The Biophysics of Protein-Lipid Interactions in Influenza, Malaria, and Muscular Dystrophy
- Joshua Zimmerberg, MD, PhD, Head, Section on Integrative Biophysics
- Paul S. Blank, PhD, Staff Scientist
- Svetlana Glushakova, MD, PhD, Staff Scientist
- Matthias Garten, PhD, Visiting Fellow
- Sourav Haldar, PhD, Visiting Fellow
- Ludmila Bezrukov, MS, Chemist
- Hang Waters, MS, Biologist
- Elena Mekhedov, MA, Contractor
- Tatyana I. Tenkova-Heuser, PhD, Contractor
- Glen Humphrey, PhD, Guest Researcher
- John E. Heuser, MD, Senior Biophysicist
- Jennifer Petersen, PhD, Electron Microscopist
- Katherine Chang, BA, Postbaccalaureate Fellow
- Alexandra Sjaarda, BA, Postbaccalaureate Fellow
- Blaise A. Stearns, BS, Postbaccalaureate Fellow
Fusion and fission, the instances when organelles gain or lose their identities, are the essence of complex membrane dynamics in living cells and are key elements of synapses and other dynamic cellular trafficking networks. Without fusion and fission, enveloped viruses and parasites could not enter cells, replicate, or exit cells, nor would inflammatory cells respond and kill such invaders or deal with sick cells. Our earliest work concentrated on model membrane systems, the physical properties and theoretical pathways required for membrane fusion to occur, and the discovery that tension spreads headgroups for hemifusion, then pulls open fusion pores to allow coalescence of adherent bilayers. However, while able to focus on basic membrane biophysical properties and help develop a theoretical framework for understanding membrane interactions, model systems are a simplification that ignores the important roles of proteins. Including the role of proteins in these fundamental biophysical processes was both fruitful and informative, culminating in what we believed to be a canonical framework for understanding both fusion and fission. We introduced a simple paradigm: proteins act as catalysts (bilayer topoisomerases) for lowering the high energy barriers to membrane remodeling steps. A few amino acids of a specialized protein domain can reversibly enter the hydrophobic membrane matrix or cover the headgroups as inclusions or scaffolds, respectively, and thus transiently alter the thermodynamics of the system by specific protein-lipid interactions. By combining quantitative light microscopy with electrophysiology and with reconstitution of fusion and fission in lipid bilayer membranes, we constructed hypotheses with predicted fusion intermediates whose dimensions were deduced by continuum theory and fits to experiments. The predicted sizes were detectable by cryo-electron microscopy, so we labored to achieve the highest-resolution electron microscopy of hydrated membrane fusion events in order to understand how proteins catalyze the new configurations of lipids that ultimately mediate these processes.
By successfully installing a new technology at NIH, the Volta Phase Plate, we were able to visualize the predicted hemifusion diaphragm mediated by the hemagglutinin (HA) of the influenza virus (IFV), and the measurements of its dimensions fit the predictions of continuum theory. However, another result was unexpected: HA catalyzed the breakage of membranes, leading to free membrane edges—often in great profusion. To understand why this was unexpected, one must consider the physical forces that act on lipids in solution: membranes avoid edges. The lipid bilayer is self-assembling because its free energy of cohesion (which derives in part from enthalpic attractive forces between hydrocarbon chains and in part from the entropic hydrophobic effect that minimizes interfacial area) automatically ensures stability of the lipid bilayer. Formally, the edge of an otherwise lamellar membrane has a large linear tension, i.e., should be a high-energy region that the membrane seeks to minimize. Nevertheless, we observe that 'free edges' do indeed outnumber hemifusion diaphragms for certain lipid compositions of target membranes. Such edges only occur in close vicinity to activated HA molecules, indicating that edges are triggered to form by the same event that triggers full fusion, namely, the amphipathic helix of HA being ejected from HA and binding to the target bilayer. We can only presume for the moment that the HA fusion peptide somehow stabilizes the observed membrane edges, i.e., drastically lowers bilayer line-tension. This observation and the resultant hypothesis form the basis of our future work on the influenza virus.
