Morphological switch to a resistant subpopulation in response to viral infection in the bloom-forming coccolithophore Emiliania huxleyi.

Recognizing the life cycle of an organism is key to understanding its biology and ecological impact. Emiliania huxleyi is a cosmopolitan marine microalga, which displays a poorly understood biphasic sexual life cycle comprised of a calcified diploid phase and a morphologically distinct biflagellate haploid phase. Diploid cells (2N) form large-scale blooms in the oceans, which are routinely terminated by specific lytic viruses (EhV). In contrast, haploid cells (1N) are resistant to EhV. Further evidence indicates that 1N cells may be produced during viral infection. A shift in morphology, driven by meiosis, could therefore constitute a mechanism for E. huxleyi cells to escape from EhV during blooms. This process has been metaphorically coined the 'Cheshire Cat' (CC) strategy. We tested this model in two E. huxleyi strains using a detailed assessment of morphological and ploidy-level variations as well as expression of gene markers for meiosis and the flagellate phenotype. We showed that following the CC model, production of resistant cells was triggered during infection. This led to the rise of a new subpopulation of cells in the two strains that morphologically resembled haploid cells and were resistant to EhV. However, ploidy-level analyses indicated that the new resistant cells were diploid or aneuploid. Thus, the CC strategy in E. huxleyi appears to be a life-phase switch mechanism involving morphological remodeling that is decoupled from meiosis. Our results highlight the adaptive significance of morphological plasticity mediating complex host-virus interactions in marine phytoplankton.

Altered lysosome distribution is an early neuropathological event in neurological forms of Gaucher disease.

In the lysosomal storage disorder Gaucher disease (GD), glucosylceramide (GlcCer) accumulates due to the defective activity of glucocerebrosidase. A subset of GD patients develops neuropathology. We now show mislocalization of Limp2-positive puncta and a large reduction in the number of Lamp1-positive puncta, which are associated with impaired tubulin. These changes occur at an early stage in animal models of GD, prior to development of overt symptoms and considerably earlier than neuronal loss. Altered lysosomal localization and cytoskeleton disruption precede the neuroinflammatory pathways, axonal dystrophy and neuronal loss previously characterized in neuronal forms of GD.

Involvement of Rho GAP GRAF1 in maintenance of epithelial phenotype.

Adhesion of epithelial cell to each other and to extracellular matrix, as well as cell migration ability and cytoskeleton organization undergo significant alterations in the course of neoplastic transformation, but regulatory mechanisms involved in these processes are not fully understood. Here, we studied the role of a Rho GAP protein GRAF1 (GTPase Regulator Associated with Focal adhesion kinase-1) in the regulation of the epithelial phenotype in cells of breast derived, non-malignant, MCF10A cell line. GRAF1 was shown to be localized to cell-cell junctions, and its depletion resulted in accelerated cell migration velocity, elongation of the cells and cell colonies, impaired monolayer integrity and significant disruption of desmosomes with a loss of associated keratin filaments. These processes were accompanied by formation of larger focal adhesions, an increased number of contractile actin stress fibers, reduction in epithelial markers and increase in mesenchymal markers such as epithelial-mesenchymal transition (EMT)-specific transcription factors Snail-1 and Snail-2, as well as N-cadherin, and vimentin. Moreover, unlike control cells, GRAF1 knocked-down cells demonstrated anchorage-independent growth in soft agar. GRAF1 expression in several highly invasive breast cancer cell lines was low, as compared to the non-malignant MCF10A cells, while overexpressing of GRAF1 in the malignant BT-549 cell line led to a decrease of mesenchymal markers, especially the Snail-1 and 2. Altogether, our analysis suggests that GRAF1 plays a role in the maintenance of normal epithelial phenotype and its depletion leads to an EMT-like process that might be involved in neoplastic transformation.

Supramolecular Assembly and Coalescence of Ferritin Cages Driven by Designed Protein-Protein Interactions.

