Joshua Zimmerberg, MD PhD - US grants

This project is aimed at the understanding of the physico-chemical mechanisms of membrane remodeling during physiological and pathogenic events. There are two components: [unreadable] [unreadable] 1. Intracellular malaria parasites leave their host erythrocytes to infect neighbouring cells after each cycle of asexual replication. No method is currently available for the direct quantification of parasite release. To quantify parasite release process, human erythrocytes infected with Plasmodium falciparum were injected into sealed chambers at optimal density, where they progressed 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 with haemozoin, and 3) fragments of the ruptured membranes. These footprints are stable for hours, allowing precise identification using differential interference contrast (DIC) microscopy. The relative rate of parasite release is defined as the percent of such footprints out of all schizonts injected and incubated into chamber at 37C for two hours. The method is highly reproducible, easy to perform, and does not require expensive equipment. Additionally, this method allows one to analyse cell and release site morphology, which adds information about the release process and the quality of the culture. The method is used here to show that swelling of schizonts caused by protein-free media inhibits parasite release.[unreadable] [unreadable] In conclusion, a novel method is described to count sites of parasite release by microscopy. Besides the direct estimation of parasite release from infected erythrocytes, this method provides a morphological evaluation of normal infected cells approaching the end of the plasmodial life cycle, or pathological forms accumulated as the result of experimental intervention in the parasite release process. One may now accurately estimate the relative parasite release rate at the time of cycle transition, without any obligatory coupling to parasite invasion.[unreadable] [unreadable] 2. The shape of enveloped viruses depends critically on an internal protein matrix, yet it remains unclear how the matrix proteins control the geometry of the envelope membrane. We found that matrix proteins, purified form Newcastle Disease Virus, adsorb on a phospholipid bilayer and condense into fluid-like domains that cause membrane deformation and budding of spherical vesicles, as seen by fluorescent and electron microscopy. Measurements of the electrical admittance of the membrane resolved the gradual growth and rapid closure of a bud, followed by its separation to form a free vesicle. The vesicle size distribution, confined by intrinsic curvature of budding domains, but broadened by their merger, matched the virus size distribution. Thus, matrix proteins implement domain-driven mechanism of budding, which suffices to control the shape of these proteolipid vesicles.

Based on amphiphiles, osmotic stress, and protease inhibitors, we hypothesize that egress is pressure driven through folding and fragmentation of the enzymatically altered erythrocyte membrane. Osmotic pressure could build up in either the parasitophorous vacuole (PV) or the host cell cytoplasm. We propose and test the idea that parasites easily pass through a hemoglobin-depleted erythrocyte cytoplasm and breach a weakened erythrocyte membrane. The vacuole swells several minutes before parasite egress. Starting adjacent to the parasites space, vacuole swelling later extends in all directions. At the same time, the visible area of the erythrocyte compartment shrinks, suggesting redistribution of water between the erythrocyte cytosol and vacuole. Eventually, dissociated parasites leave the host cell by breaching first the PVM (presumably when PV critical volume is reached) and then the erythrocyte membrane. Thus, dependence of egress on osmotic pressure can be described in terms of erythrocyte hydration, which affects the swelling and rupture of the vacuole. To test the relationship between erythrocyte hydration and parasite egress, we used dehydrated erythrocytes from donors homozygous for sickle hemoglobin gene (HbSS versus normal HbAA). Sickle erythrocytes do support P. falciparum replication, but dehydrated sickle cells did not allow normal egress. The decreased erythrocyte volume may contribute to malarial protection in individuals with sickle erythrocytes. Notably, decreased erythrocyte volume is a characteristic for individuals with thalassemia and iron deficiency. Alternatively, one may speculate that P. vivax gained an advantage by targeting reticulocytes, the largest circulating erythrocytes in the host. The negative effect of erythrocyte dehydration on parasite egress, demonstrated here, and on invasion emphasizes the general importance of host cell hydration for the asexual cycle of malaria parasites. In this project, we found two new essential steps in the program of Plasmodium falciparum egress from erythrocytes. First, the parasitophorous vacuole swells as the erythrocyte shrinks, suggesting ion and water redistribution between these two compartments of infected cells. At the end of the cycle, vacuole swelling apparently provides the space for parasite dissociation prior to egress, leading to vacuole rupture. In the midst of erythrocyte