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Home > Section on Cellular and Membrane Biophysics

The Regulation or Disturbance of Protein/Lipid Interactions in Influenza, Malaria, Diabetes, Muscular Dystrophy, Brain Trauma, and Obesity

Joshua Zimmerberg, MD, PhD
  • Joshua Zimmerberg, MD, PhD, Head, Section on Cellular and Membrane Biophysics
  • Paul S. Blank, PhD, Staff Scientist
  • Svetlana Glushakova, PhD, Staff Scientist
  • Atsuko Kimura, PhD, Research Fellow, Special Volunteer
  • Vladimir A. Lizunov, MS, Research Fellow
  • Petr Chlanda, PhD, Visiting Fellow
  • Sourav Haldar, PhD, Visiting Fellow
  • Ivonne Morales-Benavides, PhD, Visiting Fellow
  • Brad Busse, PhD, Postdoctoral Intramural Research Training Award Fellow
  • Glen Humphrey, PhD, Guest Researcher
  • Ludmila Bezrukov, MS, Chemist
  • Hang Waters, MS, Biologist
  • Jane E. Farrington, MS, Contractor
  • Elena Mekhedov, MA, Contractor
  • Rea Ravin, PhD, Contractor
  • Mariam Ghochani, MS, Graduate Student

Eukaryotic life must create the many shapes and sizes of the system of internal membranes and organelles that inhabit the variety of cells in nature. The membranes must remodel so that cells can secrete signaling macromolecules, express surface transporters, import macromolecular cargo, store energy, repair a damaged plasmalemma, and deal with infectious agents such as viruses and parasites. Such basic membrane mechanisms must be highly regulated and highly organized in various hierarchies in space and time to allow the organism to thrive despite environmental challenges, genetic instability, an unpredictable food supply, and physical trauma. We are using our expertise and the techniques we have perfected over the years to address several different biological problems that have in common the underlying regulation or disturbance of protein/lipid interactions. Our overall goal is to determine the physico-chemical mechanisms of membrane remodeling in cells and to understand the mechanisms of cellular secretion and endocytosis at the physical, biophysical, and chemical levels, including the concentration and diffusion of key vesicular components prior to and after fusion or fission.

This year, we focused on six topics: (i) the relationship between erythrocyte parameters and the multiplicity of infection for lab and field strains of Plasmodium falciparum; (ii) the role of intracellular perforin in the egress pathway of malaria gametocytes from human erythrocytes; (iii) the potential role of intrabilayer cavitation in blast-induced traumatic brain injury; (iv) the translocation of human GLUT4 to the membrane in fat bodies of transgenic fruit flies, Drosophila melanogaster, as stimulated by insulin; (v) a quantitative description of the trapping by clusters of channels, receptors, and transporters in a membrane; and (iv) trapping of diffusing particles by clusters of absorbing disks on a reflecting wall with disk centers on sites of a square lattice.

The relationship between erythrocyte parameters and the multiplicity of infection for lab and field strains of P. falciparum

The mechanisms by which alpha-thalassemia and sickle cell traits confer protection against severe Plasmodium falciparum malaria are not yet fully elucidated. We hypothesized that hemoglobinopathic erythrocytes reduce the intra-erythrocytic multiplication of P. falciparum, potentially delaying the development of life-threatening parasite densities until parasite clearing immunity is achieved. To test the hypothesis, we developed a novel in vitro assay to quantify the number of merozoites released from an individual schizont, termed the "intra-erythrocytic multiplication factor" (IMF). P. falciparum (3D7 line) schizonts produce variable numbers of merozoites in all erythrocyte types tested, with median IMFs of 27, 27, 29, 23, and 23 in control, HbAS (from heterozygous sickle-cell blood), HbSS (from homozygous sickle-cell blood), and alpha-thalassemia– and beta-thalassemia–trait erythrocytes, respectively. IMF correlated strongly with mean corpuscular hemoglobin concentration and varied significantly with mean corpuscular volume and hemoglobin content. Reduction of IMFs in thalassemia-trait erythrocytes was confirmed using clinical parasite isolates with different IMFs. Mathematical modeling of the effect of IMF on malaria progression indicates that the lower IMF in thalassemia trait erythrocytes limits parasite density and anemia severity over the first two weeks of parasite replication. HbAS and HbSS erythrocytes are associated with equal or higher IMF values; additional factors must thus account for the lower parasite densities found in African children carrying HbS. In conclusion, P. falciparum IMF, a parasite heritable virulence trait, correlates with erythrocyte indices and is reduced in thalassemia-trait erythrocytes. Parasite IMF should be examined in other low-indices erythrocytes.

