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a membrane
remodeling during viral infection,
parasite
invasion, and apoptosis and components and
kinetics in
exocytosis
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Joshua Zimmerberg, MD, PhD, Head, Section on Cellular and Membrane Biophysics Paul S. Blank, PhD, Staff Scientist Subrata
Biswas, PhD, Visiting Fellow Mukesh
Kumar, PhD, Visiting Fellow Samuel Hess, PhD, Postdoctoral Fellow Victoria Chang, AB, Postbaccalaureate Fellow Ivan V. Polozov, BS,
Postbaccalaureate Fellow Dan Yin, BA, Postbaccalaureate Fellow Laura Bakas, PhD, Guest Researcher Alexander Chanturiya,
PhD, Guest Researcher Vadim A. Frolov, PhD, Guest Researcher Glen Humphrey, PhD, Guest Researcher Shu-Rong Yin,
MD, Guest Researcher Elena M. Kapnik, MS, Biologist Vladimir A. Lizunov, MS, Student |
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In an effort to understand viral and parasite
infection, exocytosis, and apoptosis, we study membrane mechanics,
intracellular molecules, membranes, viruses, organelles, and cells. To this
end, we have organized various techniques and approaches into an interdisciplinary
attack on the mechanisms of membrane remodeling, using the physics of
continuum bilayers and direct observations of biological fusion, analytic and
numerical calculations of membrane energetics, and experiments on
phospholipid bilayers, purified proteins, cell expression systems, purified
organelles, cell surface complexes, and the actual physiological and
pathogenic events of fertilization and viral infection. In a collaboration
over the past year, we discovered that a novel frame-shifted protein encoded
by the influenza genome causes the same apoptotic
pores as those formed by Bax and truncated Bcl and Bid, that direct intra-preterminal membrane
recordings in the squid giant synapse show a different type of channel, that
molecular shape determines solid amphiphile
macroscopic structure from nanodiscs to punctuated icosohedra, and that the N-ethyl maleimide-sensitive
fusion protein NSF is not required for the fusion of purified secretory
granules to each other. We also discovered why membrane bending is predicted
to lower the energy for protein insertion into membranes. We have begun to
investigate the role of membrane microdomains in
membrane fusion. Apoptotic
pores in planar lipid bilayers caused by a new pro-death protein encoded by a
frame-shifted influenza genome Chanturiya, Bakas,
Karpunin; in collaboration with Yewdell
The apoptotic pore A
frame-shifted region of the influenza A virus PB1 gene encodes a novel
mitochondrial protein, termed PB1-F2, that can induce cell death. Many pro-apoptotic proteins are believed to act at the
mitochondrial outer membrane to form an apoptotic
pore with lipids. We studied the interaction of isolated, synthetic PB1-F2
peptide with planar phospholipid bilayer membranes. The presence of nanomolar concentrations of peptide in the bathing
solution induced a transmembrane conductance that increased in a
potential-dependent manner. Positive potential on the side of protein
addition resulted in a several-fold increase in the rate of change of
membrane conductance. Membranes treated with sPB1-F2 became permeable to
monovalent cations, chloride, and, to a lesser extent, divalent ions. Despite
varying experimental conditions, we failed to detect distinctive conductance
levels typical of large, stable pores, protein channels, or even partially
proteinaceous pores. Rather, membrane conductance induced by sPB1-F2
fluctuated and visited almost all conductance values. Consistent with a
decrease in the line tension of a lipidic pore, sPB1-F2 also dramatically
decreased bilayer stability in an electric field. Given that we see similar
membrane-destabilizing profiles with pro-apoptotic proteins (e.g., Bax)
and the cytoplasmic helix of HIV gp41, we suggest that the basis for
sPB1-F2-induced cell death may be the permeabilization
and destabilization of mitochondrial membranes,
leading to macromolecular leakage and apoptosis. Chanturiya AN, Basaez G, Schubert U, Henklein
P, Yewdell JW, Zimmerberg
J. PB1-F2, an influenza A virus-encoded proapoptotic mitochondrial protein,
creates variably sized pores in planar lipid membranes. J Virol 2004;78:6304-6312. Membrane pores in the presynaptic terminal of
the squid giant synapse Zimmerberg; in collaboration
with Hardwick, Jonas, Kaczmarek Neuronal death is often preceded by functional
alterations at nerve terminals. The BCL-2 family protein BCL-xL is an anti-apoptotic protein
found in the adult nervous system, including the giant presynaptic terminal
of the squid stellate ganglion, where it is known to form ion channels in the
outer mitochondrial membrane. The protein’s
naturally occurring N-truncated protease cleavage product delta-N BCL-xL is pro-apoptotic.
