MEMBRANE REMODELING DURING VIRAL INFECTION, PARASITE INVASION, AND APOPTOSIS; COMPONENTS AND KINETICS IN EXOCYTOSIS
, Head, Section on Membrane and Cellular Biophysics
Paul S. Blank, PhD, Staff Scientist
Svetlana Glushakova, PhD, Staff Scientist
Vadim A. Frolov, PhD, Senior Research Fellow
Andrea Fera, PhD, Postdoctoral Fellow
Vladimir A. Lizunov, PhD, Visting Fellow
Julia Mazar, PhD, Visting Fellow
Pavel Bashkirov, MS, Guest Researcher
Alexander Chanturiya, PhD, Guest Researcher
Jane E. Farrington, MS, Guest Researcher
Glen Humphrey, PhD, Guest Researcher
Shurong Yin, PhD, Guest Researcher
Elena M. Kapnik, MS, Biologist
Gulcin Pekkurnaz, MS, Predoctoral Fellow
Anna Shnyrova, MS, Predoctoral Fellow
Ludmila Bezrukov, MS, Contractor
In an effort to understand viral and parasite infection, exocytosis, and apoptosis, we study membrane mechanics, intracellular molecules, membranes, viruses, organelles, and cells. This year, we report on four projects. For malaria, we studied in detail the mechanisms by which cells are released in a synchronous fashion, and we developed a quantitative assay for release. Second, in enveloped viral disease, we discovered how the protein forming the matrix of the virus interacts with membranes to cause the membrane to bud. Third, using fluorescence photoactivation localization microscopy (FPALM), we imaged the distribution of tens of thousands of hemagglutinin (HA) molecules with sub-diffraction resolution (30–40 nm) in live and fixed fibroblasts. In live cells, we observed and quantified the dynamics of HA molecules within clusters to determine an effective diffusion coefficient. Fourth, we calculated the effect of an externally applied lateral tension on the line tension between two domains of different thickness in a lipid bilayer membrane.
A quantitative assay for malarial parasite release
After each cycle of asexual reproduction, intracellular malaria parasites leave their host erythrocytes in order to infect neighboring cells. No method is currently available for the direct quantification of parasite release. To rectify this deficiency, we developed a method by which human erythrocytes infected with Plasmodium falciparum are injected into sealed chambers at optimal density; from there, they progress through the end of the erythrocyte cycle. Each event of parasite release inside the chamber at the site of erythrocyte rupture leaves on the chamber wall a footprint composed of (1) separated parasites, (2) a digestive vacuole containing haemozoin, and (3) fragments of the ruptured membranes. The footprints are stable for hours, allowing precise identification with differential interference contrast (DIC) microscopy. The relative rate of parasite release is defined as the percent of footprints out of all schizonts injected into the chamber and incubated there at 37ºC for two hours. The method is highly reproducible, easy to perform, and does not require expensive equipment. In addition, it permits analysis of cell and release site morphology, thereby yielding information about the release process and the quality of the culture. We used the method to show that the swelling of schizonts caused by protein-free media inhibits parasite release.
In conclusion, we have developed a novel method to count, by microscopy, sites of parasite release. Besides allowing direct estimation of parasite release from infected erythrocytes, the method allows a morphological evaluation of (1) normal infected cells approaching the end of the plasmodial life cycle or (2) pathological forms accumulated as the result of experimental intervention in the parasite release process. It is now possible to estimate accurately the relative parasite release rate at the time of cycle transition, without any obligatory coupling to parasite invasion.
Glushakova S, Yin D, Gartner N, Zimmerberg J. Quantification of malaria parasite release from infected erythrocytes: inhibition by protein-free media. Malar J 2007;6:61.
How the protein forming the matrix of the virus interacts with membranes to cause them to bud
The shape of enveloped viruses depends critically on an internal protein matrix, yet how the matrix proteins control the geometry of the envelope membrane remains unclear. As seen by fluorescent and electron microscopy, we found that matrix proteins, purified from Newcastle Disease virus, adsorb on a phospholipid bilayer and condense into fluid-like domains that cause membrane deformation and budding of spherical vesicles. Measurements of the electrical admittance of the membrane resolved the gradual growth and rapid closure of a bud, followed by the bud’s separation to form a free vesicle. The vesicle size distribution, confined by the intrinsic curvature of budding domains but broadened by their merger, matched the virus size distribution. Thus, matrix proteins implement a domain-driven mechanism of budding, which suffices to control the shape of these proteolipid vesicles.
Shnyrova AV, Ayllon J, Mikhalev II, Villar E, Zimmerberg J, Frolov VA. Vesicle formation by self-assembly of membrane-bound matrix proteins into a fluidlike budding domain. J Cell Biol 2007;179:627-33.
Zimmerberg J. Membrane biophysics. Curr Biol 2006;16:R272-6.