A genetically encoded system for expression of supramolecular protein assemblies (SMPAs) based on a fusion construct between ferritin and citrine (YFP) was transferred from a mammalian to a bacterial host. The assembly process is revealed to be independent of the expression host, while dimensions and level of order of the assembled structures were influenced by the host organism. An additional level of interactions, namely, coalescence between the preformed SMPAs, was observed during the purification process. SAXS investigation revealed that upon coalescence, the local order of the individual SMPAs was preserved. Finally, the chaotropic agent urea effectively disrupted both the macroscopic coalescence and the interactions at the nanoscale until the level of the single ferritin cage.

Zooplankton may serve as transmission vectors for viruses infecting algal blooms in the ocean.

Marine viruses are recognized as a major driving force regulating phytoplankton community composition and nutrient cycling in the oceans. Yet, little is known about mechanisms that influence viral dispersal in aquatic systems, other than physical processes, and that lead to the rapid demise of large-scale algal blooms in the oceans. Here, we show that copepods, abundant migrating crustaceans that graze on phytoplankton, as well as other zooplankton can accumulate and mediate the transmission of viruses infecting Emiliania huxleyi, a bloom-forming coccolithophore that plays an important role in the carbon cycle. We detected by PCR that >80% of copepods collected during a North Atlantic E. huxleyi bloom carried E. huxleyi virus (EhV) DNA. We demonstrated by isolating a new infectious EhV strain from a copepod microbiome that these viruses are infectious. We further showed that EhVs can accumulate in high titers within zooplankton guts during feeding or can be adsorbed to their surface. Subsequently, EhV can be dispersed by detachment or via viral-dense fecal pellets over a period of 1 day postfeeding on EhV-infected algal cells, readily infecting new host populations. Intriguingly, the passage through zooplankton guts prolonged EhV's half-life of infectivity by 35%, relative to free virions in seawater, potentially enhancing viral transmission. We propose that zooplankton, swimming through topographically adjacent phytoplankton micropatches and migrating daily over large areas across physically separated water masses, can serve as viral vectors, boosting host-virus contact rates and potentially accelerating the demise of large-scale phytoplankton blooms.

Hijacking of an autophagy-like process is critical for the life cycle of a DNA virus infecting oceanic algal blooms.

Marine photosynthetic microorganisms are the basis of marine food webs and are responsible for nearly 50% of the global primary production. Emiliania huxleyi forms massive oceanic blooms that are routinely terminated by large double-stranded DNA coccolithoviruses. The cellular mechanisms that govern the replication cycle of these giant viruses are largely unknown. We used diverse techniques, including fluorescence microscopy, transmission electron microscopy, cryoelectron tomography, immunolabeling and biochemical methodologies to investigate the role of autophagy in host-virus interactions. Hallmarks of autophagy are induced during the lytic phase of E. huxleyi viral infection, concomitant with up-regulation of autophagy-related genes (ATG genes). Pretreatment of the infected cells with an autophagy inhibitor causes a major reduction in the production of extracellular viral particles, without reducing viral DNA replication within the cell. The host-encoded Atg8 protein was detected within purified virions, demonstrating the pivotal role of the autophagy-like process in viral assembly and egress. We show that autophagy, which is classically considered as a defense mechanism, is essential for viral propagation and for facilitating a high burst size. This cellular mechanism may have a major impact on the fate of the viral-infected blooms, and therefore on the cycling of nutrients within the marine ecosystem.

Accumulation of ordered ceramide-cholesterol domains in farber disease fibroblasts.

