshz@helix.nih.gov.