While most medical research aimed at reducing the impact of the influenza virus is focused on vaccines, anti-viral therapeutic strategies should not lag behind. Influenza is estimated to result in between 9.3 million to 49.0 million illnesses, between 140,000 to 960,000 hospitalizations and between 12,000 to 79,000 deaths annually. One crucial stage of infection is the moment when the virus first assembles on the cellular membrane to bud out, starting a new life cycle of replication. Some of the unique lipids that abound in cellular functions are known as the phosphoinositides. These lipids, such as phosphoinositolphosphates PIP, PIP2, and PIP3, have an essential role in exocytosis that has not yet been elucidated. They are found in clusters, yet no one can define the mechanism of lipid clustering or the dependence of lipid clustering on membrane proteins. For these questions we turned to high-resolution light microscopy of HA, a known membrane protein involved in membrane fusion, and we discovered that HA clusters PIP2.
Also this year, we continued research on the physiology of the malaria parasite Plasmodium falciparum. Despite some progress in the combating malaria, there were 219 million cases of malaria in 2017, up from 217 million cases in 2016. The latest estimate of deaths from this disease is still very high, i.e. over 445,000 deaths per year in 2016 and 2017, mostly in children under the age of 5. With no vaccine, and drug resistance climbing, we are also focused on the unique membrane biology of the parasites that cause malaria, to find new targets for therapy. In our work, methods have been compiled allowing a comprehensive quantitative evaluation of parasite replication in erythrocytes. Last year, we showed that the Plasmodium translocon of exported proteins (PTEX) in the parasite vacuolar membrane critically transports proteins from the parasite to the erythrocytic cytosol and membrane to create protein infrastructure important for virulence. The components of PTEX are stored within the dense granule, which is secreted from the parasite during invasion. We showed that EXP2 (exported protein 2), one component of PTEX, also formed the nutrient channel of the parasite vacuole membrane. We described a protein, RON3, from another invasion organelle, the rhoptry, that is also secreted during invasion. We found that RON3 is required for the protein transport function of the PTEX and for glucose transport from the red blood cell cytoplasm to the parasite, a function thought to be mediated by PTEX component EXP2. Other parasite vacuole proteins include highly expressed single-pass transmembrane proteins such as EXP1 (exported protein 1). We found that EXP1 is required for the EXP2–based nutrient-permeable channel activity of the parasite vacuole.
Towards the goal of understanding the pathophysiology of mild blast induced TBI (bTBI), we also devoted research time to identifying the physical forces associated with the primary injury phase.
Mechanisms of poly-phosphoinositide clustering by the influenza viral hemagglutinin, HA
Although the lateral organization of proteins and lipids (clustering) in the cell plasma membrane (PM) is crucial to diverse fundamental cellular processes, there is considerable disagreement on the organizational mechanisms that govern such clustering, e.g., first, confinement by cytoskeleton-based fences, second, protein-specific partitioning into liquid-ordered lipid rafts, or third, tethering of groups of molecules to the underlying actin cytoskeleton, among others. One reason a mechanistic understanding of the organizing principles has remained elusive is that such nanoscale molecular assemblies are highly dynamic, requiring recordings of individual molecules at a higher temporal bandwidth than hitherto possible to gain a better understanding of the physicochemical principles that regulate membrane clustering. In addition to physiological processes, the pathophysiological basis of disease states is increasingly focused on clusters. HA localized to the PM of host cells clusters spontaneously and is crucial for fusion, viral budding, and infection; high HA density on resultant virions is needed for entry into and fusion with the next host cell. Yet, even this model system generates conflicting data on the mechanism of lipid clustering with HA, and there is not even qualitative agreement as to which lipids cocluster with HA. In contrast to other mechanisms of protein-lipid interactions, such as ordering of molecules into lipid rafts, lipid confinement by protein fences, tethering of lipid motion, or buffering by fixed binding sites, our findings describe and explain spatial PIP2 distributions and how they change in time via a distinctly dynamic mechanism: a potential gradient resulting from binding sites that are themselves both mobile and clustered.
