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MEMBRANE REMODELING DURING PATHOGENESISAND THE MECHANISM OF EXOCYTOSIS
     
Joshua Zimmerberg, Ph.D., M.D., Head, Section on Membrane and Cellular Biophysics
Paul Blank, Ph.D., Staff Scientist
Svetlana Glushakova, Ph.D., Staff Scientist
Gorka Basanez, Ph.D., Research Fellow
Subrata Biswas, Ph.D., Postdoctoral Fellow
Mukesh Kumar, Ph.D., Postdoctoral Fellow
Tao Li, Ph.D., Postdoctoral Fellow
Amrisha Verma, Ph.D., Postdoctoral Fellow
Anil Verma, Ph.D., Postdoctoral Fellow
Jane Farrington, Technician
Ludmila Bezrukov, M.S., Guest Researcher
Alex Chanturiya, Ph.D., Guest Researcher
Vadim Frolov, Ph.D., Guest Researcher
Glen Humphrey, Ph.D., Guest Researcher
Tim Whalley, Ph.D., Guest Researcher
Shurong Yin, M.D., Guest Researcher
Cory Alfers, Postbaccalaureate Fellow
Andrew Babcock, Postbaccalaureate Fellow
Brian Bradow, Postbaccalaureate Fellow
Jennifer Galanis, Predoctoral Fellow
Samuel Hess, Predoctoral Fellow
Dan Yin, Postbaccalaureate Fellow
Joshua Zimmerberg
 
Membrane fusion is a critical for membrane assembly, mitosis, Golgi trafficking, secretion, enveloped viral infection, insulin release, histamine release, and fertilization while membrane fission allows for endocytosis and parasite entry. We attempt to understand the functional pathway of membrane events at the level of physical forces in order to assign roles to putative proteins, mutants, and second messengers. We have detected an intermediate providing ionic continuity between the two aqueous spaces separated before membrane fusion, the fusion pore, which we have continued to study with electrophysiological, molecular, and cellular techniques. We have extended the new methods developed in the study of the fusion pore to the measurement of a fission pore as the object through which parasites, in particular P. falciparum, infect cells. We have also discovered a profound and potent destabilization of lipid membranes by protein molecules that promote apoptosis. In addition, we focus on the mechanisms of exocytosis, specifically the cortical fusion of secretory vesicles of the sea urchin egg. By combining physiological and molecular analyses with a variety of purified membrane isolates containing secretory vesicles that fuse to the plasma membrane or to each other, we are characterizing the final steps of calcium-triggered exocytosis.

Molecules Mediating Apoptosis Make Lipidic Pores
Basanez, Chanturiya, Galanis; in collaborating with Sharpe, Hardwick
During apoptotic cell death, cells usually release apoptogenic proteins such as cytochrome c from the mitochondrial intermembrane space. During apoptosis, Bax-type proteins cause permeabiliza-tion of the outer mitochondrial membrane to release mitochondrial apoptogenic factors into the cytosol via a poorly understood mechanism. We have proposed that Bax and Delta-N76BclxL (the Bax-like cleavage fragment of Bcl-xL) function by forming pores that are at least partially composed of lipids (lipidic pore formation). Given that the membrane monolayer must bend during lipidic pore formation, we explored the effect of intrinsic membrane monolayer curvature on pore formation. Nonlamellar lipids with positive intrinsic curvature such as lysophospholipids promoted membrane permeabilization, whereas nonla-mellar lipids with negative intrinsic curvature such as diacylglycerol and phosphatidylethanol-amine inhibited membrane permeabilization. The differential effects of nonlamellar lipids on membrane permeabilization did not correlate with lipid-induced changes in membrane binding or insertion of Bax or Delta-N76Bcl-xL. Taken together, these results are consistent with (but do not prove) the notion that Bax-type proteins change the bending propensity of the membrane to form pores consisting at least in part of lipids in a structure of net positive monolayer curvature.