Farber disease is an inherited metabolic disorder caused by mutations in the acid ceramidase gene, which leads to ceramide accumulation in lysosomes. Farber disease patients display a wide variety of symptoms with most patients eventually displaying signs of nervous system dysfunction. We now present a novel tool that could potentially be used to distinguish between the milder and more severe forms of the disease, namely, an antibody that recognizes a mixed monolayer or bilayer of cholesterol:C16-ceramide, but does not recognize either ceramide or cholesterol by themselves. This antibody has previously been used to detect cholesterol:C16-ceramide domains in a variety of cultured cells. We demonstrate that levels of cholesterol:C16-ceramide domains are significantly elevated in fibroblasts from types 4 and 7 Farber disease patients, and that levels of the domains can be modulated by either reducing ceramide or cholesterol levels. Moreover, these domains are located in membranes of the endomembrane system, and also in two unexpected locations, namely, the mitochondria and the plasma membrane. This study suggests that the ceramide that accumulates in severe forms of Farber disease cells is sequestered to distinct membrane subdomains, which may explain some of the cellular pathology observed in this devastating lysosomal storage disease.

The expression of the beta cell-derived autoimmune ligand for the killer receptor nkp46 is attenuated in type 2 diabetes.

NK cells rapidly kill tumor cells, virus infected cells and even self cells. This is mediated via killer receptors, among which NKp46 (NCR1 in mice) is prominent. We have recently demonstrated that in type 1 diabetes (T1D) NK cells accumulate in the diseased pancreas and that they manifest a hyporesponsive phenotype. In addition, we found that NKp46 recognizes an unknown ligand expressed by beta cells derived from humans and mice and that blocking of NKp46 activity prevented diabetes development. Here we investigated the properties of the unknown NKp46 ligand. We show that the NKp46 ligand is mainly located in insulin granules and that it is constitutively secreted. Following glucose stimulation the NKp46 ligand translocates to the cell membrane and its secretion decreases. We further demonstrate by using several modalities that the unknown NKp46 ligand is not insulin. Finally, we studied the expression of the NKp46 ligand in type 2 diabetes (T2D) using 3 different in vivo models and 2 species; mice and gerbils. We demonstrate that the expression of the NKp46 ligand is decreased in all models of T2D studied, suggesting that NKp46 is not involved in T2D.

Genetic deletion of Cadm4 results in myelin abnormalities resembling Charcot-Marie-Tooth neuropathy.

The interaction between myelinating Schwann cells and the axons they ensheath is mediated by cell adhesion molecules of the Cadm/Necl/SynCAM family. This family consists of four members: Cadm4/Necl4 and Cadm1/Necl2 are found in both glia and axons, whereas Cadm2/Necl3 and Cadm3/Necl1 are expressed by sensory and motor neurons. By generating mice lacking each of the Cadm genes, we now demonstrate that Cadm4 plays a role in the establishment of the myelin unit in the peripheral nervous system. Mice lacking Cadm4 (PGK-Cre/Cadm4(fl/fl)), but not Cadm1, Cadm2, or Cadm3, develop focal hypermyelination characterized by tomacula and myelin outfoldings, which are the hallmark of several Charcot-Marie-Tooth neuropathies. The absence of Cadm4 also resulted in abnormal axon-glial contact and redistribution of ion channels along the axon. These neuropathological features were also found in transgenic mice expressing a dominant-negative mutant of Cadm4 lacking its cytoplasmic domain in myelinating glia Tg(mbp-Cadm4dCT), as well as in mice lacking Cadm4 specifically in Schwann cells (DHH-Cre/Cadm4(fl/fl)). Consistent with these abnormalities, both PGK-Cre/Cadm4(fl/fl) and Tg(mbp-Cadm4dCT) mice exhibit impaired motor function and slower nerve conduction velocity. These findings indicate that Cadm4 regulates the growth of the myelin unit and the organization of the underlying axonal membrane.

The cytoskeletal adapter protein 4.1G organizes the internodes in peripheral myelinated nerves.

