Nanoscale Dynamics of Actin Filaments in the Red Blood Cell Membrane Skeleton

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15-18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ is not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence (TIRF) and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200-300 nm apart interspersed with dimmer F-actin staining regions. Single molecule STORM imaging of Alexa-647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, non-random distribution of F-actin nodes. Treatment of RBCs with LatA and CytoD indicates F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape and membrane deformability.

Localized Cardiolipin Synthesis is Required for the Assembly of MreB During the Polarized Cell Division of Chlamydia trachomatis

Pathogenic Chlamydia species are coccoid bacteria that use the rod-shape determining protein MreB to direct septal peptidoglycan synthesis during their polarized cell division process. How the site of polarized budding is determined in this bacterium, where contextual features like membrane curvature are seemingly identical, is unclear. We hypothesized that the accumulation of the phospholipid, cardiolipin (CL), in specific regions of the cell membrane induces localized membrane changes that trigger the recruitment of MreB to the site where the bud will arise. To test this, we ectopically expressed cardiolipin synthase (Cls) and observed a polar distribution for this enzyme in Chlamydia trachomatis. In early division intermediates, Cls was restricted to the bud site where MreB is localized and peptidoglycan synthesis is initiated. The localization profile of Cls throughout division mimicked the distribution of lipids that stain with NAO, a dye that labels CL. Treatment of Chlamydia with 3-,6-dinonylneamine (diNN), an antibiotic targeting CL-containing membrane domains, resulted in redistribution of Cls and NAO-staining phospholipids. In addition, MreB localization was altered by diNN treatment, suggesting an upstream regulatory role for CL-containing membranes in directing the assembly of MreB. This hypothesis is consistent with the observation that the clustered localization of Cls is not dependent upon MreB function or peptidoglycan synthesis. Furthermore, expression of a CL-binding protein at the inner membrane of C. trachomatis dramatically inhibited bacterial growth supporting the importance of CL in the division process. Our findings implicate a critical role for localized CL synthesis in driving MreB assembly at the bud site during the polarized cell division of Chlamydia.

Embedding of HIV Egress within Cortical F-Actin

F-Actin remodeling is important for the spread of HIV via cell–cell contacts; however, the mechanisms by which HIV corrupts the actin cytoskeleton are poorly understood. Through live cell imaging and focused ion beam scanning electron microscopy (FIB-SEM), we observed F-Actin structures that exhibit strong positive curvature to be enriched for HIV buds. Virion proteomics, gene silencing, and viral mutagenesis supported a Cdc42-IQGAP1-Arp2/3 pathway as the primary intersection of HIV budding, membrane curvature and F-Actin regulation. Whilst HIV egress activated the Cdc42-Arp2/3 filopodial pathway, this came at the expense of cell-free viral release. Importantly, release could be rescued by cell–cell contact, provided Cdc42 and IQGAP1 were present. From these observations, we conclude that a proportion out-going HIV has corrupted a central F-Actin node that enables initial coupling of HIV buds to cortical F-Actin to place HIV at the leading cell edge. Whilst this initially prevents particle release, the maturation of cell–cell contacts signals back to this F-Actin node to enable viral release & subsequent infection of the contacting cell.

An ancestral interaction module promotes oligomerization in divergent mitochondrial ATP synthases

Mitochondrial ATP synthase forms stable dimers arranged into oligomeric assemblies that generate the inner-membrane curvature essential for efficient energy conversion. Here, we report cryo-EM structures of the intact ATP synthase dimer from Trypanosoma brucei in ten different rotational states. The model consists of 25 subunits, including nine lineage-specific, as well as 36 lipids. The rotary mechanism is influenced by the divergent peripheral stalk, conferring a greater conformational flexibility. Proton transfer in the lumenal half-channel occurs via a chain of five ordered water molecules. The dimerization interface is formed by subunit-g that is critical for interactions but not for the catalytic activity. Although overall dimer architecture varies among eukaryotes, we find that subunit-g together with subunit-e form an ancestral oligomerization motif, which is shared between the trypanosomal and mammalian lineages. Therefore, our data defines the subunit-g/e module as a structural component determining ATP synthase oligomeric assemblies.