Super-resolution of the dynamics and distribution of HA molecules within clusters in living cells by using FPALM
We use a fusion protein expressed in fibroblasts as a model for the exocytotic proteins. Organization in biological membranes spans many orders of magnitude in length scale, but limited resolution in far-field light microscopy has impeded distinction between numerous biomembrane models. One well-studied example of a heterogeneously distributed membrane protein is HA from influenza virus, which is associated with controversial cholesterol-rich lipid rafts. Using FPALM, we imaged distributions of tens of thousands of HA molecules with sub-diffraction resolution (30–40 nm) in live and fixed fibroblasts. HA molecules form irregular clusters on length scales from 30 nm up to many micrometers, consistent with results from electron microscopy. In live cells, we observed and quantified the dynamics of HA molecules within clusters in order to determine an effective diffusion coefficient. We interpreted the results in terms of several established models of biological membranes.
Hess ST, Gould TJ, Gudheti MV, Maas SA, Mills KD, Zimmerberg J. Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories. Proc Natl Acad Sci USA 2007;104:17370-5.
Externally applied lateral tension increases line tension between two domains of different thickness
Last year, we described the creation of macroscopic raft domains in lipid membranes. Further, we described 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 extremely rapidly (characteristic times are in the submillisecond and millisecond ranges, respectively), and the system consists of nanodomains of about 5 to 50 nm average radius. Thereafter, the mobility of the 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 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 co-exist with a new global phase. The theoretical critical value of line tension needed for rapid formation of large rafts is in accord with the experimental estimate from the curvatures of budding domains in giant unilamellar vesicles.
This year, we calculated the effect of an externally applied lateral tension on the line tension between two domains of different thickness in a lipid bilayer membrane, treating the thick domain as a liquid-ordered phase in order to model a raft in a biological membrane. To model the surrounding region, we considered the thin domain as a liquid-disordered phase. In our model, the monolayers elastically distort at the domain boundary, thus creating a smooth rather than steplike boundary to avoid exposure of the hydrophobic interior of the thick raft to water. The energy of the distortion is described by the fundamental deformations of splay and tilt. The energy per unit length of boundary yields the line tension of the raft. Applying lateral tension alters the fundamental deformations such that line tension increases. The increase in line tension is larger when the spontaneous curvature of a raft is greater than that of the surround; if the spontaneous curvature of the raft is less than that of the surround, the increase in line tension attributable to the application of lateral tension is more modest.
Akimov SA, Kuzmin PI, Zimmerberg J, Cohen FS. Lateral tension increases the line tension between two domains in a lipid bilayer membrane. Phys Rev E Stat Nonlin Soft Matter Phys 2007;75:011919.
Frolov VA, Chizmadzhev YA, Cohen FS, Zimmerberg J. Entropic traps in the kinetics of phase separation in multicomponent membranes stabilize nanodomains. Biophys J 2006;91:189-205.
Publications Related to Other Works
Bakas L, Chanturiya A, Herlax V, Zimmerberg J. Paradoxical lipid dependence of pores formed by the Escherichia coli alpha-Hemolysin in planar phospholipid bilayer membranes. Biophys J 2006;91:3748-55.
Chen SS, Fitzgerald W, Zimmerberg J, Kleinman HK, Margolis L. Cell-cell and cell-extracellular matrix interactions regulate embryonic stem cell differentiation. Stem Cells 2007;25:553-61.
Chen SS, Revoltella RP, Zimmerberg J, Margolis L. Differentiation of rhesus monkey embryonic stem cells in three-dimensional collagen matrix. Methods Mol Biol 2006;330:431-43.
Chen X, Araç D, Wang TM, Gilpin CJ, Zimmerberg J, Rizo J. SNARE-mediated lipid mixing depends on the physical state of the vesicles. Biophys J 2006;90:2062-74.
Chernomordik LV, Zimmerberg J, Kozlov MM. Membranes of the world unite! J Cell Biol 2006;175:201-7.
Lizunov V, Zimmerberg J. Cellular biophysics: bacterial endospore, membranes and random fluctuation. Curr Biol 2006;16:R1025-8.
Zimmerberg J, Akimov SA, Frolov V. Synaptotagmin: fusogenic role for calcium sensor? Nat Struct Mol Biol 2006;13:301-3.
Zimmerberg J, Gawrisch K. The physical chemistry of biological membranes. Nat Chem Biol 2006;2:564-7.
Zimmerberg J, Kozlov MM. How proteins produce cellular membrane curvature. Nat Rev Mol Cell Biol 2006;7:9-19.
Zimmerberg J, Wu L-G, Reese TS. Fusion pore. In: Squire L, Albright T, Bloom F, Gage F, Spitzer N, eds. The New Encyclopedia of Neuroscience. Elsevier, in press.
COLLABORATORS
Sergey Akimov, MS, Frumkin Institute of Electrochemistry, Russian Academy of Sciences, Moscow, Russia
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
Klaus Gawrisch, PhD, Laboratory of Membrane Biochemistry and Biophysics, NIAAA, Bethesda, MD
Samuel T. Hess, PhD, University of Maine, Orono, ME
Michael Kozlov, PhD, Tel Aviv University School of Medicine, 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
José Rizo-Rey, PhD, University of Texas Southwestern Medical Center at Dallas, Dallas, TX
For further information, contact joshz@mail.nih.gov.