Application of delta-N Bcl-xL to mitochondria
inside the squid presynaptic terminal rapidly induced the opening of large multiconductance channels in mitochondrial membranes. The
maximal conductance of the channels was significantly larger than that of
control recordings or that obtained with full-length BCL-xL.
Mutants of delta-N (DN) Bcl-xL lacking a C-terminal
mitochondrial anchor region, or in which the death-producing BH3 domain was
mutated, failed to induce channel activity. Moreover,
delta-N Bcl-xL failed to produce channel activity
when applied to plasma membranes, suggesting that a component of
mitochondrial membranes is necessary for its actions. NADH, an inhibitor of
the outer mitochondrial membrane voltage-dependent anionic channel (VDAC),
reduced the occurrence of the large conductance openings. Pharmacological and
genetic experiments on yeast mitochondria lacking VDAC indicated that the
NADH-sensitive DN Bcl-xL-induced channel activity
results from a functional interaction between DN Bcl-xL
and VDAC. Furthermore, exposure of squid ganglia to hypoxia, a death stimulus
to neurons, rapidly induced proteolysis of full-length BCL-xL, which was blocked by the protease inhibitor zVAD, suggesting that proteolysis of BCL-2 family
proteins may contribute to the induction of large conductance activity in the
outer mitochondrial membrane. Jonas EA, Hickman JA, Chachar
M, Polster BM, Brandt TA, Fannjiang Y, Ivanovska I, Basaez G, Kinnally KW, Zimmerberg J, Hardwick JM, Kaczmarek LK.
Proapoptotic N-truncated BCL-xL protein activates
endogenous mitochondrial channels in living synaptic terminals. Proc Natl Acad Sci USA 2004;101:13590-13595. A general scheme for shape control of
surfactants Lizuno; in collaboration with Dubois, Kuzmin, Zemb When crystallized at
various ratios in the absence of added salt, mixtures of cationic and anionic
surfactants form micron-sized colloids. We proposed and tested a general
mechanism to explain how the ration controls the shape of the resulting
colloidal structure, which can vary from nanodiscs
to punctured planes. During co-crystallization, excess (nonstoichiometric)
surfactant accumulates on edges or pores rather than being incorporated into
crystalline bilayers; molecular segregation then produces a sequence of
shapes controlled solely by the initial mole ratio. Using freeze-fracture
electron microscopy, we identified three of these states and their
corresponding coexistence regimes. Fluorescence confocal microscopy directly
showed the segregation of cationic and anionic components within the
aggregate. The observed shapes were consistently reproduced upon thermal
cycling, demonstrating that the icosahedral shape corresponds to the
existence of a local minimum of bending energy for facetted icoshedra when the optimal amount of excess segregated
material is present. Dubois
M, Lizunov VA, Meister A,
Gulik-Krzywicki T, Verbavatz J, Perez E, Zimmerberg J, Zemb T. Shape
control through molecular segregation in giant surfactant aggregates. Proc Natl Acad Sci USA 2004; 101:15082-15087. Modeling membrane deformations upon protein
insertion Frolov; in collaboration with Chizmadzhev, Dunina-Barkovskaya, Kozlov The effects of calcium and the ensuing fusion
depend on protein moieties embedded into the lipid bilayer. To model this
aspect of exocytosis, we considered the elastic behavior of a flat lipid
monolayer embedding cylindrical inclusions oriented obliquely with respect to
the monolayer plane. An oblique inclusion models a fusion peptide, a part of
a specialized protein capable of inducing merger of biological membranes in
the course of fundamental cellular processes. Although the crucial importance
of the fusion peptides for membrane merger is well established, the molecular
mechanism of their action remains unknown. Our analysis aimed at revealing
mechanical deformations and stresses of lipid monolayers induced by the
fusion peptides, which can potentially destabilize
the monolayer structure and enhance membrane fusion. We calculated the
deformation of a monolayer embedding a single oblique inclusion and subject
to a lateral tension and analyzed the membrane-mediated interactions between
two inclusions, taking into account bending of the monolayer and tilt of the
hydrocarbon chains with respect to a line drawn perpendicular to the surface.
