Pasi Laurinmäki

Molecular Mechanisms of Membrane Deformation by I-BAR Domain Proteins

BACKGROUND Generation of membrane curvature is critical for the formation of plasma membrane protrusions and invaginations and for shaping intracellular organelles. Among the central regulators of membrane dynamics are the BAR superfamily domains, which deform membranes into tubular structures. In contrast to the relatively well characterized BAR and F-BAR domains that promote the formation of plasma membrane invaginations, I-BAR domains induce plasma membrane protrusions through a poorly understood mechanism. RESULTS We show that I-BAR domains induce strong PI(4,5)P(2) clustering upon membrane binding, bend the membrane through electrostatic interactions, and remain dynamically associated with the inner leaflet of membrane tubules. Thus, I-BAR domains induce the formation of dynamic membrane protrusions to the opposite direction than do BAR and F-BAR domains. Strikingly, comparison of different I-BAR domains revealed that they deform PI(4,5)P(2)-rich membranes through distinct mechanisms. IRSp53 and IRTKS I-BARs bind membranes mainly through electrostatic interactions, whereas MIM and ABBA I-BARs additionally insert an amphipathic helix into the membrane bilayer, resulting in larger tubule diameter in vitro and more efficient filopodia formation in vivo. Furthermore, FRAP analysis revealed that whereas the mammalian I-BAR domains display dynamic association with filopodia, the C. elegans I-BAR domain forms relatively stable structures inside the plasma membrane protrusions. CONCLUSIONS These data define I-BAR domain as a functional member of the BAR domain superfamily and unravel the mechanisms by which I-BAR domains deform membranes to induce filopodia in cells. Furthermore, our work reveals unexpected divergence in the mechanisms by which evolutionarily distinct groups of I-BAR domains interact with PI(4,5)P(2)-rich membranes.

Gold-embedded photosensitive liposomes for drug delivery: triggering mechanism and intracellular release.

Liposomes embedded with gold nanoparticles show light-triggered contents release. We investigated the mechanism of the light-induced changes and functionality of the light-induced release in the cells. The real time small angle X-ray scattering (SAXS) analysis revealed time-dependent phase transitions in distearoylphosphatidylcholine (DSPC)/dipalmitoylphosphatidylcholine (DPPC) liposomes upon heating. Similar changes were observed when gold nanoparticle-embedded liposomes were exposed to the UV light: gold nanoparticles absorb light energy and transfer it to heat, thereby causing lipid phase transition from gel phase to rippled phase, and further to fluid phase. Without UV light exposure the gold nanoparticles did not affect the liposomal bilayer periodicity. The light-triggered release of hydrophilic fluorescent probe (calcein) from the gold nanoparticle-loaded liposomes was demonstrated with fluorescence-activated cell sorting after liposome internalization into the ARPE-19 cells. The liposome formulations did not decrease the cell viability in vitro. In conclusion, the light-triggered release from the liposomes is functional in the cells, and the release is triggered by thermal phase changes in the lipid bilayers.

Electron Cryotomography of Tula Hantavirus Suggests a Unique Assembly Paradigm for Enveloped Viruses

ABSTRACT Hantaviruses (family Bunyaviridae) are rodent-borne emerging viruses that cause a serious, worldwide threat to human health. Hantavirus diseases include hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. Virions are enveloped and contain a tripartite single-stranded negative-sense RNA genome. Two types of glycoproteins, GN and GC, are embedded in the viral membrane and form protrusions, or “spikes.” The membrane encloses a ribonucleoprotein core, which consists of the RNA segments, the nucleocapsid protein, and the RNA-dependent RNA polymerase. Detailed information on hantavirus virion structure and glycoprotein spike composition is scarce. Here, we have studied the structures of Tula hantavirus virions using electron cryomicroscopy and tomography. Three-dimensional density maps show how the hantavirus surface glycoproteins, membrane, and ribonucleoprotein are organized. The structure of the GN-GC spike complex was solved to 3.6-nm resolution by averaging tomographic subvolumes. Each spike complex is a square-shaped assembly with 4-fold symmetry. Spike complexes formed ordered patches on the viral membrane by means of specific lateral interactions. These interactions may be sufficient for creating membrane curvature during virus budding. In conclusion, the structure and assembly principles of Tula hantavirus exemplify a unique assembly paradigm for enveloped viruses.