The role of intracellular perforin in the egress pathway of malaria gametocytes from human erythrocytes

Egress of malaria parasites from the host cell requires the concerted rupture of its enveloping membranes. We therefore investigated the role of the plasmodial perforin-like protein PPLP2 in the egress of P. falciparum from erythrocytes. PPLP2 is expressed in blood stage schizonts and mature gametocytes. The protein localizes in vesicular structures, which in activated gametocytes discharge PPLP2 in a calcium-dependent manner. PPLP2 bears a MACPF domain, and recombinant PPLP2 has hemolytic activities towards erythrocytes. PPLP2–deficient (PPLP2−) merozoites show normal egress dynamics during the erythrocytic replication cycle, but activated PPLP2− gametocytes were unable to leave erythrocytes and stayed trapped within these cells. While the parasitophorous vacuole membrane ruptured normally, the activated PPLP2− gametocytes were unable to permeabilize the erythrocyte membrane and release the erythrocyte cytoplasm. As a consequence, transmission of PPLP2− parasites to the Anopheles vector was reduced. Pore-forming equinatoxin II rescued both PPLP2− gametocyte exflagellation and parasite transmission. The pore sealant Tetronic 90R4, on the other hand, caused trapping of activated wild-type gametocytes within the enveloping erythrocytes, thus mimicking the PPLP2− loss-of-function phenotype. We propose that the hemolytic activity of PPLP2 is essential for gametocyte egress because it permeabilizes the erythrocyte membrane and thus depletes the erythrocyte cytoplasm.

The potential role of intrabilayer cavitation in blast-induced traumatic brain injury

A transient period of negative hydrostatic pressure is one of the more intriguing aspects of estimated pressure profiles in the skull during a bomb blast. We hypothesized that one source of traumatic brain injury could be interleaflet cavitation in lipid bilayer membranes, or intramembrane cavitation (IMC) for short. The project focuses on the thermodynamics of IMC, namely, on the minimum work required to form an intramembrane cavity. The minimum work can be separated into two parts, one depending on the volume and number of gas molecules in the bubble and another on the bubble geometry. Minimization of the second part at a fixed bubble volume determines the optimized bubble shape. In homogeneous cavitation, this part is proportional to the bubble surface area, and the bubble is therefore spherical. In contrast, in IMC the second part is no longer a simple function of the bubble area, and the optimized cavity is not spherical because of the finite elasticity of the membrane. Using a simplified assumption about the cavity shape, the geometry-dependent term is derived and minimized at a fixed cavity volume. We found that the optimized cavity is almost spherical at large bubble volumes, while at small volumes the cavity has a lens-like shape. The optimized shape is used to analyze the minimum work of IMC.

The translocation of human GLUT4 to the membrane in fat bodies of a transgenic Drosophila melanogaster, as stimulated by insulin