Myelinating Schwann cells regulate the localization of ion channels on the surface of the axons they ensheath. This function depends on adhesion complexes that are positioned at specific membrane domains along the myelin unit. Here we show that the precise localization of internodal proteins depends on the expression of the cytoskeletal adapter protein 4.1G in Schwann cells. Deletion of 4.1G in mice resulted in aberrant distribution of both glial adhesion molecules and axonal proteins that were present along the internodes. In wild-type nerves, juxtaparanodal proteins (i.e., Kv1 channels, Caspr2, and TAG-1) were concentrated throughout the internodes in a double strand that flanked paranodal junction components (i.e., Caspr, contactin, and NF155), and apposes the inner mesaxon of the myelin sheath. In contrast, in 4.1G(-/-) mice, these proteins "piled up" at the juxtaparanodal region or aggregated along the internodes. These findings suggest that protein 4.1G contributes to the organization of the internodal axolemma by targeting and/or maintaining glial transmembrane proteins along the axoglial interface.

Reconstructing adhesion structures in tissues by cryo-electron tomography of vitrified frozen sections.

Cryo-electron tomography enables three-dimensional insights into the macromolecular architecture of cells in a close-to-life state. However, it is limited to thin specimens, <1.0 µm in thickness, typically restricted to the peripheral areas of intact eukaryotic cells. Analysis of tissue ultrastructure, on the other hand, requires physical sectioning approaches, preferably cryo-sectioning, following which electron tomography (ET) may be performed. Nevertheless, cryo-electron microscopy of vitrified sections is a demanding technique and typically cannot be used to examine thick sections, >80-100 nm, due to surface crevasses. Here, we explore the potential use of cryo-ET of vitrified frozen sections (VFSs) for imaging cell adhesions in chicken smooth muscle and mouse epithelial tissues. By investigating 300-400 nm thick sections, which are collected on the EM grid and re-vitrified, we resolved fine 3D structural details of the membrane-associated dense plaques and flanking caveoli in smooth muscle tissue, and desmosomal adhesions in stratified epithelium. Technically, this method offers a simple approach for reconstructing thick volumes of hydrated frozen sections.

Involvement of the Rho-mDia1 pathway in the regulation of Golgi complex architecture and dynamics.

In mammalian cells, the Golgi apparatus is a ribbon-like, compact structure composed of multiple membrane stacks connected by tubular bridges. Microtubules are known to be important to Golgi integrity, but the role of the actin cytoskeleton in the maintenance of Golgi architecture remains unclear. Here we show that an increase in Rho activity, either by treatment of cells with lysophosphatidic acid or by expression of constitutively active mutants, resulted in pronounced fragmentation of the Golgi complex into ministacks. Golgi dispersion required the involvement of mDia1 formin, a downstream target of Rho and a potent activator of actin polymerization; moreover, constitutively active mDia1, in and of itself, was sufficient for Golgi dispersion. The dispersion process was accompanied by formation of dynamic F-actin patches in the Golgi area. Experiments with cytoskeletal inhibitors (e.g., latrunculin B, blebbistatin, and Taxol) revealed that actin polymerization, myosin-II-driven contractility, and microtubule-based intracellular movement were all involved in the process of Golgi dispersion induced by Rho-mDia1 activation. Live imaging of Golgi recovery revealed that fusion of the small Golgi stacks into larger compartments was repressed in cells with active mDia1. Furthermore, the formation of Rab6-positive transport vesicles derived from the Golgi complex was enhanced upon activation of the Rho-mDia1 pathway. Transient localization of mDia1 to Rab6-positive vesicles was detected in cells expressing active RhoA. Thus, the Rho-mDia1 pathway is involved in regulation of the Golgi structure, affecting remodeling of Golgi membranes.

Intracellular localization of organized lipid domains of C16-ceramide/cholesterol.

