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Membrane Remodeling During Viral Infection, Parasite Invasion, and Apoptosis; Components and Kinetics in Exocytosis

Joshua Zimmerberg, MD, PhD, Head, Section on Cellular and Membrane Biophysics
Pavel Bashkirov, MS, Guest Researcher
Ludmila Bezrukov, MS, Contractor
Paul S. Blank, PhD, Staff Scientist
Alexander Chanturiya, PhD, Guest Researcher
Jane E. Farrington, MS, Guest Researcher
Vadim A. Frolov, PhD, Senior Research Fellow
Svetlana Glushakova, PhD, Staff Scientist
Glen Humphrey, PhD, Guest Researcher
Vladimir A. Lizunov, MS, Visiting Fellow
Julia Mazar, PhD, Visiting Fellow
Gulcin Pekkurnaz, MS, Postdoctoral Fellow
Ivan Polozov, PhD, Guest Researcher
Anna Shnyrova, MS, Predoctoral Fellow

We study membrane mechanics, intracellular molecules, membranes, viruses, organelles, and cells in order to understand viral and parasite infection, exocytosis, apoptosis, the mechanism of immune protection by stem cells and their cytotoxic potential, and immune dysfunction in microgravity.

One of our goals is to determine the physico-chemical mechanisms of membrane remodeling in cells. These processes underlie many pathological processes, such as E. coli toxicosis, malarial infection, viral infection, and the failure of apoptosis in cancer. Using a method we developed to isolate large quantities of directly accessible plasma membrane from attached cells, we determined the relative extractability of cholesterol from cells and cellular membranes. We also study stem cell apoptosis, determining that the mechanism by which human bone marrow stromal cells (BMSCs) are immunosuppressive and escape cytotoxic lymphocytes (CTLs) involves FasL-mediated apoptosis. In space research, we found that human lymphoid tissue could not be activated in either simulated microgravity of rotation in a NASA-designed bioreactor or in actual microgravity of the International Space Station.

We strive to understand the mechanisms of exocytosis at physical, biophysical, and chemical levels. This process of protein secretion is the climax of the secretory pathway, and operates in both constitutive and triggered ways. The process of endocytosis is equally important to retrieve membrane components. This year, we focused on two components, dynamin in endocytosis, and Exo70 in insulin-stimulated exocytosis. The GTPase dynamin is critical to membrane fission during endocytosis; our aim was to determine how dynamin uses the energy of GTP hydrolysis for membrane remodeling. By monitoring the ionic permeability through lipid nanotubes (NT), we found that dynamin was capable of squeezing them to extremely small radii, depending on the NT lipid composition. However, long dynamin scaffolds did not produce fission: instead, fission followed GTPase-dependent cycles of assembly and disassembly of short dynamin scaffolds and involved a stochastic process dependent on the curvature stress imposed by dynamin. Fission happened spontaneously upon NT release from the scaffold, without leakage. Our calculations revealed that local narrowing of NT could induce cooperative lipid tilting, leading to self-merger of the inner monolayer of NT (hemifission), consistent with the absence of leakage. We propose that dynamin transmits GTP's energy to periodic assembling of a limited curvature scaffold that brings lipids to an unstable intermediate.

An adhesion-based method for plasma membrane isolation: evaluating cholesterol extraction from cells and their membranes.