shrinkage, the tension of erythrocyte membrane decreases without loss of integrity. Second, parasite egress requires host cell membrane poration prior to host cell membrane rupture. Membrane poration is observed in erythrocytes that are not swollen, thus it does not result from critical membrane stretching. Perhaps either release of protein from erythrocytes or an influx of ions into the host cells, or both, is needed for the asexual parasite cycle to complete. Alternatively, host cell membrane poration could serve to weaken a barrier that parasites must breach to egress. Similarities in parasite egress mechanisms between two families of the phylum Apicomplexa, Plasmodium, and Toxo- plasma are emerging: both type of parasites make pores in host cell membrane and activate host cell calpain prior to egress. Because P. falciparum has multiple experimental limitations, Toxoplasma, a more conventional organism, emerges as a model for Apicomplexan biology. Regardless, egress of parasites is a vital step of diseases devastating humanity. Our appreciation of a more complex egress program provides more targets for novel antimalarials, just as it may help to explain the selective advantage that the sickle trait confers upon its carriers. 2) Electron tomography (ET) provides a three-dimensional (3D) view of cellular ultrastructure at nanoscale spatial resolution, and thus gives unique insight into the supramolecular basis of biological processes. In this project we investigated an alternative approach to electron tomography that yields 3D reconstructions of thicker (1m) sections at resolutions comparable to conventional ET of thin sections. The ability to perform 3D reconstructions from larger volumes is particularly attractive for studying unicellular eukaryotic microorganisms, some of which are sufficiently small to be contained within just a few serial sections. Our approach is also valuable for reconstructing entire mammalian cells using serial thick-section tomography. Scanning transmission electron tomography using a tightly focused electron probe can overcome some of the limitations imposed by conventional ET. First, the ability to focus the probe dynamically in STEM enables in-focus imaging of very large specimen areas even at the highest tilt angles. Second, because in STEM there are no image-forming lenses after the specimen, the resolution in images of thick specimens is not degraded by chromatic aberration. Generating high resolution STEM tomograms from entire cells that span several micrometers in depth can be accomplished by imaging serial 1-2 μm-thick sections. Here we demonstrate the feasibility of reconstructing an entire human erythrocyte infected with important human pathogen Plasmodium falciparum, the causative agent of malaria, from only four consecutive 1 μm-thick sections. Tomogram slices of one infected erythrocyte revealed the parasite during the process of schizogany, i.e. multiple nuclear divisions and formation of new parasites. At this stage of the parasite cycle active morphogenesis multiplies or produces de novo intracellular organelles for up to 32 new parasites within one schizont. The dynamics of morphogenesis is poorly understood because of the laborious procedure of 3D reconstructions of the serial thin sectioning of cells with complex architecture. The spatial resolution achievable with the new method allows us to identify the major organelles of schizont in thick sections, such as nuclei, rhoptries, pigment vacuole, rough endoplasmic reticulum, Golgi complex, apicoplast, and lipid body. Three layers of membranes surrounding the schizont are clearly identifiable: 1) the parasite plasma membrane, caught at the onset of invagination to form new parasites, 2) the membrane of the parasitophorus vacuole and 3) the erythrocyte membrane. Parasite-derived membrane structures such as tubular extensions of the vacuolar membrane, Maurers clefts and circular clefts, are visible inside erythrocyte cytoplasm as well. Thus, a new ultrastructural method is now available to study the complex dynamics of malaria parasite development inside human erythrocyte. The significance of this technique is that 10 nm resolution is sufficient to discern organelles, and the process of organellogenesis during schizogeny is currently obscured by the amorphous nature of standard ultrastructural views. This new technique is rapid enough to allow a series of schizonts to be studied and the morphological sequence of events established, as was recently achieved for parasite release. Already new objects are emerging whose identity is unclear and will require elaboration of labeling techniques for markers. Thus STEM tomography using axial detection for imaging thick sections of biological specimens at a spatial resolution around 5-10 nm, which is comparable to the spatial resolution of traditional ET from thin sections (typically 3-8 nm) is both feasible and advantageous. The demand for high-resolution, large-volume imaging of biological specimens has been answered so far by the large scale application of traditional ET of thin sections. The present study suggests that it will be possible to reconstruct conveniently and efficiently entire mammalian cells through serial thick-section STEM tomography.