Structure and host-cell interaction of SH1, a membrane-containing, halophilic euryarchaeal virus

The Archaea, and the viruses that infect them, are the least well understood of all of the three domains of life. They often grow in extreme conditions such as hypersaline lakes and sulfuric hot springs. Only rare glimpses have been gained into the structures of archaeal viruses. Here, we report the subnanometer resolution structure of a recently isolated, hypersalinic, membrane-containing, euryarchaeal virus, SH1, in which different viral proteins can be localized. The results indicate that SH1 has a complex capsid formed from single β-barrels, an important missing link in hypotheses on viral capsid protein evolution. Unusual, symmetry-mismatched spikes seem to play a role in host adsorption. They are connected to highly organized membrane proteins providing a platform for capsid assembly and potential machinery for host infection.

Structure of the archaeal head-tailed virus HSTV-1 completes the HK97 fold story

It has been proposed that viruses can be divided into a small number of structure-based viral lineages. One of these lineages is exemplified by bacterial virus Hong Kong 97 (HK97), which represents the head-tailed dsDNA bacteriophages. Seemingly similar viruses also infect archaea. Here we demonstrate using genomic analysis, electron cryomicroscopy, and image reconstruction that the major coat protein fold of newly isolated archaeal Haloarcula sinaiiensis tailed virus 1 has the canonical coat protein fold of HK97. Although it has been anticipated previously, this is physical evidence that bacterial and archaeal head-tailed viruses share a common architectural principle. The HK97-like fold has previously been recognized also in herpesviruses, and this study expands the HK97-like lineage to viruses from all three domains of life. This is only the second established lineage to include archaeal, bacterial, and eukaryotic viruses. Thus, our findings support the hypothesis that the last common universal ancestor of cellular organisms was infected by a number of different viruses.

Isolation, Identification, and Characterization of Novel Arenaviruses, the Etiological Agents of Boid Inclusion Body Disease

ABSTRACT Boid inclusion body disease (BIBD) is a progressive, usually fatal disease of constrictor snakes, characterized by cytoplasmic inclusion bodies (IB) in a wide range of cell types. To identify the causative agent of the disease, we established cell cultures from BIBD-positive and -negative boa constrictors. The IB phenotype was maintained in cultured cells of affected animals, and supernatants from these cultures caused the phenotype in cultures originating from BIBD-negative snakes. Viruses were purified from the supernatants by ultracentrifugation and subsequently identified as arenaviruses. Purified virus also induced the IB phenotype in naive cells, which fulfilled Kochs postulates in vitro. One isolate, tentatively designated University of Helsinki virus (UHV), was studied in depth. Sequencing confirmed that UHV is a novel arenavirus species that is distinct from other known arenaviruses including those recently identified in snakes with BIBD. The morphology of UHV was established by cryoelectron tomography and subtomographic averaging, revealing the trimeric arenavirus spike structure at 3.2-nm resolution. Immunofluorescence, immunohistochemistry, and immunoblotting with a polyclonal rabbit antiserum against UHV and reverse transcription-PCR (RT-PCR) revealed the presence of genetically diverse arenaviruses in a large cohort of snakes with BIBD, confirming the causative role of arenaviruses. Some snakes were also found to carry arenavirus antibodies. Furthermore, mammalian cells (Vero E6) were productively infected with UHV, demonstrating the potential of arenaviruses to cross species barriers. In conclusion, we propose the newly identified lineage of arenaviruses associated with BIBD as a novel taxonomic entity, boid inclusion body disease-associated arenaviruses (BIBDAV), in the family Arenaviridae.

