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

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
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 for cells to secrete signaling macromolecules, express surface transporters, import macromolecular cargo, store energy, and repair damaged plasmalemma. 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, such as infections by other organisms, unpredictable food supply, and physical trauma. We aim to use the expertise and the techniques we have perfected over the years to address a number of different biological problems that have in common the underlying regulation or disturbance of protein/lipid interactions.

An overarching issue in a basic understanding of protein/lipid interactions is to distinguish between several alternate hypotheses. Do the acylated domains of proteins attract cholesterol through tail interactions, or does the lipid pull the protein into the headgroup region to directly perturb the physical properties of the membrane? Conversely, do the lipid associations play a role in a) clustering even more of the same protein or b) attracting sphingolipids? Do the physical principles controlling remodeling apply to the biogenesis of lipid droplets, with their drastically different composition and structure? We recently found that regulation of lipid/protein interactions in fission and in domains pivots on constant crosstalk between molecules to retain the robustness of self-assembly yet direct topological change. Correspondingly, we find that insulin, an extracellular hormone, can reach through a complex signaling cascade to regulate the diffusion of a membrane protein monomer in or out of a membrane domain. Though systemic, insulin exerts its effects at the plasma membrane, where it remodels membranes by augmenting fusion, inhibiting endocytosis, diminishing domains resulting from fusion, and shifting individual glucose transporters out of formed domains. Type II diabetes is characterized and preceded by a failure of cells to respond to insulin, and we have recapitulated this phenotype in isolated adipose cells cultured from patients with metabolic syndrome. Conflicting signals from different levels of control exhaust many regulatory factors, causing some fat cells to shut down entirely, forcing higher and higher levels of insulin secretion.

To identify therapeutic targets in infectious diseases, we focus on ways that disease-causative agents uniquely affect membrane remodeling and organization. In malaria, two membranes surrounding parasites are remodeled after invasion to aid replication, then ruptured two days later during the parasite’s egress from infected erythrocytes. Having discovered that intracellular Ca²⁺ concentration controls this egress, we will test our hypotheses that the one of intracellular targets of this divalent cation is a channel in the modified parasitophorous vacuolar membrane surrounding mature parasites, and that the parasite directs the reorganization of both the parasitophorous vacuolar membrane and the erythrocyte plasma membrane.

Calcium continuously rises during the egress program of the malaria parasite.

A steady rise in cytoplasmic free Ca²⁺ is found to precede parasite egress. The increase is independent of extracellular Ca²⁺ for at least the last two hours of the cycle, but depends on Ca²⁺ release from internal stores. Intracellular BAPTA chelation of Ca²⁺ within the last 45 minutes of the cycle inhibits egress prior to parasitophorous vacuole swelling and erythrocyte membrane poration, two characteristic morphological transformations preceding parasite egress. Inhibitors of the parasite endoplasmic reticulum (ER) Ca²⁺-ATPase accelerate parasite egress, indicating that Ca²⁺ stores within the ER are sufficient to support egress. Markedly accelerated egress of apparently viable parasites was achieved in mature schizonts using the Ca²⁺ ionophore A23187. Ionophore treatment overcomes the BAPTA–induced block of parasite egress, confirming that free Ca²⁺ is essential for egress initiation. Ionophore treatment of immature schizonts had an adverse effect of inducing parasitophorous vacuole swelling and killing the parasites within the host cell.

We conclude that the parasite egress program requires intracellular free Ca²⁺ for egress initiation, vacuole swelling, and host cell cytoskeleton digestion. The evidence that parasitophorous vacuole swelling, a stage of unaffected egress, depends on a rise in intracellular Ca²⁺ suggests a mechanism for ionophore-inducible egress and a new target for Ca²⁺ in the program liberating parasites from the host cell. We proposed a regulatory pathway for egress that depends on increases in intracellular free Ca²⁺.