The lipid phosphatidylinositol 4,5-bisphosphate (PIP2) forms nanoscopic clusters in cell plasma membranes; however, the processes determining PIP2 mobility and thus its spatial patterns are not fully understood. Using superresolution imaging of living cells, we found that PIP2 is tightly colocalized with and modulated by overexpression of the influenza viral protein hemagglutinin (HA). Within and near clusters, HA and PIP2 follow a similar spatial dependence, which can be described by an HA–dependent potential gradient; PIP2 molecules move as if they are attracted to the center of clusters by a radial force of 0.079 0.002 pN in HAb2 cells. The measured clustering and dynamics of PIP2 are inconsistent with the unmodified forms of the raft, tether, and fence models. Rather, we found that the spatial PIP2 distributions and how they change in time are explained by a novel, to our knowledge, dynamic mechanism: a radial gradient of PIP2 binding sites that are themselves mobile. The model may be useful for understanding other biological membrane domains whose distributions display gradients in density while maintaining their mobility.
Membranes during invasion of Plasmodium falciparum, the causative agent of malaria
We focus on our continued research on the physiology of the deadly malaria parasite Plasmodium falciparum. By developing, publishing, and promulgating new methods to study the biology of the malaria parasite, our work has impacted the field by transforming qualitative imaging into quantitative measures, by providing, e.g., the first recordings of P. falciparum egress and invasion of erythrocytes, and by describing new phenomena such as shape transformation of infected cells, which signals the egress initiation and membrane transformation upon egress. We developed several noninterventional methods that permit fine-staging of cell phenotype and quantification of the parasite replication cycle, as it naturally progresses from parasite invasion of erythrocytes to parasite egress from the host cells.
Intracellular malaria parasites grow in a vacuole delimited by the parasitophorous vacuolar membrane (PVM). The membrane fulfills critical roles for survival of the parasite in its intracellular niche such as in protein export and nutrient acquisition. Using a conditional knockout, this year we demonstrated that the abundant integral PVM protein EXP1 is essential for parasite survival but that this is independent of its previously postulated function as a glutathione S-transferase. Patch-clamp experiments indicated that EXP1 is critical for the nutrient-permeable channel activity at the PVM. Loss of EXP1 abolished the correct localization of EXP2, a pore-forming protein required for the nutrient-permeable channel activity and protein export at the PVM. Unexpectedly however, loss of EXP1 affected only the nutrient-permeable channel activity of the PVM but not protein export. Parasites with low levels of EXP1 became hypersensitive to low nutrient conditions, indicating that EXP1 is indeed needed for nutrient uptake and experimentally confirming the long-standing hypothesis that the channel activity measured at the PVM is required for parasite nutrient acquisition. Hence, EXP1 is specifically required for the functional expression of EXP2, the nutrient-permeable channel. Both EXP1 and EXP2 are critical for the metabolite supply of malaria parasites.
The survival of Plasmodium spp. within the host red blood cell (RBC) depends on the function of a membrane protein complex, termed the Plasmodium translocon of exported proteins (PTEX), that exports certain parasite proteins, collectively referred to as the exportome, across the parasitophorous vacuolar membrane (PVM) that encases the parasite in the host RBC cytoplasm. The core of PTEX consists of three proteins: EXP2, PTEX150, and the HSP101 ATPase; of these three proteins, only EXP2 is a membrane protein. Studying the PTEX–dependent transport of members of the exportome, we discovered that exported proteins, such as ring-infected erythrocyte surface antigen (RESA), failed to be transported in parasites in which the parasite rhoptry protein RON3 was conditionally disrupted. RON3–deficient parasites also failed to develop beyond the ring stage, and glucose uptake was significantly reduced. These findings provide evidence that RON3 influences two translocation functions, namely, transport of the parasite exportome through PTEX and the transport of glucose from the RBC cytoplasm to the parasitophorous vacuolar (PV) space, where it can enter the parasite via the hexose transporter (HT) in the parasite plasma membrane.