Given that the hallmark of glucose metabolism is insulin-stimulated delivery of the glucose transporter-4 (GLUT4) to the plasma membrane (PM) and the hallmark of membrane protein organization is its domain structure, we continue to examine insulin's effect on GLUT4 organization in the PM of adipose cells. The fruit fly Drosophila melanogaster is an excellent model system for studying genes that control development and disease. However, its applicability to physiological systems is less clear because of the metabolic differences between insects and mammals. Insulin signaling has been studied in mammals because of its relevance to diabetes and other diseases, but there are many parallels between mammalian and insect pathways. For example, deletion of Drosophila Insulin-Like Peptides resulted in 'diabetic' flies with elevated circulating sugar levels. Whether this situation reflects failure of sugar uptake into peripheral tissues, as seen in mammals, is unclear and depends on whether flies harbor the machinery to mount mammalian-like insulin-dependent sugar uptake responses. We investigated whether Drosophila fat cells are competent to respond to insulin with mammalian-like regulated trafficking of sugar transporters. We generated transgenic Drosophila expressing the human glucose transporter-4 (GLUT4), the sugar transporter expressed primarily in insulin-responsive tissues. After expression in fat bodies, GLUT4 intracellular trafficking and localization were monitored by confocal and total internal reflection fluorescence (TIRF) microscopy. We found that fat-body cells responded to insulin with increased GLUT4 trafficking and translocation to the plasma membrane. While the amplitude of these responses was relatively weak in animals reared on a standard diet, it was greatly enhanced in animals reared on sugar-restricted diets, suggesting that flies fed standard diets are insulin-resistant. Our findings demonstrate that flies are competent to mobilize translocation of sugar transporters to the cell surface in response to insulin and suggest that Drosophila fat cells are primed for a response to insulin and that these pathways are down-regulated when animals are exposed to constant high levels of sugar. The studies are the first to use TIRF microscopy for monitoring insulin-signaling pathways in Drosophila and to demonstrate its utility in tracking, in insects, signaling pathways with tagged sugar transporters.

A quantitative description of the trapping by clusters of channels, receptors, and transporters in a membrane

Various membrane functional units such as receptors, transporters, and channels, whose action necessarily involves capturing diffusing molecules, are often organized into multimeric complexes forming clusters on the cell and organelle membranes. Such functional units themselves are usually oligomers of several integral proteins, which have their own symmetry. Depending on the symmetry, the clusters form on different packing lattices. Moreover, local membrane inhomogeneities, e.g., the so-called membrane domains, rafts, stalks, etc., lead to different patterns even within the structures on the same packing lattice. Units in the cluster compete for diffusing molecules and screen each other. We proposed a general approach that allows one to quantify the screening effects. The approach is used to derive simple approximate formulas that give the trapping rates of diffusing molecules by clusters of absorbers on lattices of different packing symmetries. The obtained results describe smooth variation of the trapping rate from the sum of the rates of individual absorbers forming the cluster to the effective collective rate. The latter shows how the trapping efficiency of an individual absorber declines as the number of absorbers in the cluster increases and/or the inter-absorber distance decreases. Numerical tests demonstrate good agreement between the rates predicted by the theory and obtained from Brownian dynamics simulations for clusters of different shapes and sizes.

Trapping of diffusing particles by clusters of absorbing disks on a reflecting wall with disk centers on sites of a square lattice

A feature of cell membrane proteins is that they are almost always clustered. Recently, we noticed that most clusters are not circular but vary in size and shape. However, for a single protein the shape is stereotypical. We thus hypothesized that the shape and size of a cluster might affect its function. To start, we analyzed a simple approximate formula, derived for the rate constant that describes steady-state flux of diffusing particles through a cluster of perfectly absorbing disks on the otherwise reflecting flat wall, assuming that the disk centers occupy neighboring sites of a square lattice. A distinctive feature of trapping by a disk cluster is that disks located at the cluster periphery shield the disks in the center of the cluster. This competition of the disks for diffusing particles makes it impossible to find an exact analytical solution for the rate constant in the general case. To derive the approximate formula, we use a recently suggested approach (Berezhkovskii et al., J Chem Phys 2012;136:211102), which is based on the replacement of the disk cluster by an effective uniform partially absorbing spot. The formula shows how the rate constant depends on the size and shape of the cluster. To check the accuracy of the formula, we compared its predictions with the values of the rate constant obtained from Brownian dynamics simulations. The comparison made for 18 clusters of various shapes and sizes shows good agreement between the theoretical predictions and numerical results.