Lipid microdomains, also called lipid rafts, consisting of sphingolipids and cholesterol, play important roles in membrane trafficking and in signaling. Despite years of study of the composition, size, half-life and dynamic organization of these domains, many open questions remain about their precise characteristics. To address some of these issues, we have developed a new experimental approach involving the use of specific monoclonal antibodies as recognition tools. One such antibody was raised against a homogeneous, mixed, ordered monolayer phase comprised of 60:40 mol% cholesterol:C16-ceramide, and has been used previously to demonstrate the existence of C16-ceramide/cholesterol domains in the membranes of cultured cells. We now use a combination of quantitative fluorescence microscopy, immuno-transmission electron microscopy and immuno-scanning cryo-electron microscopy, optimized for the study of intracellular lipid antigens. In a variety of cultured cells, C16-ceramide/cholesterol structural domains were found at high levels in late endosomes and in the trans-Golgi network, but were not found at statistically significant levels in early endosomes, lysosomes or the endoplasmic reticulum. We discuss the relevance of these results to understanding the role of lipid lateral organization in biological membranes.

N-WASP is required for membrane wrapping and myelination by Schwann cells.

During peripheral nerve myelination, Schwann cells sort larger axons, ensheath them, and eventually wrap their membrane to form the myelin sheath. These processes involve extensive changes in cell shape, but the exact mechanisms involved are still unknown. Neural Wiskott-Aldrich syndrome protein (N-WASP) integrates various extracellular signals to control actin dynamics and cytoskeletal reorganization through activation of the Arp2/3 complex. By generating mice lacking N-WASP in myelinating Schwann cells, we show that N-WASP is crucial for myelination. In N-WASP-deficient nerves, Schwann cells sort and ensheath axons, but most of them fail to myelinate and arrest at the promyelinating stage. Yet, a limited number of Schwann cells form unusually short internodes, containing thin myelin sheaths, with the occasional appearance of myelin misfoldings. These data suggest that regulation of actin filament nucleation in Schwann cells by N-WASP is crucial for membrane wrapping, longitudinal extension, and myelination.

Microtubule organization in the final stages of cytokinesis as revealed by cryo-electron tomography.

The completion of cytokinesis is dominated by the midbody, a tightly-packed microtubule (MT)-based bridge that transiently connects the two daughter cells. Assembled from condensed, spindle-MTs and numerous associated proteins, the midbody gradually narrows down until daughter cell partitioning occurs at this site. Although described many years ago, detailed understanding of the abscission process remains lacking. Applying cryo-electron tomography to purified midbodies, in combination with fluorescence microscopy, we present here new insight into MT organization within the midbody. We find that the midbody is spatially divided into a core bundle of MTs that traverses the electron-dense overlap region (continuous MTs), surrounded by MTs that terminate within the overlap region (polar MTs). Residual continuous MTs remained intact up to the verge of abscission, whereas the residual polar MTs lost their organization and retreated from the overlap region at late cytokinesis stages. A detailed localization of the microtubule-bundling protein PRC1 supports the above notion. Our study thus provides a detailed account of the abscission process and suggests that the midbody, having acquired a distinct MT architecture as compared to the preceding central spindle, actively facilitates the final stage of cytokinesis.

The neurexin superfamily of Caenorhabditis elegans.

The neurexin superfamily is a group of transmembrane molecules mediating cell-cell contacts and generating specialized membranous domains in polarized epithelial and nerves cells. We describe here the domain organization and expression of the entire, core neurexin superfamily in the nematode Caenorhabditis elegans, which is composed of three family members. One of the superfamily members, nrx-1, is an ortholog of vertebrate neurexin, the other two, itx-1 and nlr-1, are orthologs of the Caspr subfamily of neurexin-like genes. Based on reporter gene analysis, we find that nrx-1 is exclusively expressed in most if not all cells of the nervous system and localizes to presynaptic specializations. itx-1 and nrx-1 reporter genes are expressed in non-overlapping patterns within and outside the nervous system. ITX-1 protein co-localizes with ß-G-spectrin to a subapical domain within intestinal cells. These studies provide a starting point for further functional analysis of this family of proteins.

A glial signal consisting of gliomedin and NrCAM clusters axonal Na+ channels during the formation of nodes of Ranvier.