Physical forces that drive CNS cell excitation during mild blast-induced traumatic brain injury
We developed a system that couples a pneumatic blast to a microfluidic channel to precisely and reproducibly deliver shear transients to dissociated human central nervous system (CNS) cells, on a time scale comparable to an explosive blast but with minimal pressure transients. Using fluorescent beads, we characterized the shear transients experienced by the cells, and demonstrated that the system is capable of accurately and reproducibly delivering uniform shear transients with minimal pressure across the cell-culture volume. The system is compatible with high-resolution, time-lapse optical microscopy. Using this system, we demonstrated that blast-like shear transients produced with minimal pressure transients and sub-millisecond rise times activate calcium responses in dissociated human CNS cultures. Cells respond with increased cytosolic free calcium to a threshold shear stress between 8-25 Pa; the propagation of this calcium response is a result of purinergic signaling. We propose that this system models, in vitro, the fundamental injury wave produced by shear forces consequent to blast shock waves passing through density inhomogeneity in human CNS.
Additional Funding
- 2018 Deputy Director for Intramural Research (DDIR) Innovator’s Award
- NIH Intramural-to-Russia (I-to-R) Program Award
- Office of AIDS Research (OAR) Award
Publications
- Garten M, Nasamu AS, Niles JC, Zimmerberg J, Goldberg DE, Beck JR. EXP2 is a nutrient-permeable channel in the vacuolar membrane of Plasmodium and is essential for protein export via PTEX. Nat Microbiol 2018;3:1090-1098.
- Glushakova S, Beck JR, Garten M, Busse BL, Nasamu AS, Tenkova-Heuser T, Heuser J, Goldberg DE, Zimmerberg J. Rounding precedes rupture and breakdown of vacuolar membranes minutes before malaria parasite egress from erythrocytes. Cell Microbiol 2018;20:e12868.
- Mesén-Ramírez P, Bergmann B, Tran TT, Garten M, Stäcker J, Naranjo-Prado I, Höhn K, Zimmerberg J, Spielmann T. EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites. PLoS Biology 2019;17:e3000473.
- Ravin R, Morgan NY, Blank PS, Ravin N, Guerrero-Cazares H, Quinones-Hinojosa A, Zimmerberg J. Response to blast-like shear stresses associated with mild blast-induced brain injury. Biophys J 2019;117:1167-1178.
- Curthoys NM, Mlodzianoski MJ, Parent M, Butler MB, Raut P, Wallace J, Lilieholm J, Mehmood K, Maginnis MS, Waters H, Busse B, Zimmerberg J, Hess ST. Influenza hemagglutinin modulates phosphatidylinositol 4,5-bisphosphate membrane clustering. Biophys J 2019;116:893-909.
Collaborators
- Oleg Batishchev, PhD, A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, Russia
- Josh Beck, PhD, Iowa State University, Ames, IA
- Michael J. Blackman, PhD, The Francis Crick Institute and London School of Hygiene & Tropical Medicine, London, United Kingdom
- Nikki Curthoys, PhD, University of Maine, Orono, ME
- Andrew Demidowich, MD, PhD, Section on Growth and Obesity, NICHD, Bethesda, MD
- Rick M. Fairhurst, MD, PhD, Laboratory of Malaria and Vector Research, NIAID, Bethesda, MD
- Vadim Frolov, PhD, Universidad del País Vasco, Bilbao, Spain
- Daniel Goldberg, MD, PhD, Washington University St. Louis, St. Louis, MO
- Samuel T. Hess, PhD, University of Maine, Orono, ME
- Mary Kraft, PhD, University of Illinois at Urbana-Champaign, Urbana, IL
- Louis H. Miller, MD, Laboratory of Malaria & Vector Research, NIAID, Bethesda, MD
- Richard Pastor, PhD, Laboratory of Membrane Biophysics, NHLBI, Bethesda, MD
- Thomas S. Reese, MD, Laboratory of Neurobiology, NINDS, Bethesda, MD
- Anna Shnyrova, PhD, Universidad del País Vasco, Bilbao, Spain
- Tobias Spielmann, PhD, Bernhard-Nocht-Institut für Tropenmedizin, Hamburg, Germany
- Peter K. Weber, PhD, Lawrence Livermore National Laboratory, Livermore, CA
- Jack Yanovski, MD, PhD, Section on Growth and Obesity, NICHD, Bethesda, MD
Contact
For more information, email zimmerbj@mail.nih.gov or visit http://irp.nih.gov/pi/joshua-zimmerberg.
