1. The highly conserved cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) contains three cysteines, post-translationally modified by covalently bound fatty acids. While viral HA acylation is crucial in virus replication, its physico-chemical role is unknown. We used virus-like particles (VLP) to study the effect of acylation on morphology, protein incorporation, lipid composition, and membrane fusion. De-acylation interrupted HA-M1 interactions since de-acylated mutant HA fail to incorporate an M1 layer within spheroidal VLP, and filamentous particles incorporated increased numbers of neuraminidase (NA). While HA acylation did not influence VLP shape, lipid composition, or HA lateral spacing, acylation significantly affected envelope curvature. Compared to wild type HA, de-acylated HA is correlated with released particles with flatter envelope curvature in the absence of the matrix (M1) protein layer. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations, and was found to be comparable to the curvature values derived from VLP size distributions. Cell-cell fusion assays show a strain-independent failure of fusion pore enlargement amongst H2 (A/Japan/305/57), H3 (A/Aichi/2/68), H3 (A/Udorn/72). In contradistinction, acylation made no difference in the low pH-dependent fusion of isolated VLPs to liposomes: fusion pores formed and expanded as demonstrated by the presence of complete fusion products observed using cryo-electron tomography (cryo-ET). We propose that the primary mechanism of action of acylation is to control membrane curvature and to modify HAs interaction with M1 protein, while the stunting of fusion by deacylated HA acting in isolation may be balanced by other viral proteins, which help lower the energetic barrier to pore expansion. 2. While many parasites develop within host cells to avoid antibody responses and to utilize host cytoplasmic resources, elaborate egress processes have evolved to minimize the time between escaping and invading the next cell. In human erythrocytes, malaria parasites perforate their enclosing erythrocyte membrane shortly before egress. Here, we show that these pores clearly function as an entry pathway into infected erythrocytes for compounds that inhibit parasite egress. The natural glycosaminoglycan heparin surprisingly inhibited malaria parasite egress, trapping merozoites within infected erythrocytes. Labeled heparin neither bound to nor translocated through the intact erythrocyte membrane during parasite development, but fluxed into erythrocytes at the last minute of the parasite lifecycle. This short encounter was sufficient to significantly inhibit parasite egress and dispersion. Heparin blocks egress by interacting with both the surface of intra-erythrocytic merozoites and the inner aspect of erythrocyte membranes, preventing the rupture of infected erythrocytes but not parasitophorous vacuoles, and independently interfering with merozoite disaggregation. Since this action of heparin recapitulates that of neutralizing antibodies,membrane poration presentsa briefopportunity for a new strategy to inhibit egress. 3. The integrity and stability of biological membranes are critical to their biological functions; losses in these properties are associated with disease. For example, fragile sarcolemmal-associated muscular dystrophies, a class of genetic disorders, are correlated with a deficiency in key proteins leading to increased death of muscle cells, presumably through cellular membrane breakage. Damage to lipid bilayer membranes initiates from defect sites, referred to as pores, which provide an aqueous pathway through the membrane. Stability to pore formation can be described using pore edge line tension, which is an energetic parameter describing the energy barrier height at a supercritical point beyond which the pore irreversibly expands, while before it the pore spontaneously reseals. In contradistinction, here we show that line tension is insufficient to explain how membranes with branched-chain lipids resist breaking, since they have approximately the same pore edge line tension as membranes with non-branched lipids. Planar phospholipid bilayer membranes containing isoprenoid (branched hydrocarbon tail) lipids exhibit significantly higher stability to pore formation than membranes containing non-branched lipids, both by conventional electroporation measurements and by molecular dynamics simulation of pore closure kinetics. Importantly, muscle fibers from dysferlinopathic mice were significantly resistant to wounding after treatment with branched-chain lipids. We propose that this increased stability is due to increased membrane viscosity and decreased probability of pore formation (nucleation), and suggest a metastable theory of pore formation wherein both system thermodynamics and pore growth kinetics are considered. 4. 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. Towards the goal of understanding the pathophysiology of mild blast induced TBI (bTBI), and identifying the physical forces associated with the primary injury phase, 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 have characterized the shear transients experienced by the cells, and demonstrate that the system is capable of accurately and reproducibly delivering uniform shear transients with minimal pressure across the cell culture volume. This system is compatible with high resolution, time-lapse optical microscopy. Using this system, we demonstrate 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.
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