Saltatory conduction requires high-density accumulation of Na(+) channels at the nodes of Ranvier. Nodal Na(+) channel clustering in the peripheral nervous system is regulated by myelinating Schwann cells through unknown mechanisms. During development, Na(+) channels are first clustered at heminodes that border each myelin segment, and later in the mature nodes that are formed by the fusion of two heminodes. Here, we show that initial clustering of Na(+) channels at heminodes requires glial NrCAM and gliomedin, as well as their axonal receptor neurofascin 186 (NF186). We further demonstrate that heminodal clustering coincides with a second, paranodal junction (PNJ)-dependent mechanism that allows Na(+) channels to accumulate at mature nodes by restricting their distribution between two growing myelin internodes. We propose that Schwann cells assemble the nodes of Ranvier by capturing Na(+) channels at heminodes and by constraining their distribution to the nodal gap. Together, these two cooperating mechanisms ensure fast and efficient conduction in myelinated nerves.

DAP-kinase-mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl-XL and induction of autophagy.

Autophagy, an evolutionarily conserved process, has functions both in cytoprotective and programmed cell death mechanisms. Beclin 1, an essential autophagic protein, was recently identified as a BH3-domain-only protein that binds to Bcl-2 anti-apoptotic family members. The dissociation of beclin 1 from its Bcl-2 inhibitors is essential for its autophagic activity, and therefore should be tightly controlled. Here, we show that death-associated protein kinase (DAPK) regulates this process. The activated form of DAPK triggers autophagy in a beclin-1-dependent manner. DAPK phosphorylates beclin 1 on Thr 119 located at a crucial position within its BH3 domain, and thus promotes the dissociation of beclin 1 from Bcl-XL and the induction of autophagy. These results reveal a substrate for DAPK that acts as one of the core proteins of the autophagic machinery, and they provide a new phosphorylation-based mechanism that reduces the interaction of beclin 1 with its inhibitors to activate the autophagic machinery.

Anomalous features of EMT during keratinocyte transformation.

During the evolution of epithelial cancers, cells often lose their characteristic features and acquire a mesenchymal phenotype, in a process known as epithelial-mesenchymal transition (EMT). In the present study we followed early stages of keratinocyte transformation by HPV16, and observed diverse cellular changes, associated with EMT. We compared primary keratinocytes with early and late passages of HF1 cells, a cell line of HPV16-transformed keratinocytes. We have previously shown that during the progression from the normal cells to early HF1 cells, immortalization is acquired, while in the progression to late HF1, cells become anchorage independent. We show here that during the transition from the normal state to late HF1 cells, there is a progressive reduction in cytokeratin expression, desmosome formation, adherens junctions and focal adhesions, ultimately leading to poorly adhesive phenotype, which is associated with anchorage-independence. Surprisingly, unlike "conventional EMT", these changes are associated with reduced Rac1-dependent cell migration. We monitored reduced Rac1-dependent migration also in the cervical cancer cell line SiHa. Therefore we can conclude that up to the stage of tumor formation migratory activity is eliminated.

A central role for Necl4 (SynCAM4) in Schwann cell-axon interaction and myelination.

Myelination in the peripheral nervous system requires close contact between Schwann cells and the axon, but the underlying molecular basis remains largely unknown. Here we show that cell adhesion molecules (CAMs) of the nectin-like (Necl, also known as SynCAM or Cadm) family mediate Schwann cell-axon interaction during myelination. Necl4 is the main Necl expressed by myelinating Schwann cells and is located along the internodes in direct apposition to Necl1, which is localized on axons. Necl4 serves as the glial binding partner for axonal Necl1, and the interaction between these two CAMs mediates Schwann cell adhesion. The disruption of the interaction between Necl1 and Necl4 by their soluble extracellular domains, or the expression of a dominant-negative Necl4 in Schwann cells, inhibits myelination. These results suggest that Necl proteins are important for mediating axon-glia contact during myelination in peripheral nerves.