Chemical imaging of lipid domains in cell membranes

To directly probe plasma membrane (PM) chemistry of hemagglutinin (HA) domains, we established a collaboration with Mary Kraft, the pioneer of imaging by high-resolution secondary ion mass spectrometry (SIMS) of lipid bilayers. We determined conditions for preserving PM molecular organization and measured the distributions of metabolically incorporated ¹⁵N-sphingolipids in the PM of mouse fibroblast cells stably expressing HA, detecting 100nm–1μ diameter sphingolipid patches in the PM. Sphingolipid domains are strongly perturbed by disruption of the cytoskeleton, but not in response to cholesterol depletion, and exist independently of PM–localized HA (50% colocalization). The sphingolipid domains were temperature-insensitive and hardly circular—their origin was not a tail-interaction–dominated phase separation with typical line tensions. We also succeeded in metabolically labeling fibroblasts expressing HA with ¹⁵N-sphingolipid such that 90% of the cellular sphingolipids contained one ¹⁵N isotope, and 60% of the cellular cholesterol contained one ¹⁸O isotope. After stabilizing the lipids using chemical fixation, as measured by direct real-time observation of fluorescent PM sphingomyelin during fixation of live cells, cells were imaged by scanning electron microscopy (SEM) followed by SIMS. Membrane domains with elevated ¹⁵N-enrichment, and thus high ¹⁵N-sphinoglipid abundance, were visible, confirming our previous measurements. Surprisingly, the ¹⁸O-enrichment images of the same cell did not reveal cholesterol-enriched domains. We could detect no significant difference in the ¹⁸O-cholesterol abundance within the sphingolipid domain and non-domain regions. Thus PM sphingolipid domains are not significantly enriched in cholesterol. The ¹⁸O-cholesterol instead appears to be evenly distributed within the plasma membrane. The significance of this work lies in the fact that it reports for the first time that cholesterol and sphingolipids have been directly imaged in the plasma membrane of an intact cell without the use of potentially perturbing labels.

Interaction of the cytoskeleton with the clusters of influenza hemagglutinin

HA clusters co-localize with actin. Individual molecular trajectories in live cells show restricted HA mobility on actin. However, HA does not directly bind to actin as it is mobile on timescales much shorter than those of actin remodeling. The actin-binding protein cofilin was excluded from regions within several hundred nanometers of HA clusters, suggesting that HA controls a cytoskeletal agent and thus the concept of membrane protein–cytoskeletal crosstalk. While the idea of the cytoskeleton organizing membrane proteins is not new, here HA is organizing the cytoskeleton. The angular dependence is not random, consistent with the hypothesis that HA diffusion is constrained by boundary reflection owing to diffusional barrier fences, implying a relatively immobile set of pickets. Yet, even the restricted HA moves. Either there is another, as yet undetected, picket or this model is wrong. To test which is correct, we will carry out a pull-down of HA–binding components in these cells, and perform proteomic mass spectrometry. We are aware that the cell lysis conditions (detergent, sonication and temperature) will be critical for these assays and may influence the proteins identified as binding partners. We will consider using osmotic stress to keep weak binding partners together throughout the pull-down procedure.

The mechanism by which insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments

Membrane remodeling and domain organization are at the pathophysiological core of type II diabetes and its precursor, insulin resistance, because glucose transport is the rate-limiting step in glucose uptake in both adipose and muscle cells. The glucose transporter-4 (GLUT4) is the key molecule responsible for insulin-stimulated glucose uptake, and its ability to function is determined by its translocation (redistribution) from an intracellular compartment to the cell surface in response to insulin. A signaling cascade induced by insulin affects multiple stages of GLUT4 translocation: intracellular trafficking of GLUT4 storage vesicles (GSV), docking (tethering) of GSV to the plasma membrane (PM), and GSV fusion with PM that finally delivers GLUT4 to the cell surface.