Influenza remains a top killer of human beings throughout the world, in part because of the influenza virus's rapid binding to cells and its uptake into compartments hidden from the immune system. To attack the influenza virus during this time of hiding, we need to understand the physical forces that allow the internalized virus to infect the cell. In particular, we need to know how the protective coats of protein outside and inside the viral surface reacts to the changes in acid that come soon after internalization. In addition, Influenza virus assembles on the plasma membrane where viral proteins localize to form a bud encompassing the viral genome, which ultimately pinches off to give rise to newly formed infectious virions. Upon entry, the virus faces the opposite taskfusion with the endosomal membrane and disassembly to deliver the viral genome to the cytoplasm. There are at least four influenza proteinshemagglutinin (HA), neuraminidase (NA), matrix 1 protein (M1), and the M2 ion channelthat are known to directly interact with the cellular membrane and modify membrane curvature in order to both assemble and disassemble membrane-enveloped virions. 1. The virus that causes flu, influenza, is coated with spike proteins called hemagglutinin (HA) that play a large role in determining its immunogenicity and in the vaccine to flu. This HA protein plays a crucial role during infection after viral endocytosis, undergoing a conformational change that drives the membrane fusion of viral and endosomal membranes at the low pH of the endosome. Although membrane fusion is widely thought to proceed through an intermediate called hemifusion, in fact the hemifusion structure had never been determined. In this project, influenza virus-like particles carrying wild-type HA or an HA hemifusion mutant (G1S) and liposome mixtures were studied at low pH by cryo-electron tomography. For the first time in virology, the Volta phase plate was used, which improves the signal-to-noise ratio close to focus. We determined two distinct hemifusion structures: a hemifusion diaphragm and a novel structure termed a lipidic junction. Liposomes with lipidic junctions were ruptured with membrane edges stabilized by haemagglutinin. The rupture frequency and hemifusion diaphragm diameter were not affected by G1S mutation, but decreased when the cholesterol level in the liposomes was close to physiological concentrations. We propose that haemagglutinin induces a merger between the viral and target membranes by one of two independent pathways: a ruptureinsertion pathway leading to the lipidic junction and a hemifusion-stalk pathway leading to a fusion pore. The latter is relevant under the conditions of influenza virus infection of cells. Cholesterol concentration functions as a pathway switch because of its negative spontaneous curvature in the target bilayer, as determined by continuum analysis. 2. In cells, influenza virus is taken up from a pH-neutral extracellular milieu into an endosome, whose contents then acidify.. At a pH of about six, the viral matrix protein (M1) that coats the inner monolayer of the viral lipid envelope M1 interacts with the viral ribonucleoprotein (RNP) in a putative priming stage; at this stage, the interactions of the M1 scaffold coating the lipid envelope are intact. The M1 coat disintegrates as acidification continues to a pH of about five to clear a physical path for the viral genome to transit from the viral interior to the cytoplasm. This year we investigated the physicochemical mechanism of M1's pH-dependent disintegration. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. The energy of the interaction of M1 molecules with each other in M1 dimers was about 10 times as weak as that of the interaction of M1 molecules with the lipid bilayer. Acidification drives conformational changes in M1 molecules due to changes in the M1 charge, leading to alterations in their electrostatic interactions. Dropping the pH from 7.1 to 6.0 did not disturb the M1 layer; dropping it lower partially desorbed M1 because of increased repulsion between M1 monomers still stuck to the membrane. Lipid vesicles coated with M1 demonstrated pH-dependent rupture of the vesicle membrane, presumably because of the tension generated by this repulsive force. Thus, the disruption of the vesicles coincident with M1 protein scaffold disintegration at pH 5 likely stretches the lipid membrane to the point of rupture, promoting fusion pore widening for RNP release. 3. Blast-induced traumatic brain injury (bTBI) continues to be a worldwide health problem. bTBI can be complex, resulting from one or more physical phases of the blast phenomenon. Even those experiencing low-level blast explosions, such as those produced by explosives used to breach fortifications, can develop neurocognitive symptoms without evidence of neurotrauma. The cellular mechanisms of this phenomenon are unknown. The primary phase of bTBI, characterized by organ-shockwave interaction, is unique to blast exposure. Understanding the mechanisms and pathology arising from the primary phase of bTBI is limited, in part, because of the limited availability of in vitro models simulating the blast shockwave. Therefore, it is critical to develop experimental methods to study the primary phase of bTBI. To better study the primary phase of bTBI, we developed a pneumatic device that simulates an explosive blast by producing pressure transients similar to those observed in a free field explosion and is compatible with real-time fluorescence microscopy of cultured cells; this device can produce blast-like pressure transients with and without accompanying shear forces. Using Ca2+ ion-selective fluorescent indicators, changes in intracellular free calcium following simulated blast were detected. We previously showed that a) cultured human brain cells are indifferent to transient shockwave pressures known to cause mild bTBI, b) when sufficient shear forces are simultaneously induced with the shockwave pressure, central nervous system (CNS) cells respond with increased intracellular Ca2+ that propagates from cell to cell; and c) cell survival is unaffected 20 hours after shockwave exposure. In this years project, we report the cell type responsible for the waves of increased intracellular free Ca2+ in dissociated human CNS cultures, and that these calcium waves primarily propagate through astrocyte-dependent, purinergic signaling pathways that are blocked by P2 antagonists. Human astrocytes, compared to rat astrocytes, had an increased calcium response and prolonged calcium wave propagation kinetics, suggesting that in our model system rat CNS cells are less responsive to simulated blast. Furthermore, in response to simulated blast, human CNS cells have increased expressions of a reactive astrocyte marker, glial fibrillary acidic protein (GFAP) and a protease, matrix metallopeptidase 9 (MMP-9). The conjoint increased expression of GFAP and MMP-9 and a purinergic ATP (P2) receptor antagonist reduction in calcium response identifies both potential mechanisms for sustained changes in brain function following primary bTBI and therapeutic strategies targeting abnormal astrocyte activity.