Techniques currently used for the isolation of plasma membranes include zonal or density gradient centrifugation, the "ripflip" method developed for the microscopic observation of small pieces of the cytoplasmic side of plasma membranes, and methods based on the adhesion of negatively charged cells to a positively charged surface such as polylysine-coated polyacrylamide or glass beads. We isolated large quantities of directly accessible cytoplasmic surface of the plasma membranes suitable for both microscopy and biochemical analysis by adhering cells to an adsorbed layer of polylysine on glass plates, followed by hypotonic lysis with ice-cold distilled water. Optimal conditions were established for all preparation steps, including polylysine coating, cell adhesion, and membrane washing, ensuring both high purity and high yield of the membrane preparation. This method permits (1) the creation of isolated plasma membranes without chemical (high-salt) or mechanical (vortexing or sonication) treatments, (2) direct lipid extraction on glass plates, and (6) the study of both biochemical and structural properties of isolated plasma membranes. Methyl-beta-cyclodextrin (MbCD) is used to alter the cholesterol content of cells and, in particular, the cholesterol content of the plasma membrane. The cholesterol content of cellular membranes and the relationships between cholesterol-enriched domains and physiological function is an active area of research. Although questions remain about the detailed kinetics, dependence on composition and temperature, and specific and nonspecific effects, exposure of cells to MbCD reduces cellular cholesterol. However, reduction in cellular cholesterol following MbCD treatment might not quantitatively reflect the changes in plasma membrane cholesterol content. Having developed the method to isolate plasma membranes in quantities suitable for biochemical examination, we compared MbCD cholesterol depletion from intact cells with that from their plasma membranes. MbCD treatment extracted cholesterol from the plasma membrane of HAB2 cells, a hemagglutinin-expressing fibroblast cell line, and intact HAB2 cells in a temperature-dependent manner; the reduction in plasma membrane cholesterol content was not, however, proportional to the decrease observed using intact cells. Nearly complete removal of plasma membrane cholesterol can be achieved by extraction at physiological temperature (37°C). At 4°C, MbCD extracts less cholesterol from the plasma membrane. These data indicate that one cannot predict the loss of cholesterol from the plasma membrane based on the loss determined from intact cells. Treatment with 10 mM MbCD for 30 min at 37°C did not deplete cholesterol from all membrane fractions but essentially depleted all cholesterol from the plasma membrane.

The roles of cholesterol in membrane heterogeneity/domains (rafts vs. nonrafts) and the association and function of proteins to specialized domains are of considerable interest. MbCD is often used to selectively deplete cholesterol from low- and high-density membrane fractions. Not only are MbCD concentration and exposure time important parameters in perturbing the cholesterol content of the membrane, but extraction temperature is also critical. The method to isolate and evaluate biochemical quantities of pure plasma membrane will benefit those studies for which cholesterol perturbation needs to be minimized. The combined effects of MbCD concentration, exposure time, and temperature extraction can now be evaluated easily.

Cytotoxicity mediated by the Fas ligand (FasL) – activated apoptotic pathway in stem cells

Although it is now clear that human bone marrow stromal cells (BMSCs) can be immunosuppressive and escape cytotoxic lymphocytes (CTLs) in vitro and in vivo, the mechanisms of this phenomenon remain controversial. We tested the hypothesis that BMSCs suppress immune responses by Fas-mediated apoptosis of activated lymphocytes and found that both Fas and FasL are expressed by primary BMSCs. Jurkat cells or activated lymphocytes were each killed by BMSCs after 72 h of co-incubation. In comparison, the cytotoxic effect of BMSCs on non-activated lymphocytes and on caspase-8(/) Jurkat cells was extremely low. Fas/Fc fusion protein strongly inhibited BMSC-induced lymphocyte apoptosis. Although we detected a high level of Fas expression in BMSCs, stimulation of Fas with anti-Fas antibody did not result in the expected BMSC apoptosis, regardless of concentration, suggesting a disruption of the Fas activation pathway. Thus BMSCs may have an endogenous mechanism to evade Fas-mediated apoptosis. Cumulatively, these data provide a parallel between adult stem/progenitor cells and cancer cells, consistent with the idea that stem/progenitor cells can use FasL to prevent lymphocyte attack by inducing lymphocyte apoptosis during the regeneration of injured tissues. We hypothesize that BMSC-mediated cytotoxicity of lymphocytes involves the FasL-activated apoptotic machinery. FasL is a type II transmembrane protein belonging to the tumor necrosis factor (TNF) family. FasL interacts with its receptor, Fas (CD95/APO-1), and triggers a cascade of subcellular events culminating in apoptotic cell death. FasL and Fas are key regulators of apoptosis in the immune system. In addition, FasL is expressed by cells in immune-privileged sites, such as cancer cells, neurons, eyes, cytotrophoblasts of the placenta, and reproductive organs. In neurons, FasL expression specifically protects against T cell–mediated cytotoxicity.

The discovery that FasL is also expressed by a variety of tumor cells raises the possibility that FasL may mediate immune privilege in human tumors. Activated T cells expressing Fas are sensitive to Fas-mediated apoptosis. Thus, up-regulation of FasL expression by tumor cells may enable tumorigenesis by targeting apoptosis in infiltrating lymphocytes.