In contrast to a straightforward prediction that the oblique inclusions should
induce tilt of the lipid chains, our analysis showed that the monolayer
accommodates the oblique inclusion solely by bending. We find that the
interaction between two inclusions varied nonmonotonically
with the interinclusion distance and decayed at large
separations as the square of their distance, similar to the electrostatic
interaction between two electric dipoles in two dimensions. We predict that
this long-range interaction will dominate the other interactions previously
considered in the literature. Frolov VA, Lizunov VA, Dunina-Barkovskaya AY, Samsonov
AV, Zimmerberg J. Shape bistability
of a membrane neck: a toggle switch to control vesicle content release. Proc
Natl Acad Sci USA
2003;100:8698-8703. Kozlovsky Y, Zimmerberg J, Kozlov MM.
Orientation and interaction of oblique cylindrical inclusions embedded in a
lipid monolayer: a theoretical model for viral fusion peptides. Biophys J 2004;87:999-1012. Zimmerberg J,
McLaughlin S. Membrane curvature: how BAR domains bend bilayers. Curr Biol 2004;14:R250-252.
Role of the N-ethylmaleimide-sensitive fusion
protein in egg exocytosis Bezrukov, Blank, Humphrey; in
collaboration with Rahamimoff, Whalley The role of cytosolic ATPases
such as N-ethylmaleimide (NEM)–sensitive fusion
protein (NSF) in membrane fusion is controversial. We examined the physiology
and biochemistry of ATP and NSF in the cortical system of the echinoderm egg
to determine if NSF is an essential factor in membrane fusion during Ca2+-triggered
exocytosis. Neither exocytosis in vitro nor homotypic cortical vesicle
(CV) fusion required soluble proteins or nucleotides, and both occurred in
the presence of nonhydrolyzable analogs of ATP.
While sensitive to thiol-specific reagents, CV exocytosis is not restored by
the addition of cytosolic NSF, and fusion and NSF function are differentially
sensitive to thiol-specific agents. To test participation of tightly bound,
nonexchangeable NSF in CV-CV fusion, we cloned the sea urchin homolog and
developed a species-specific antibody for Western blots and physiological
analysis. Despite being functionally inhibitory, the antibody had no effect
on CV exocytosis or homotypic fusion. NSF is detectable in intact cortices,
cortices from which CVs had been removed, and in
isolated CVs treated with ATP-gamma-S and egg
cytosol to reveal NSF binding sites. In contrast, isolated CVs, though all capable of Ca2+-triggered
homotypic fusion, contain less than one hexamer of NSF per CV. Thus, NSF is
not a required component of the CV fusion machinery. Coorssen JR,
Blank PS, Albertorio F, Bezrukov
L, Kolosova I, Chen X, Backlund PS, Zimmerberg J.
Regulated secretion SNARE density, vesicle fusion and calcium dependence. J
Cell Sci 2003;116:2087-2097. Whalley T, Timmers K, Coorssen J, Bezrukov L, Kingsley DH, Zimmerberg
J. Membrane fusion of secretory vesicles of the sea urchin egg in the absence
of NSF. J Cell Sci 2004;117:2345-2356. Membrane microdomains
in viral fusion Blank,
Chang, Cherif, Hess, Kapnik,
Kumar, Polozov, Yin We have embarked on a new subproject
to investigate the role of membrane microdomains in
the fusion of membranes critical to syncytium formation and infection by the
influenza virus. We find that, by dint of its transmembrane domain, the virus
selects and concentrates microdomain lipids for
both viral assembly and viral fusion. From the perspective of both physical
chemistry and cell membrane biology, we are investigating various hypotheses
that posit the reason behind formation of the domain. COLLABORATORS Laura
Bakas, PhD, Instituto de
Investigaciones Bioquímicas La Plata (INIBIOLP), La Plata, Argentina Yuri Chizmadzhev, PhD, Frumkin
Institute of Electrochemistry, Russian Monique
Dubois, PhD, Service de Chimie Moléculaire, CEA
Saclay, Gif-sur-Yvette, France Antonina
Dunina-Barkovskaya, PhD, Marie J. Hardwick,
PhD, The Elizabeth Jonas, PhD, Len Kaczmarek, PhD, Michael Kozlov,
PhD, Peter Kuzmin,
MS, Frumkin Institute of
Electrochemistry, Russian Rami Timothy Whalley,
PhD, Jonathan Yewdell,
MD, PhD, Laboratory of Viral Diseases, NIAID, Thomas
Zemb, PhD, Service de
Chimie Moléculaire, CEA Saclay, Gif-sur-Yvette, France For
further information, contact joshz@helix.nih.gov |
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