1. EXP2 is a nutrient-permeable channel in the vacuolar membrane of Plasmodium and is essential for protein export via PTEX. Plasmodium spp. causes over 200 million annually reported malaria cases with more than 450,000 deaths, mostly in children under the age of 5. The blood stage of the parasite is responsible for the symptoms of malaria. Understanding how the parasite establishes a red blood cell infection and acquires nutrients is critical to devise new medicine to a parasite that repeatedly developed resistance to frontline treatments. Blood stage malaria parasites reside within a parasitophorous vacuolar membrane (PVM) formed when invading its host cell. Establishment of infection requires the parasite to export effector proteins into the red blood cell cytosol, as well as to import nutrients past the PVM. Protein export is achieved by a protein complex, the Plasmodium translocon of exported proteins (PTEX). Its putative membrane spanning pore complex consists of the protein EXP2, shares sequence homology with nutrient permeable pores of other apicomplexans suggesting a potential dual role of the protein in nutrient uptake and protein export. Using regulated gene expression we showed that EXP2 is essential for protein export. Further, EXP2 expression correlates with the occurrence of a previously characterized nutrient permeable PVM channel of unknown molecular identity in cell-attached patch clamp experiments. To show that EXP2 indeed constitutes the nutrient permeable PVM channel, charged amino acid residues of EXP2 were truncated. This diminished the response of the nutrient-permeable channel to applied voltages, thus identifying EXP2 as the channel-forming protein. These results put EXP2 in the center of focus for understanding nutrient import and protein export past the PVM in blood stage malaria, and therefore how to disrupt it. The realization represents an important step in understanding the interaction of the malaria parasite with its host cell. 2. Plasmepsins IX and X are essential and druggable mediators of malaria parasite egress and invasion. Proteases of the malaria parasite Plasmodium falciparum have long been investigated as drug targets. The P. falciparum genome encodes 10 aspartic proteases called plasmepsins, which are involved in diverse cellular processes. Most have been studied extensively but the functions of plasmepsins IX and X (PMIX and PMX) were unknown. We aimed to decipher the role of two putative proteases, plasmepsin 9 and 10 (PMIX and PMX), in parasite replicative cycle in human erythrocytes. Using our new quantitative assays based on light microscopy we showed that PMX is essential for both egress and invasion, controlling maturation of the subtilisin-like serine protease SUB1 in exoneme secretory vesicles. PMIX, in contrast, is essential for erythrocyte invasion, acting on rhoptry secretory organelle biogenesis. This study has identified compounds with potent antimalarial activity targeting PMX, including a compound known to have oral efficacy in a mouse model of malaria. 3. Rounding precedes rupture and breakdown of vacuolar membranes minutes before malaria parasite egress from erythrocytes. Because Plasmodium falciparum replicates inside of a parasitophorous vacuole (PV) within a human erythrocyte, parasite egress requires the rupture of two limiting-membranes. Parasite Ca2+, kinases, and proteases contribute to efficient egress; their coordination in space and time is not known. In this project, the kinetics of parasite egress were linked to specific steps with specific compartment markers, using live cell microscopy of parasites expressing PV-targeted fluorescent proteins, and specific egress inhibitors. Several minutes before egress, under control of parasite Ca2+i the parasitophorous vacuole began rounding. Then after 1.5 minutes, under control of PfPKG and SUB1, there was abrupt rupture of the PV membrane and release of vacuolar contents. Over the next 6 minutes there was progressive vacuolar membrane deterioration simultaneous with erythrocyte membrane distortion, lasting until the final minute of the egress program when newly-formed parasites mobilized, erythrocyte membranes permeabilized and then ruptured a dramatic finale to the parasite cycle of replication. The new stage discovered in this project has features that suggest the possibility of a new target for antimalarial drug development.