In the present work, we show that BMSCs can mediate immunosuppressive activity by FasL–induced killing of activated lymphocytes. Thus, BMSCs have properties of immune-privileged cells.

Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station.

The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity completely lose their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction.

Formation of an endocytic vesicle is completed by scission of a thin membrane neck connecting the vesicle and the plasma membrane

Although scission is intuitively associated with cutting and resealing, even transient permeabilization of cellular membranes could be damaging, especially for small vesicles whose content can quickly escape through even minute pores. Thus, fission of neck membranes is more likely to proceed via hemifission to avoid leakage. The neck fission involves extensive bending deformations, which are generated by specialized protein machinery assembled on the membrane neck, where the GTPase dynamin is a key component. Crucial for many cell processes featuring fission, dynamin family members form dense collars around necks of budding vesicles and dividing organelles. Assembly of this collar triggers cooperative GTP hydrolysis, which is thought to force membrane remodeling. However, the pathway that links the GTPase activity of dynamin with membrane rearrangements during fission is obscure.

The dynamics of membrane remodeling by short dynamin assemblies formed in the presence of GTP is difficult to characterize because the corresponding membrane transformations are expected to be fast and highly localized. In cellular systems, similar membrane transformations are studied by electrophysiology, revealing a rich dynamic behavior of the membrane necks of endocytic vesicles. We applied such techniques to resolve dynamin's interaction with nanotubes (NT) pulled from lipid membranes. We measured the ionic permeability of the tube's interior to estimate average changes in the diameter of the tube and to resolve the kinetics of tube fission. We discovered that self-assembling dynamin scaffolds caused dramatic narrowing of the NTs, their final radius depending on the tube rigidity. However, fission required partial disassembly of long dynamin scaffolds triggered by GTP hydrolysis. GTPase cycles of dynamin were coupled to assembly and disassembly of short dynamin coats, producing membrane curvature and fission in a stochastic lipid-dependent manner. These results suggest that dynamin acts as a catalyst for membrane remodeling, bringing membrane NTs to the point of spontaneous fission by creating regulated curvature constraints.

Membrane fission converges to a highly localized and fast restructuring of the lipid bilayer. Using sensitive time-resolved conductance measurements, we identified the key steps for fission of NT mediated by dynamin. Theoretical analysis of these data revealed that the fission is catalyzed in two critical steps: GTP-independent scaffolding of membrane curvature by dynamin followed by GTP-dependent disassembly of the scaffold, allowing lipid to complete membrane remodeling. Self-assembly of the dynamin scaffold induces NT narrowing until the scaffold reaches a length sufficient to trigger GTP hydrolysis. Depending on the curvature imposed on the NT, membrane detachment from the dynamin scaffold upon GTP hydrolysis can cause spontaneous hemifission followed by complete fission. This step is apparently stochastic: hemifission probability depends on the energy barrier for the hemifission transformation and on the time frame during which the dynamin scaffold holds its rigidity upon GTP hydrolysis. On the NT, the scaffold softens 10 s after hydrolysis. If fission does not happen within this time, the NT expands with the softening of the scaffold, whereupon a new squeezing cycle is initiated. Consistent with this scheme, cyclic assembly of fluorescently labeled dynamin in the presence of GTP has been visualized directly. Several sequential squeezing attempts might be needed to trigger hemifission of the NT.

Our experiments reveal that dynamin activity is crucially modulated by lipid composition: final diameters of dynamin-coated tubes (without GTP) are not dictated by dynamin alone, but also depend on lipid composition. If dynamin scaffold had completely dominated the energetics of tube formation, then it would have satisfied its optimal protein packing constraints by forming the same diameter tube. If, however, the energy of scaffold polymerization is comparable to the energy of membrane bending, then the final tubule diameter will depend on lipid composition. Hence, dynamin-like proteins may function as GTP-dependent curvature agents.