Untargeted Lipidomics of Vesicular Stomatitis Virus-Infected Cells and Viral Particles

The viral lifecycle is critically dependent upon host lipids. Enveloped viral entry requires fusion between viral and cellular membranes. Once an infection has occurred, viruses may rely on host lipids for replication and egress. Upon exit, enveloped viruses derive their lipid bilayer from host membranes during the budding process. Furthermore, host lipid metabolism and signaling are often hijacked to facilitate viral replication. We employed an untargeted HILIC-IM-MS lipidomics approach and identified host lipid species that were significantly altered during vesicular stomatitis virus (VSV) infection. Many glycerophospholipid and sphingolipid species were modified, and ontological enrichment analysis suggested that the alterations to the lipid profile change host membrane properties. Lysophosphatidylcholine (LPC), which can contribute to membrane curvature and serve as a signaling molecule, was depleted during infection, while several ceramide sphingolipids were augmented during infection. Ceramide and sphingomyelin lipids were also enriched in viral particles, indicating that sphingolipid metabolism is important during VSV infection.

Revisiting I-BAR Proteins at Central Synapses

Dendritic spines, the distinctive postsynaptic feature of central nervous system (CNS) excitatory synapses, have been studied extensively as electrical and chemical compartments, as well as scaffolds for receptor cycling and positioning of signaling molecules. The dynamics of the shape, number, and molecular composition of spines, and how they are regulated by neural activity, are critically important in synaptic efficacy, synaptic plasticity, and ultimately learning and memory. Dendritic spines originate as outward protrusions of the cell membrane, but this aspect of spine formation and stabilization has not been a major focus of investigation compared to studies of membrane protrusions in non-neuronal cells. We review here one family of proteins involved in membrane curvature at synapses, the BAR (Bin-Amphiphysin-Rvs) domain proteins. The subfamily of inverse BAR (I-BAR) proteins sense and introduce outward membrane curvature, and serve as bridges between the cell membrane and the cytoskeleton. We focus on three I-BAR domain proteins that are expressed in the central nervous system: Mtss2, MIM, and IRSp53 that promote negative, concave curvature based on their ability to self-associate. Recent studies suggest that each has distinct functions in synapse formation and synaptic plasticity. The action of I-BARs is also shaped by crosstalk with other signaling components, forming signaling platforms that can function in a circuit-dependent manner. We discuss another potentially important feature—the ability of some BAR domain proteins to impact the function of other family members by heterooligomerization. Understanding the spatiotemporal resolution of synaptic I-BAR protein expression and their interactions should provide insights into the interplay between activity-dependent neural plasticity and network rewiring in the CNS.

Correlating biological activity to thermo-structural analysis of the interaction of CTX with synthetic models of macrophage membranes

The important pharmacological actions of Crotoxin (CTX) on macrophages, the main toxin in the venom of Crotalus durissus terrificus, and its important participation in the control of different pathophysiological processes, have been demonstrated. The biological activities performed by macrophages are related to signaling mediated by receptors expressed on the membrane surface of these cells or opening and closing of ion channels, generation of membrane curvature and pore formation. In the present work, the interaction of the CTX complex with the cell membrane of macrophages is studied, both using biological cells and synthetic lipid membranes to monitor structural alterations induced by the protein. Here we show that CTX can penetrate THP-1 cells and induce pores only in anionic lipid model membranes, suggesting that a possible access pathway for CTX to the cell is via lipids with anionic polar heads. Considering that the selectivity of the lipid composition varies in different tissues and organs of the human body, the thermostructural studies presented here are extremely important to open new investigations on the biological activities of CTX in different biological systems.