Additional Funding

  • Jain Foundation Award
  • NICHD Director’s Award (Co-Principal Investigator with Jack Yanovski)

Publications

  1. Glushakova S, Balaban A, McQueen PG, Coutinho R, Miller JL, Nossal R, Fairhurst RM, Zimmerberg J. Hemoglobinopathic erythrocytes affect the intraerythrocytic multiplication of Plasmodium falciparum in vitro. J Infect Dis 2014;210:1100-1109.
  2. Wirth CC, Glushakova S, Scheuermayer M, Repnik U, Garg S, Schaack D, Kachman MM, Weißbach T, Zimmerberg J, Dandekar T, Griffiths G, Chitnis CE, Singh S, Fischer R, Pradel G. Perforin-like protein PPLP2 permeabilizes the red blood cell membrane during egress of Plasmodium falciparum gametocytes. Cell Microbiol 2014;16:709-733.
  3. Berezhkovskii AM, Dagdug L, Lizunov VA, Zimmerberg J, Bezrukov SM. Trapping by clusters of channels, receptors, and transporters: quantitative description. Biophys J 2014;106:500-509.
  4. Crivat G, Lizunov VA, Li CR, Stenkula KG, Zimmerberg J, Cushman SW, Pick L. Insulin stimulates translocation of human GLUT4 to the membrane in fat bodies of transgenic Drosophila melanogaster. PLoS One 2013;8:e77953.
  5. Gudheti MV, Curthoys NM, Gould TJ, Kim D, Gunewardene MS, Gabor KA, Gosse JA, Kim CH, Zimmerberg J, Hess ST. Actin mediates the nanoscale membrane organization of the clustered membrane protein influenza hemagglutinin. Biophys J 2013;104:2182-2192.

Collaborators

  • Alexander Berezhkovskii, PhD, Mathematical and Statistical Computing Laboratory, CIT, NIH, Bethesda, MD
  • Sergey Bezrukov, PhD, Program in Physical Biology, NICHD, Bethesda, MD
  • Georgeta Crivat, PhD, University of Maryland, College Park, MD
  • Nikki Curthoys, PhD, University of Maine, Orono, ME
  • Samuel W. Cushman, PhD, Diabetes Branch, NIDDK, Bethesda, MD
  • Leonardo Dagdug, PhD, Universidad Autónoma Metropolitana Iztapalapa, Mexico City, Mexico
  • Rick M. Fairhurst, MD, PhD, Laboratory of Malaria and Vector Research, NIAID, Bethesda, MD
  • Vadim Frolov, PhD, Universidad del País Vasco, Bilbao, Spain
  • Klaus Gawrisch, PhD, Laboratory of Membrane Biochemistry and Biophysics, NIAAA, Bethesda, MD
  • Hugo Guerrero-Cazares, MD, The Johns Hopkins University, Baltimore, MD
  • Samuel T. Hess, PhD, University of Maine, Orono, ME
  • Mary Kraft, PhD, University of Illinois at Urbana-Champaign, Urbana, IL
  • Caroline R. Li, PhD, University of Maryland, College Park, MD
  • Philip G. McQueen, PhD, Mathematical and Statistical Computing Laboratory, CIT, NIH, Bethesda, MD
  • Jeffery Miller, MD, Molecular Medicine Branch, NIDDK, Bethesda, MD
  • Ralph J. Nossal, PhD, Program in Physical Biology, NICHD, Bethesda, MD
  • Leslie Pick, PhD, University of Maryland, College Park, MD
  • Gabriele Pradel, PhD, Institute of Molecular Biotechnology, RWTH Aachen University, Aachen, Germany
  • Alfredo Quinones-Hinojosa, MD, The Johns Hopkins University, Baltimore, MD
  • Shay M. Rappaport, PhD, Program in Physical Biology, NICHD, Bethesda, MD
  • Thomas S. Reese, MD, Laboratory of Neurobiology, NINDS, Bethesda, MD
  • Sandra L. Schmid, PhD, The University of Texas Southwestern Medical Center, Dallas, TX
  • Anna Shnyrova, PhD, Universidad del País Vasco, Bilbao, Spain
  • Karin G. Stenkula, PhD, Diabetes Branch, NIDDK, Bethesda, MD

Contact

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

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