Rachel Santarella-Mellwig

Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Hepatitis C Virus Replication

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.

Mouse Rif1 is a key regulator of the replication-timing programme in mammalian cells

The eukaryotic genome is replicated according to a specific spatio‐temporal programme. However, little is known about both its molecular control and biological significance. Here, we identify mouse Rif1 as a key player in the regulation of DNA replication timing. We show that Rif1 deficiency in primary cells results in an unprecedented global alteration of the temporal order of replication. This effect takes place already in the first S‐phase after Rif1 deletion and is neither accompanied by alterations in the transcriptional landscape nor by major changes in the biochemical identity of constitutive heterochromatin. In addition, Rif1 deficiency leads to both defective G1/S transition and chromatin re‐organization after DNA replication. Together, these data offer a novel insight into the global regulation and biological significance of the replication‐timing programme in mammalian cells.

Inflammation-Induced Emergency Megakaryopoiesis Driven by Hematopoietic Stem Cell-like Megakaryocyte Progenitors

Infections are associated with extensive platelet consumption, representing a high risk for health. However, the mechanism coordinating the rapid regeneration of the platelet pool during such stress conditions remains unclear. Here, we report that the phenotypic hematopoietic stem cell (HSC) compartment contains stem-like megakaryocyte-committed progenitors (SL-MkPs), a cell population that shares many features with multipotent HSCs and serves as a lineage-restricted emergency pool for inflammatory insults. During homeostasis, SL-MkPs are maintained in a primed but quiescent state, thus contributing little to steady-state megakaryopoiesis. Even though lineage-specific megakaryocyte transcripts are expressed, protein synthesis is suppressed. In response to acute inflammation, SL-MkPs become activated, resulting in megakaryocyte protein production from pre-existing transcripts and a maturation of SL-MkPs and other megakaryocyte progenitors. This results in an efficient replenishment of platelets that are lost during inflammatory insult. Thus, our study reveals an emergency machinery that counteracts life-threatening platelet depletions during acute inflammation.

Coordination of Kinase and Phosphatase Activities by Lem4 Enables Nuclear Envelope Reassembly during Mitosis

Mitosis in metazoa requires nuclear envelope (NE) disassembly and reassembly. NE disassembly is driven by multiple phosphorylation events. Mitotic phosphorylation of the protein BAF reduces its affinity for chromatin and the LEM family of inner nuclear membrane proteins; loss of this BAF-mediated chromatin-NE link contributes to NE disassembly. BAF must reassociate with chromatin and LEM proteins at mitotic exit to reform the NE; however, how its dephosphorylation is regulated is unknown. Here, we show that the C. elegans protein LEM-4L and its human ortholog Lem4 (also called ANKLE2) are both required for BAF dephosphorylation. They act in part by inhibiting BAFs mitotic kinase, VRK-1, in vivo and in vitro. In addition, Lem4/LEM-4L interacts with PP2A and is required for it to dephosphorylate BAF during mitotic exit. By coordinating VRK-1- and PP2A-mediated signaling on BAF, Lem4/LEM-4L controls postmitotic NE formation in a function conserved from worms to humans.

The Nup107-160 Nucleoporin Complex Promotes Mitotic Events via Control of the Localization State of the Chromosome Passenger Complex

The human Nup107-160 nucleoporin complex plays a major role in formation of the nuclear pore complex and is localized to kinetochores in mitosis. Here we report that Seh1, a component of the Nup107-160 complex, functions in chromosome alignment and segregation by regulating the centromeric localization of Aurora B and other chromosome passenger complex proteins. Localization of CENP-E is not affected by Seh1 depletion and analysis by electron microscopy showed that microtubule kinetochore attachments are intact. Seh1-depleted cells show impaired Aurora B localization, which results in severe defects in biorientation and organization of the spindle midzone and midbody. Our results indicate that a major function of the Nup107 complex in mitosis is to ensure the proper localization of the CPC at the centromere.

Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development

Summary Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. Here, we show that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. We demonstrate that NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. We delineate a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. We finally show that this unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation.

Bio-identity and fate of albumin-coated SPIONs evaluated in cells and by the C. elegans model