Herpesvirus Nuclear Egress across the Outer Nuclear Membrane

Herpesvirus capsids are assembled in the nucleus and undergo a two-step process to cross the nuclear envelope. Capsids bud into the inner nuclear membrane (INM) aided by the nuclear egress complex (NEC) proteins UL31/34. At that stage of egress, enveloped virions are found for a short time in the perinuclear space. In the second step of nuclear egress, perinuclear enveloped virions (PEVs) fuse with the outer nuclear membrane (ONM) delivering capsids into the cytoplasm. Once in the cytoplasm, capsids undergo re-envelopment in the Golgi/trans-Golgi apparatus producing mature virions. This second step of nuclear egress is known as de-envelopment and is the focus of this review. Compared with herpesvirus envelopment at the INM, much less is known about de-envelopment. We propose a model in which de-envelopment involves two phases: (i) fusion of the PEV membrane with the ONM and (ii) expansion of the fusion pore leading to release of the viral capsid into the cytoplasm. The first phase of de-envelopment, membrane fusion, involves four herpes simplex virus (HSV) proteins: gB, gH/gL, gK and UL20. gB is the viral fusion protein and appears to act to perturb membranes and promote fusion. gH/gL may also have similar properties and appears to be able to act in de-envelopment without gB. gK and UL20 negatively regulate these fusion proteins. In the second phase of de-envelopment (pore expansion and capsid release), an alpha-herpesvirus protein kinase, US3, acts to phosphorylate NEC proteins, which normally produce membrane curvature during envelopment. Phosphorylation of NEC proteins reverses tight membrane curvature, causing expansion of the membrane fusion pore and promoting release of capsids into the cytoplasm.

Ions and lipids drive aggregation of surface-functionalized gold nanoparticles on lipid membranes

The control of the aggregation of biomedical nanoparticles (NP) in physiological conditions is crucial as clustering may change completely the way they interact with the biological environment. Here we show that Au nanoparticles, functionalized by an anionic, amphiphilic shell, with an overall diameter of 7 nm, spontaneously aggregate in fluid zwitterionic lipid bilayers. We use Molecular Dynamics and enhanced sampling techniques to disentangle the short-range and long-range driving forces of aggregation. At short inter-particle distances, ion-mediated, charge-charge interactions (ion bridging) stabilize the formation of large NP aggregates, as confirmed by cryo-electron microscopy. Lipid depletion and membrane curvature are the main membrane deformations driving long-range NP-NP attraction. Ion bridging, lipid depletion, and membrane curvature stem from the configurational flexibility of the nanoparticle shell. Our simulations show, more in general, that the aggregation of same-charge membrane inclusions can be expected as a result of intrinsically nanoscale effects taking place at the NP-NP and NP-bilayer soft interfaces.

Phosphodiesterase Type 5 Inhibitors Greatly Affect Physicochemical Properties of Model Lipid Membranes

Although phosphodiesterase type 5 inhibitors are widely used and well-studied drugs, the potential benefits of their application in the treatment of various diseases and new drug delivery systems, including liposome forms, are still being discussed. In this regard, the role of the lipid matrix of cell membranes in the pharmacological action of the inhibitors is of special interest. It was shown that sildenafil, vardenafil, and tadalafil caused a significant decrease in the boundary potential of model membranes composed of palmitoyloleoylphosphatidylcholine or its mixture with cholesterol, by 70–80 mV. The reduction in the membrane dipole potential induced by inhibitors led to a 20–25% increase in the conductance of cation-selective pores formed by the antimicrobial peptide gramicidin A. The addition of sildenafil or vardenafil also led to a significant decrease in the temperature of the main phase transition of dipalmytoylphosphatidylcholine, by about 1.5 °C, while tadalafil did not change the melting temperature. Sildenafil, vardenafil, and tadalafil enhanced the pore-forming activity of the antifungal polyene antibiotic nystatin by 11, 13, and 2 times, respectively. This fact might indicate the induction of membrane curvature stress by the inhibitors. The data obtained might be of special interest for the development of lipid-mediated forms of drugs.