New Method for Culturing Plasmodium falciparum, the Causative Agent of Malaria
Li, Glushakova, Chanturiya, Yin
While the traditional method of culturing Plasmodium falciparum has been highly successful, plasmodia replicate poorly in erythrocyte densities with a hematocrit greater than 20 percent. We developed a new method of culturing the major malaria parasite by using a hollow fiber bioreactor that preserves healthy erythrocytes at a hematocrit of up to 100 percent. Plasmodium falciparum replicated equally well at all densities studied. The method proved advantageous for large-scale preparation of parasitized erythrocytes (and potentially immunogens thereof) in that high yields (about 1010 in four days, about 1011 in a week) could be prepared with less cost and labor than conventional methods. Concomitantly, secreted proteins are concentrated by molecular sieving during culture, perhaps contributing to the parasitemic limit of 8 to 12 percent with 3D7 strain. Our finding that Plasmodium falciparum can replicate at packed densities suggests that the method may be useful for studying the pathogenesis of the fatal disease cerebral malaria, which features densely packed blood cells in brain microvasculature.

Mechanism of Influenza Virus Entry during Infection
Frolov, Kumar, Verma, Biswas, Alfers, Babcock, Bradow, Farrington, Yin; in collaboration with Reese
All enveloped viruses inject their genome into the cytoplasm by fusing their envelope membrane with that of the host. In cases such as influenza virus infection, the fusion follows endocytic uptake of a virus particle into a cellular vesicle, which then transports the viral particle into the cell. The size, shapes, and membrane composition of the vesicle affect viral fusion efficiency. Our experimental strategy is to approach vesicle formation and transport and then fusion as sequential stages of a single process. We developed high-resolution admittance techniques in combination with low light–level fluorescent microscopy to detect the formation of individual cellular vesicles and the pinching off of such vesicles into the cellular interior. We resolved the evolution, from initial budding to pinching off, of single vesicles capable of carrying individual influenza virus particles. Determination of the lipid composition of such vesicles and the detection of the transport of actual virus particles in the vesicles are in progress. By developing a system to monitor the fusion of a single virus particle with a model membrane (with a controlled composition), we were able to resolve individual fusion events following viral particle uptake. Our experiments revealed that acidification of the virus interior (normally going through the M2 channel in the viral membrane) is crucial for the fusion pore expansion and thus for the RNA release and ultimate infection. The antivirus drugs amantadine and remantadine, known inhibitors of the M2 channel, do not affect fusion pore formation by the viral fusion protein hemagglutinin, but they do prevent fusion pore expansion. Thus, a further pH-sensitive viral protein besides the classical fusion protein hemagglutinin is involved in the completion of fusion.

Kinetics and Calcium Dynamics of Exocytosis
Blank, Hess; in collaboration with Rahamimoff
We are investigating the mechanisms of calcium-triggered exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents. Although the relationship between exocytosis and calcium is fundamental both to synaptic and nonneuronal secretory function, analysis is problematic because of the temporal and spatial properties of calcium and the fact that vesicle transport, priming, retrieval, and recycling are coupled. By analyzing the kinetics of sea urchin egg secretory vesicle exocytosis in vitro, we can resolve the final steps of exocytosis. These steps are modeled as a three-state system: activated, committed, and fused, in which interstate transitions are determined by the probabilities that an active fusion complex commits (alpha) and that a committed fusion complex results in fusion, p. The number of committed complexes per vesicle docking site is a Poisson distribution with mean n-bar. Experimentally, p and n-bar increase with increasing calcium concentration, whereas alpha and the p/n-bar ratio remain constant, reducing the kinetic description to only one calcium-dependent, controlling variable, n-bar. On average, the calcium dependence of n-bar describes the calcium dependence of the maximum rate (Rmax) and the time to reach Rmax (Tpeak). Thus, the nonlinear relationship between the free calcium concentration and the rate of exocytosis can be explained solely by the calcium dependence of the distribution of fusion complexes at vesicle docking sites.

Biochemistry and Components of Exocytosis
Bezrukov, Backlund, Blank, Humphrey, Verma, Whalley; in collaboration with Yergey, Coorssen
Although Western blotting is widely used for the detection of specific proteins, it is often thought to be an inadequate technique for accurate measurements of protein concentration. In fact, the analysis of calcium-triggered exocytosis requires an unambiguous identification and quantitative assessment of the membrane surface density of specific molecules. Newly refined immunoblotting and analysis approaches permit a quantitative analysis of the SNARE protein complement (VAMP, SNAP-25, and syntaxin) of functional secretory vesicles. The method illustrates the feasibility of the routine quantification of femtomole to attomole amounts of known proteins by immunoblotting. The results indicate that sea urchin egg secretory vesicles and synaptic vesicles have a marked similarity in SNARE densities.