UNLABELLEDnNanoparticles which surface adsorb proteins in an uncontrolled and non-reproducible manner will have limited uses as nanomedicinal products. A promising approach to avoid nanoparticle non-specific interactions with proteins is to design bio-hybrids by purposely pre-forming a protein corona around the inorganic cores. Here, we investigate, in vitro and in vivo, the newly acquired bio-identity of superparamagnetic iron oxide nanoparticles (SPIONs) upon their functionalization with a pre-formed and well-defined bovine serum albumin (BSA) corona. Cellular uptake, intracellular particle distribution and cytotoxicity were studied in two cell lines: adherent and non-adherent cells. BSA decreases nanoparticle internalization in both cell lines and protects the iron core once they have been internalized. The physiological response to the nanoparticles is then in vivo evaluated by oral administration to Caenorhabditis elegans, which was selected as a model of a functional intestinal barrier. Nanoparticle biodistribution, at single particle resolution, is studied by transmission electron microscopy. The analysis reveals that the acidic intestinal environment partially digests uncoated SPIONs but does not affect BSA-coated ones. It also discloses that some particles could enter the nematodes enterocytes, likely by endocytosis which is a different pathway than the one described for the worm nutrients.nnnSTATEMENT OF SIGNIFICANCEnUnravelling meaningful relationships between the physiological impact of engineered nanoparticles and their synthetic and biological identity is of vital importance when considering nanoparticles biomedical uses and when establishing their nanotoxicological profile. This study contributes to better comprehend the inorganic nanoparticles behavior in real biological milieus. We synthesized a controlled pre-formed BSA protein corona on SPIONs to lower unspecific cell uptake and decrease nanoparticle fouling with other proteins. Such findings may be of relevance considering clinical translation and regulatory issues of inorganic nanoparticles. Moreover, we have advanced in the validation of C. elegans as a simple animal model for assessing biological responses of engineering nanomaterials. The physiological response of BSA coated SPIONs was evaluated in vivo after their oral administration to C. elegans. Analyzing ultra-thin cross-sections of the worms by TEM with single-particle precision, we could track NP biodistribution along the digestive tract and determine unambiguously their translocation through biological barriers and cell membranes.

A bacterial tubulovesicular network

ABSTRACT We report the presence of a membranous tubulovesicular network in the planctomycete bacterium Gemmata obscuriglobus. This endomembrane system interacts with membrane coat proteins and is capable of protein internalization and degradation. Taken together, this suggests that the planctomycetal bacterium could illuminate the emergence of complex endomembrane systems.

CHD4 Is a RanGTP-Dependent MAP that Stabilizes Microtubules and Regulates Bipolar Spindle Formation

BACKGROUNDnProduction of the GTP-bound form of the Ran GTPase (RanGTP) around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)-containing proteins. Several NLS proteins have been identified as spindle assembly factors, but the complexity of the process led us to search for additional proteins with distinct roles in spindle assembly.nnnRESULTSnWe identify a chromatin-remodeling ATPase, CHD4, as a RanGTP-dependent microtubule (MT)-associated protein (MAP). MT binding occurs via the region containing an NLS and chromatin-binding domains. In Xenopus egg extracts and cultured cells, CHD4 largely dissociates from mitotic chromosomes and partially localizes to the spindle. Immunodepletion of CHD4 from egg extracts significantly reduces the quantity ofxa0MTs produced around chromatin and prevents spindle assembly. CHD4 RNAi in both HeLa and Drosophila S2 cells induces defects in spindle assembly and chromosome alignment in early mitosis, leading to chromosome missegregation. Further analysis in egg extracts and in HeLa cells reveals that CHD4 is a RanGTP-dependent MT stabilizer. Moreover, the CHD4-containing NuRD complex promotes organization of MTs into bipolar spindles in egg extracts. Importantly, this function of CHD4 is independent of chromatin remodeling.nnnCONCLUSIONSnOur results uncover a new role for CHD4 as a MAP required for MT stabilization and involved in generating spindle bipolarity.

Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells

ABSTRACT The mechanism of endoplasmic reticulum (ER) morphogenesis is incompletely understood. ER tubules are shaped by the reticulons (RTNs) and DP1/Yop1p family members, but the mechanism of ER sheet formation is much less clear. Here, we characterize TMEM170A, a human transmembrane protein, which localizes in ER and nuclear envelope membranes. Silencing or overexpressing TMEM170A in HeLa K cells alters ER shape and morphology. Ultrastructural analysis reveals that downregulation of TMEM170A specifically induces tubular ER formation, whereas overexpression of TMEM170A induces ER sheet formation, indicating that TMEM170A is a newly discovered ER-sheet-promoting protein. Additionally, downregulation of TMEM170A alters nuclear shape and size, decreases the density of nuclear pore complexes (NPCs) in the nuclear envelope and causes either a reduction in inner nuclear membrane (INM) proteins or their relocalization to the ER. TMEM170A interacts with RTN4, a member of the reticulon family; simultaneous co-silencing of TMEM170A and RTN4 rescues ER, NPC and nuclear-envelope-related phenotypes, implying that the two proteins have antagonistic effects on ER membrane organization, and nuclear envelope and NPC formation. Highlighted Article: TMEM170A is a human ER and nuclear envelope transmembrane protein. Down- and overexpression induce tubular or sheet ER formation, respectively, indicating that TMEM170A is a newly discovered ER-sheet-promoting protein.