In order to understand viral and parasite infection, exocytosis, and apoptosis, we study membrane mechanics, intracellular molecules, membranes, viruses, organelles, and cells. Our interdisciplinary approach to investigating the mechanisms of membrane remodeling relies on several techniques and approaches that take advantage of the physics of continuum bilayers and direct observations of biological fusion, analytic and numeric calculations of membrane energetics, and experiments on phospholipid bilayers, purified proteins, cell expression systems, purified organelles, cell surface complexes, and the physiological and pathogenic events of fertilization, viral infection, malaria, and diabetes. In the past year, we discovered that the dominant hypothesis for membrane heterogeneity, that of membrane proteins ideally mixing and partitioning into membrane microdomains, does not hold true for proteins of the viruses that allow influenza viruses to infect cells and that the initial physiological function of insulin in adipose cells is to arrest the trafficking of mobile glucose transporter vesicles. We also discovered the pathway of release of the parasite that causes malaria as well as a new physical model for the creation of liquid-ordered membrane microdomains known as rafts.
Determination whether influenza Ha partitions into rafts with ideal mixing
Farrington, Blank, Bezrukov, Biswas, Polozov, Chang; in collaboration with Hess, Lippincott-Schwartz, Reese
Although lipid-dependent protein clustering in biomembranes mediates numerous functions, membrane models demonstrate little consensus as to cluster organization or size. We used influenza viral envelope protein hemagglutinin (HA0) to test the hypothesis that clustering results from proteins partitioning into pre-existing, fluid-ordered “raft” domains, wherein the proteins exhibit a random distribution. Using electron microscopy with immunogold labeling, we visualized Japan HA0 expressed in fibroblasts and examined the expression with fluorescence resonance energy transfer (FRET). Labeled HA coincided with electron-dense, often noncircular membrane patches. Poisson and K-test analyses revealed clustering on accessible length scales (20 to 900 nm). Membrane treatments with methyl-cyclodextrin and glycosphingolipid synthesis inhibitors did not abolish clusters but did alter their pattern, especially at the shortest lengths, as corroborated by changes in FRET efficiency. The density dependence and magnitude of the measured FRET efficiency also indicated a nonrandom distribution on molecular length scales (6 to 7 nm). Our investigations rule out the tested hypothesis for HA over the accessible length scales yet show clearly how its spatial distribution depends on lipid composition.
Hess S, Kumar M, Verma A, Farrington J, Kenworthy A, Zimmerberg J. Quantitative electron microscopy and fluorescence spectroscopy of the membrane distribution of influenza hemagglutinin. J Cell Biol 2005;169:965-976.
Kenworthy AK, Nichols BJ, Remmert CL, Hendrix GM, Kumar M, Zimmerberg J, Lippincott-Schwartz J. Dynamics of putative raft-associated proteins at the cell surface. J Cell Biol 2004;165:735-746.
Membrane transformation during malaria parasite release from human red blood cells
Glushakova, Yin, Kapnik
Three opposing pathways have been proposed for the release of malaria parasites from infected erythrocytes: coordinated rupture of the two membranes surrounding mature parasites, fusion of the erythrocyte and parasitophorus vacuolar membranes (PVM), and liberation of parasites enclosed within the vacuole from the erythrocyte followed by PVM disintegration. Rupture by cell swelling should yield erythrocyte ghosts; membrane fusion would be inhibited by inner-leaflet amphiphiles of positive intrinsic curvature, which contrariwise promote membrane rupture; without protease inhibitors, parasites would leave erythrocytes packed within the vacuole. To distinguish between the possible pathways, we visualized erythrocytes releasing P. falciparum using fluorescence microscopy of differentially labeled membranes. Release did not yield erythrocyte ghosts; positive curvature amphiphiles did not inhibit release but rather promoted it; and release of packed merozoites was shown to be an artifact. Instead, two sequential morphological stages preceded a convulsive rupture of membranes and rapid radial discharge of separated merozoites, leaving segregated internal membrane fragments and plasma membrane vesicles or blebs at the sites of parasite egress. These results, together with the modulation of release by osmotic stress, suggest a pathway of parasite release that features a biochemically altered erythrocyte membrane that folds after pressure-driven rupture of membranes.
Duray PH, Yin SR, Ito Y, Bezrukov L, Cox C, Cho MS, Fitzgerald W, Dorward D, Zimmerberg J, Margolis L. Invasion of human tissue ex vivo by Borrelia burgdorferi. J Infect Dis 2005;191:1747-1754.
Glushakova S, Yin D, Li T, Zimmerberg J. Membrane transformation during malaria parasite release from human red blood cells. Curr Biol 2005;15:1645-1650.
The halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells by insulin
Lizunov, Karpunin, Chanturiya; in collaboration with Cushman, Frolov
Insulin regulates glucose transport in muscle and adipose cells through intracellular redistribution between the cell interior and the plasma membrane (PM) of the glucose transporter 4 (GLUT4). GLUT4 content in the PM of adipose cells is determined by a dynamic equilibrium between its exocytosis and internalization. In basal adipose cells, the content of GLUT4 in the PM remains low (5 percent) as GLUT4 internalizes 10 times faster than it is delivered to the PM. Insulin considerably stimulates the rate of GLUT4 exocytosis with relatively little effect on the rate of internalization. Consequently, 50 percent of intracellular GLUT4 is translocated to the PM upon insulin activation, providing a 10-fold increase in the amount of transporter on the cell surface. GLUT4 is carried to the PM by specialized tubulovesicular compartments (referred to here as GLUT4 vesicles) tightly packed with the transporter.
We applied time-lapse total internal reflection fluorescence microscopy to dissect intermediates of the GLUT4 translocation in rat adipose cells in primary culture. Without insulin, GLUT4 vesicles rapidly moved along a microtubule network covering the entire PM, periodically stopping, most often just briefly, by loosely tethering to the PM. Insulin halted the traffic by tightly tethering vesicles to the PM, where they formed clusters and slowly fused to the PM. The slow release of GLUT4 determined the overall increase of the PM GLUT4. Thus, insulin initially recruits GLUT4 sequestered in mobile vesicles near the PM. It is likely that the primary mechanism of insulin action in GLUT4 translocation is to stimulate tethering and fusion of trafficking vesicles to specific fusion sites in the PM.
In summary, we propose that GLUT4 vesicles follow common pathways of constitutive exocytosis, exploiting microtubule tracks on their way to the PM and revealing constrained release of membrane cargo. However, the probability of tethering and fusion of these vesicles to the PM is specifically sensitive to insulin. Insulin is known to stimulate constitutive exocytosis in general, though to a lesser extent than GLUT4 exocytosis. We are currently investigating molecular mechanisms providing the specificity of insulin action on GLUT4 vesicles.
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.
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.
Lizunov VA, Matsumoto H, Zimmerberg J, Cushman SW, Frolov VA. Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells. J Cell Biol 2005;169:481-489.
Line tension and interaction energies of membrane rafts calculated from lipid splay and tilt
Zimmerberg; in collaboration with Chizmadhev, Cohen, Dubois, Kuzmin, Zemb
Exocytosis, it has been suggested, takes place in certain membrane microdomains. Membrane domains known as rafts are rich in cholesterol and sphingolipids and thought to be thicker than the surrounding membrane. If so, monolayers should elastically deform so as to avoid exposure of hydrophobic surfaces to water at the raft boundary. We calculated the energy of splay and tilt deformations necessary to avoid such hydrophobic exposure. The derived value of energy per unit length, the line tension g, depends on the elastic moduli of the raft and the surrounding membrane. It increases quadratically with the initial difference in thickness between the raft and surround and is reduced by differences, either positive or negative, in spontaneous curvature between the two. For zero spontaneous curvature, g is 1 pN for a monolayer height mismatch of 0.3 nm, in agreement with experimental measurement. Our model reveals conditions that could prevent rafts from forming as well as a mechanism that can cause rafts to remain small. Prevention of raft formation is based on our finding that the calculated line tension is negative if the difference in spontaneous curvature between a raft and its surround is sufficiently large: rafts cannot form if g is less than zero unless molecular interactions (ignored in the model) are strong enough to make the total line tension positive. Control of size is based on our finding that the height profile from raft to surround does not decrease monotonically but rather exhibits a damped, oscillatory behavior. As an important consequence, the calculated energy of interaction between rafts also oscillates as it decreases with distance of separation, creating energy barriers between closely apposed rafts. The height of the primary barrier is a complex function of the spontaneous curvatures of the raft and the surround. The barrier can kinetically stabilize the rafts against merger. Our physical theory thus quantifies conditions that allow rafts to form and moreover defines the parameters that control raft merger.
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.
Kuzmin PI, Akimov SA, Chizmadzhev YA, Zimmerberg J, Cohen FS. Line tension and interaction energies of membrane rafts calculated from lipid splay and tilt. Biophys J 2005;88:1120-1133.
Collaborators
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
Monique Dubois, PhD, CEA-Saclay, Gir-sur-Yvette, France
Vadim A. Frolov, PhD, Frumkin Institute of Electrochemistry, Russian Academy of Sciences, Moscow, Russia
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
Jennifer A. Lippincott-Schwartz, PhD, Cell Biology and Metabolism Branch, NICHD, Bethesda, MD
Rami Rahamimoff, MD, University of Jerusalem, Jerusalem, Israel
Thomas S. Reese, MD, Laboratory of Neurobiology, NINDS, Bethesda, MD
Thomas Zemb, PhD, CEA-Saclay, Gir-sur-Yvette, France
For further information, contact joshz@helix.nih.gov.