To summarize, our results reveal a tight coupling between dynamin and the lipid template. Dynamin is designed to selectively target highly curved membrane necks and probe their mechanical stability by repetitive squeezing. Fission critically depends on the geometry and mechanical parameters of the neck membrane. This dependence has a clear physiological significance: dynamin effectively cuts only those necks that are prone to fission, such as narrow necks formed at the final stages of vesicle detachment. On wider and/or more rigid necks, dynamin is expected to operate as a GTP-dependent curvature regulator. Hence, cooperation of dynamin and the lipid membrane provides a universal tool to control the behavior of the vesicle neck.

Insulin regulates fusion of GLUT4 vesicles independently of Exo70-mediated tethering

At the cellular level, glucose uptake is regulated by controlling the amount of the GLUT4 glucose transporter present in the plasma membrane. In insulin-responsive adipose and muscle cells, insulin regulates glucose uptake by promoting the exocytosis of specialized GLUT4 storage vesicles (GSV). Although many steps of the insulin signal transduction pathway have been elucidated, an understanding of the mechanism bridging insulin signaling with the actual fusion of GSV with the plasma membrane, with concomitantly enhanced glucose uptake, is still lacking. Mounting evidence suggests that insulin regulates GLUT4 exocytosis at many different levels such as intracellular sequestration of GLUT4 into GSV, traffic to the plasma membrane, and tethering and fusion. Recent reports based on the use of total internal reflection fluorescence (TIRF) microscopy and comprehensive kinetic analysis using rat adipose cells and 3T3-L1 adipocytes suggest that both tethering and fusion of GSV are primary steps regulated by insulin stimulation.

Insulin regulates GLUT4 exocytosis at multiple steps involving trafficking, tethering, and fusion. The molecular events associated with stimulated GLUT4 exocytosis are likely to take place at both the vesicular surface and the plasma membrane. In the current study, we investigated the role of the Exocyst component Exo70 on the trafficking and tethering events of GSVs. We saw no effect of overexpressing the wild-type Exo70 protein. Surprisingly, the Exo70-N mutant, reported to block the assembly of the Exocyst complex and GLUT4 exocytosis in differentiated 3T3-L1 cells, enhances the tethering in the primary adipose cells but does not affect the rate of fusion, neither in the basal nor in the insulin-stimulated state. The effect of the Exo70 mutant on the tethering in basal conditions was strikingly similar to insulin-induced tethering but was insufficient to induce fusion, indicating that insulin regulates fusion steps of GLUT4 exocytosis independently of upstream tethering, where Exo70 might be involved.

Additional Funding

Jain Foundation
DOD CNRM Breast Cancer Program
Intramural AIDS Targeted Antiviral Program (IATAP)
Center for Neuroscience & Regenerative Medicine (CNRM)

Publications

Bashkirov PV, Akimov SA, Evseev AI, Schmid SL, Zimmerberg J, Frolov VA. GTPase cycle of dynamin is coupled to membrane squeeze and release, leading to spontaneous fission. Cell 2008 135:1276-1286.
Glushakova S, Mazar J, Hohmann-Marriott MF, Hama E, Zimmerberg J. Irreversible effect of cysteine protease inhibitors on the release of malaria parasites from infected erythrocytes. Cell Microbiol 2009 11:95-105.
Hohmann-Marriott MF, Sousa AA, Azari AA, Glushakova S, Zhang G, Zimmerberg J, Leapman RD. Nanoscale 3D cellular imaging by axial scanning transmission electron tomography. Nat Meth 2009 6:729-731.
Collins RN, Zimmerberg J. Cell biology: a score for membrane fusion. Nature 2009 459:1065-1066.
Mazar J, Thomas M, Bezrukov L, Chanturia A, Pekkurnaz G, Yin S, Kuznetsov SA, Robey PG, Zimmerberg J. Cytotoxicity mediated by the Fas ligand (FasL)-activated apoptotic pathway in stem cells. J Biol Chem 2009 284:22022-22028.

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
Frederic S. Cohen, PhD, Rush Medical College, 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
Martin Hohmann-Marriott, PhD, Laboratory of Bioengineering and Physical Science, NIBIB, Bethesda, MD
Thomas S. Reese, MD, Laboratory of Neurobiology, NINDS, Bethesda, MD
Sandra Schmid, PhD, The Scripps Research Institute, La Jolla, CA

Contact

For more information, email zimmerbj@mail.nih.gov.

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