Given that the hallmark of glucose metabolism is insulin-stimulated delivery of glucose transporter-4 (GLUT4) to the plasma membrane (PM) and the hallmark of membrane protein organization is its domain structure, we examined insulin's effect on GLUT4 organization in PM of adipose cells. After delivery to the PM, all GLUT4 monomers' outside domains diffuse freely, but GLUT4 within elongated domains (sized 60–240 nm) diffuse with confinement. Insulin stimulates dissociation of GLUT4 monomers from the domains but does not stimulate monomer-domain association, thereby shifting most PM GLUT4 from clustered to dispersed states. While outside the domains, GLUT4 monomers collide frequently but do not form new domains; GLUT4 domain formation is only observed immediately upon exocytosis. Insulin also inhibits exit of GLUT4 from the PM, which occurs through endocytosis only at the domains. Thus, insulin not only regulates both exocytosis and endocytosis of GLUT4, it also regulates molecular details of its diffusion, all to control glucose homeostasis.

Dynamics of protein domains in the catalysis of membrane fission: dynamin

Biological membrane fission requires protein-driven stress. The guanosine triphosphatase (GTPase) dynamin builds up membrane stress by polymerizing into a helical collar that constricts the neck of budding vesicles. How this curvature stress mediates nonleaky membrane remodeling is actively debated. The GTPase dynamin is critical to membrane fission during endocytosis, but how does dynamin use the energy of GTP hydrolysis for membrane remodeling? Having developed model membrane assays for distinguishing membrane hemifusion from fusion pore formation, we developed a specific lipid nanotubule system to study dynamin on the small neck at a budding vesicle. By monitoring ionic permeability through these lipid nanotubes (NT), we determined that dynamin produced narrow NT whose radii depend on the NT lipid composition. Fission follows GTPase-dependent cycles of assembly and disassembly of dynamin involving a stochastic process that depends on the curvature stress imposed by dynamin. Suprisingly, NT widen immediately before fission. The absence of leakage upon fission rules out tension-driven rupture and resealing, leaving the hemifission pathway hypothesis viable. We propose that dynamin transmits GTP's energy to periodic assembling of a limited curvature scaffold that brings lipids to an unstable intermediate. The aim of this year's project is to determine the energetic pathway for dynamin assembly and the energetic landscape for dynamin-mediated fission.

Using short lipid nanotubes as substrates to directly measure geometric intermediates of the fission pathway, we found that GTP hydrolysis–mediated assembly and disassembly cycles drive dynamin polymerization into short, metastable collars that are optimal for fission. Collars as short as two-rungs can translate radial constriction to reversible hemifission via membrane wedging of the pleckstrin homology domains (PHD) of dynamin. Modeling reveals that tilting of the PHDs to conform with membrane deformations creates the lowest possible energy pathway for hemifission. This local coordination of dynamin and lipids suggests a novel paradigm of membrane remodeling in cells. The theoretical analysis reveals that a stable dynamin polymer constrains highly curved membrane structures, thereby effectively inhibiting topological transitions, just as tighter substrate binding inhibits enzymatic catalysis. These constraints are partially relaxed in short and metastable dynamin scaffolds, which not only apply elastic stress (mechano-chemical effects) but also, in coordination with lipids, participate in stochastic searching for the optimal pathway of membrane rearrangements (catalytic effects). The nanoscale coordination between the geometry of the protein scaffold, concerted membrane wedging, and the shape of the lipid bilayer results in a distinct geometric catalytic pathway to hemifission. This coordination can be supported by any membrane-inserting protein complex with a ring-like structure, providing a new structural rationale for protein-regulatory and catalytic function in membrane remodeling.