Membrane Fusion and Fission: Theoretical Considerations
Zimmerberg, Frolov; in collaboration with Chizmadzhev, Kuzmin, Huttner
While continuing our theoretical work on membrane fusion during exocytosis, we extended it to the processes of membrane fission, which is essential to endocytosis. We study the ways that physical forces and cell membrane inhomogeneities are coordinated to allow controlled and organized vesiculation in the general vacuolar system of cells, the release of infectious viral particles, and the internalization of membrane-bound material. The biochemical and biophysical mechanisms of membrane remodeling are dependent on the composition of a small piece of membrane that rolls up into a new biological entity. Consideration of the structure of these lipid microdomains is important to understanding the mechanisms of membrane budding and fission.

Many investigators believe that microdomains of ordered lipids exist in both leaflets of a lipid bilayer, explaining the effects of lipid composition on cytoplasmic leaflet signalling. Moreover, the coordination of these two leaflets into a “bilayer raft” is an attractive, albeit unproven, idea. If one accepts that the inner and outer leaflets of a microdomain can indepen-dently assemble or disassemble and that these more ordered structures have less hydrocarbon chain dispersity, then microdomains should be higher in density than non-raft areas. Thus, the area occupied by each phospholipid head group will decrease in such areas. If one leaflet exists as a raft while the other does not, then the difference in areas would cause the membrane to curve as long as there is no flip-flop of membrane compartments to relieve the asymmetry of area and as long as lipid diffusion is restricted by molecular fences. Thus, control of monolayer order could regulate membrane bending, leading to the provocative suggestion that transmembrane signalling may proceed through monolayer ordering. In a non-ordered membrane, ordering one leaflet may lead to the ordering of the trans leaflet through the same forces that would stabilize bilayer rafts. The cis leaflet order conveys information, and it can cause protein aggregation onto the newly ordered trans leaflet. For example, polymerization of specialized protein domains that bind specifically to the phosphoinositol biphosphate (PIP2) of the inner leaflet raft lipid can lead to PIP2 aggregation, which may order the internal leaflet and in turn order the outer leaflet, perhaps then aggregating extracellular (or lumenal) domains. In this way, proteins on clathrin that bind to PIP2 may cause cargo assembly into a nascent bud. It is thus possible to build a mechanism for trans-bilayer signalling that need not involve protein transmembrane domains.

If microdomains are sitting on either side of a neck, they are effectively in a ring topology; that is, if a vesicle buds out of a membrane from a large domain (a 150-nm-diameter raft has more than twice the area of a 50-nm vesicle), then the donor membrane is left with the hitherto unconsidered geometry of a ring. In other words, dynamin, which resides outside the bilayer, would have a counterpart in the bilayer, a “ring raft.” The ring could facilitate fission, as discussed above. In addition, given that rafts must adapt their three-dimensional geometry to the needs of the dynamic situation and change, for example, from tubes to cups to spheres and so forth, then it is important to consider the differential effects of curvature and geometry on ordered and disordered membrane domains. Ring rafts can also play an important role as barriers to lipid diffusion, thus facilitating fission by reducing the number of lipid molecules involved in the fission reaction. Ultimately, lipid microdomain topology must be regulated and organized by the underlying cellular architecture and organizing principles.

 
 