Structure of Chlorosomes from the Green Filamentous Bacterium Chloroflexus aurantiacus

The green filamentous bacterium Chloroflexus aurantiacus employs chlorosomes as photosynthetic antennae. Chlorosomes contain bacteriochlorophyll aggregates and are attached to the inner side of a plasma membrane via a protein baseplate. The structure of chlorosomes from C. aurantiacus was investigated by using a combination of cryo-electron microscopy and X-ray diffraction and compared with that of Chlorobi species. Cryo-electron tomography revealed thin chlorosomes for which a distinct crystalline baseplate lattice was visualized in high-resolution projections. The baseplate is present only on one side of the chlorosome, and the lattice dimensions suggest that a dimer of the CsmA protein is the building block. The bacteriochlorophyll aggregates inside the chlorosome are arranged in lamellae, but the spacing is much greater than that in Chlorobi species. A comparison of chlorosomes from different species suggested that the lamellar spacing is proportional to the chain length of the esterifying alcohols. C. aurantiacus chlorosomes accumulate larger quantities of carotenoids under high-light conditions, presumably to provide photoprotection. The wider lamellae allow accommodation of the additional carotenoids and lead to increased disorder within the lamellae.

Diblock copolymers consisting of a polymerized ionic liquid and poly(N-isopropylacrylamide). Effects of PNIPAM block length and counter ion on self-assembling and thermal properties

Amphiphilic diblock copolymers composed of a polymeric ionic liquid, PIL, and poly(N-isopropylacrylamide), PNIPAM, have been synthesized using RAFT reactions. The length of the PIL block was kept constant while the molecular mass of the PNIPAM block was varied. The PIL was poly(2-(1-butylimidazolium-3-yl)ethyl methacrylate tetrafluoroborate) which is insoluble in water owing to the bulky hydrophobic counterion. When the PNIPAM block was long enough, the polymers formed spherical micelles in water, which showed thermally responsive behaviour. Colloidally stable particles could be prepared also from the homopolymeric PIL. PNIPAM affects noticeably the properties of the PIL, and also the polycation has a strong effect on the thermal properties of PNIPAM in aqueous dispersions. As a reference, a polymer where bromide was the counter ion instead of the tetrafluoroborate ion was synthesized, providing a water soluble PIL block. The core–shell micelles formed by amphiphilic block copolymers in pure water and those by double-hydrophilic polymers in aqueous NaBF4 undergo partial structural inversion upon the thermal collapse of PNIPAM.

Biochemical and structural characterisation of membrane-containing icosahedral dsDNA bacteriophages infecting thermophilic Thermus thermophilus.

Icosahedral dsDNA viruses isolated from hot springs and proposed to belong to the Tectiviridae family infect the gram-negative thermophilic Thermus thermophilus bacterium. Seven such viruses were obtained from the Promega Corporation collection. The structural protein patterns of three of these viruses, growing to a high titer, appeared very similar but not identical. The most stable virus, P23-77, was chosen for more detailed studies. Analysis of highly purified P23-77 by thin layer chromatography for neutral lipids showed lipid association with the virion. Cryo-EM based three-dimensional image reconstruction of P23-77 to 1.4 nm resolution revealed an icosahedrally-ordered protein coat, with spikes on the vertices, and an internal membrane. The capsid architecture of P23-77 is most similar to that of the archaeal virus SH1. These findings further complicate the grouping of icosahedrally-symmetric viruses containing an inner membrane. We propose a single superfamily or order with members in several viral families.

Insights into Head-Tailed Viruses Infecting Extremely Halophilic Archaea

ABSTRACT Extremophilic archaea, both hyperthermophiles and halophiles, dominate in habitats where rather harsh conditions are encountered. Like all other organisms, archaeal cells are susceptible to viral infections, and to date, about 100 archaeal viruses have been described. Among them, there are extraordinary virion morphologies as well as the common head-tailed viruses. Although approximately half of the isolated archaeal viruses belong to the latter group, no three-dimensional virion structures of these head-tailed viruses are available. Thus, rigorous comparisons with bacteriophages are not yet warranted. In the present study, we determined the genome sequences of two of such viruses of halophiles and solved their capsid structures by cryo-electron microscopy and three-dimensional image reconstruction. We show that these viruses are inactivated, yet remain intact, at low salinity and that their infectivity is regained when high salinity is restored. This enabled us to determine their three-dimensional capsid structures at low salinity to a ∼10-Å resolution. The genetic and structural data showed that both viruses belong to the same T-number class, but one of them has enlarged its capsid to accommodate a larger genome than typically associated with a T=7 capsid by inserting an additional protein into the capsid lattice.