Cellular GLUT4 trafficking in insulin-resistant subjects

How insulin resistance develops is not clear. By elucidating the PM remodeling and organizational events that insulin triggers, and how they are differentially regulated in cells from human subjects, we hope to determine the cellular defect causing insulin resistance. Adipose cells play an important role in the regulation and maintenance of glucose and lipid metabolism and are of clinical importance in type II diabetes. Adipose tissue plays a crucial regulatory function by integrating lipid and glucose metabolism and exerting significant influence over the metabolic function of others tissues via adipokines; adipose tissue controls stored fat distribution among fat depots, muscle, and liver where increased ectopic fat storage is highly associated with systemic insulin resistance. Thus we aim to test insulin action in cells from adipose tissue. Adipose cells from insulin-resistant human subjects exhibit decreased levels of glucose transporter-4 (GLUT4) and impaired insulin signaling. We investigated the dynamics of GLUT4 trafficking and the insulin-stimulated translocation of GLUT4 in adipose cells isolated from human subjects with varying body mass indexes (BMI) and insulin sensitivities (SI). Cells were transfected with HA-GLUT4-GFP/mCherry and imaged live, using total internal reflection fluorescent microscopy to monitor GLUT4 storage vesicle (GSV) trafficking and fusion with the PM. We used confocal microscopy to assess the redistribution of HA-GLUT4-GFP to the PM, using the surface-exposed HA epitope, and to distinguish dispersed from clustered transporters. Without insulin, GSV trafficking on microtubules and fusion with the PM, and total cell-surface GLUT4, do not vary with donor subject SI. However, while insulin in cells from insulin-sensitive subjects halts GSV trafficking by stimulating tethering and fusion to PM, thereby increasing cell-surface GLUT4, the effects diminish with decreasing SI, without affecting PM GLUT4 cluster number. In a subgroup of subjects with BMIs of 25 to 35, we found that altered GLUT4 trafficking highly correlated with systemic insulin resistance, independent of BMI. We suggest that development of systemic insulin resistance is associated with maintenance of basal GLUT4 trafficking and PM clusters, but with diminished insulin-stimulated GSV tethering and fusion, and cell-surface GLUT4, independent of obesity, and that this altered insulin responsiveness in adipose cells may represent a fundamental mechanistic link between cellular and systemic dysfunction.

Additional Funding

Jain Foundation
Bench-to-Bedside Award

Publications

Lizunov VA, Lee JP, Skarulis MC, Zimmerberg J, Cushman SW, Stenkula KG. Impaired tethering and fusion of GLUT4 vesicles in insulin-resistant human adipose cells. Diabetes 2013;62:3114-3119.
Shnyrova AV, Bashkirov PV, Akimov SA, Pucadyil TJ, Zimmerberg J, Schmid SL, Frolov VA. Geometric catalysis of membrane fission driven by flexible dynamin rings. Science 2013;339:1433-1436.
Frisz JF, Klitzing HA, Lou K, Hutcheon ID, Weber PK, Zimmerberg J, Kraft ML. Sphingolipid domains in the plasma membranes of fibroblasts are not enriched with cholesterol. J Biol Chem 2013;288:16855-16861.
Glushakova S, Lizunov V, Blank PS, Melikov K, Humphrey G, Zimmerberg J. Cytoplasmic free Ca2+ is essential for multiple steps in malaria parasite egress from infected erythrocytes. Malar J 2013;12:41.
Lizunov VA, Stenkula K, Troy A, Cushman SW, Zimmerberg J. Insulin regulates Glut4 confinement in plasma membrane clusters in adipose cells. PLoS One 2013;8:e57559.

Collaborators

Pavel Bashkirov, PhD, Russian Academy of Sciences, A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, Moscow, Russia
Alexander Berezhkovskii, PhD, Mathematical and Statistical Computing Laboratory, CIT, NIH, Bethesda, MD
Sergey Bezrukov, PhD, Program in Physical Biology, NICHD, Bethesda, MD
Samuel W. Cushman, PhD, Diabetes Branch, NIDDK, 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
Jeffery Miller, MD, Molecular Medicine Branch, NIDDK, Bethesda, MD
Alfredo Quinones-Hinojosa, MD, The Johns Hopkins University, Baltimore, 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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