PUBLICATIONS

  1. Basanez G, Zhang J, Chau BN, Maksaev GI, Frolov VA, Brandt TA, Burch J, Hardwick JM, Zimmerberg J. Pro-apoptotic cleavage products of Bcl-xL form cytochrome c-conducting pores in pure lipid membranes. J Biol Chem. 2001;276:31083-31091.
  2. Basanez G, Zimmerberg J. HIV and apoptosis death and the mitochondrion. J Exp Med. 2001;193:F11-14.
  3. Blank PS, Vogel SS, Malley JD, Zimmerberg J. A kinetic analysis of calcium-triggered exocytosis. J Gen Physiol. 2001;118:145-156.
  4. Chizmadzhev YA, Kuzmin PI, Kumenko DA, Zimmerberg J, Cohen FS. Dynamics of fusion pores connecting membranes of different tensions. Biophys J. 2000;78:2241-2256.
  5. Coorssen JR, Blank PS, Albertorio F, Bezrukov L, Kolosova I, Backlund PS, Zimmerberg J. Quantitative femto- to attomole immunodetection of regulated secretory vesicle proteins critical to exocytosis. Anal Biochem. 2002;307:54-62.
  6. Desai SA, Bezrukov SM, Zimmerberg J. A voltage-dependent channel involved in nutrient uptake by red blood cells infected with the malaria parasite. Nature. 2000;406:1001-1005.
  7. Frolov VA, Cho MS, Bronk P, Reese TS, Zimmerberg J. Multiple local contact sites are induced by GPI-linked influenza hemagglutinin during hemifusion and flickering pore formation. Traffic. 2000;1:622-630.
  8. Greengard O, Poltoratskaia N, Leikina E, Zimmerberg J, Moscona A. The anti-influenza virus agent 4GU-DANA (zanamivir) inhibits cell fusion mediated by human parainfluenza virus and influenza virus HA. J Virol. 2000;74:11108-11114.
  9. Huttner WB, Zimmerberg J. Implications of lipid microdomains for membrane curvature, budding and fission. Curr Opin Cell Biol. 2001;13:478-484.
  10. Kim KM, Giedt CD, Basanez G, O’Neill JW, Hill JJ, Han YH, Tzung SP, Zimmerberg J, Hockenbery DM, Zhang KY. Biophysical characterization of recombinant human Bcl-2 and its interactions with an inhibitory ligand, antimycin A. Biochemistry. 2001;40:4911-4922.
  11. Kuzmin PI, Zimmerberg J, Chizmadzhev YA, Cohen FS. A quantitative model for membrane fusion based on low-energy intermediates. Proc Natl Acad Sci USA. 2001;98:7235-7240.
  12. Markovic I, Leikina E, Zhukovsky M, Zimmerberg J, Chernomordik L. Synchronized activation and unfolding of influenza virus hemagglutinins in multimeric fusion machine. J. Cell Biol. 2001;155:833-844.
  13. Tzung SP, Kim KM, Basanez G, Giedt CD, Simon J, Zimmerberg J, Zhang KY, Hockenbery DM. Antimycin A mimics a cell-death-inducing Bcl-2 homology domain 3. Nat Cell Biol. 2001;3:183-191.
  14. Yergey AL, Coorssen JR, Backlund PS, Blank PS, Humphrey GA, Zimmerberg J, Campbell JM, Vestal ML. De novo sequencing of peptides using MALDI/TOF-TOF. J Am Soc Mass Spectrom. 2002;13:784-791.
  15. Zimmerberg J. Are the curves in all the right places? Traffic. 2000;1:366-368.
  16. Zimmerberg J. How can proteolipids be central players in membrane fusion? Trends Cell Biol. 2001;11:233-235.
  17. Zimmerberg J, Blank PS, Kolosova I, Cho MS, Tahara M, Coorssen JR. A stage-specific preparation to study the Ca(2+)-triggered fusion steps of exocytosis: rationale and perspectives. Biochimie. 2000;82:303-314.

Collaborators
Peter Backlund, Ph.D., Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda, MD
Yuri Chizmadzhev, Ph.D., Frumkin Institute of Electrochemistry, Moscow, Russia
Jens Coorssen, Ph.D., University of Calgary, Calgary, Canada
Marie J. Hardwick, Ph.D., Johns Hopkins University, Baltimore, MD
Wieland B. Huttner, Ph.D., Max-Planck-Institut für Molekulare Zellbiologie und Genetik, Dresden, Germany
Peter Kuzmin, Ph.D., Frumkin Institute of Electrochemistry, Moscow, Russia
Rami Rahamimoff, M.D., University of Jerusalem, Jerusalem, Israel
Thomas S. Reese, M.D., Laboratory of Neurobiology, NINDS, Bethesda, MD
Juanita Sharpe, Ph.D., Surgical Neurology Branch, NINDS, Bethesda, MD
Al Yergey, Ph.D., Laboratory of Cellular and Molecular Biophysics, NICHD, Bethesda, MD