Nuclear Pores Assemble from Nucleoporin Condensates During Oogenesis.

The molecular events that direct nuclear pore complex (NPC) assembly toward nuclear envelopes have been conceptualized in two pathways that occur during mitosis or interphase, respectively. In gametes and embryonic cells, NPCs also occur within stacked cytoplasmic membrane sheets, termed annulate lamellae (AL), which serve as NPC storage for early development. The mechanism of NPC biogenesis at cytoplasmic membranes remains unknown. Here, we show that during Drosophila oogenesis, Nucleoporins condense into different precursor granules that interact and progress into NPCs. Nup358 is a key player that condenses into NPC assembly platforms while its mRNA localizes to their surface in a translation-dependent manner. In concert, Microtubule-dependent transport, the small GTPase Ran and nuclear transport receptors regulate NPC biogenesis in oocytes. We delineate a non-canonical NPC assembly mechanism that relies on Nucleoporin condensates and occurs away from the nucleus under conditions of cell cycle arrest.

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

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.

Life-cycle-coupled evolution of mitosis in close relatives of animals.

Eukaryotes have evolved towards one of two extremes along a spectrum of strategies for remodelling the nuclear envelope during cell division: disassembling the nuclear envelope in an open mitosis or constructing an intranuclear spindle in a closed mitosis1,2. Both classes of mitotic remodelling involve key differences in the core division machinery but the evolutionary reasons for adopting a specific mechanism are unclear. Here we use an integrated comparative genomics and ultrastructural imaging approach to investigate mitotic strategies in Ichthyosporea, close relatives of animals and fungi. We show that species in this clade have diverged towards either a fungal-like closed mitosis or an animal-like open mitosis, probably to support distinct multinucleated or uninucleated states. Our results indicate that multinucleated life cycles favour the evolution of closed mitosis.

The baseless mutant links protein phosphatase 2A with basal cell identity in the brown alga Ectocarpus.

The first mitotic division of the initial cell is a key event in all multicellular organisms and is associated with the establishment of major developmental axes and cell fates. The brown alga Ectocarpus has a haploid-diploid life cycle that involves the development of two multicellular generations: the sporophyte and the gametophyte. Each generation deploys a distinct developmental programme autonomously from an initial cell, the first cell division of which sets up the future body pattern. Here, we show that mutations in the BASELESS (BAS) gene result in multiple cellular defects during the first cell division and subsequent failure to produce basal structures during both generations. BAS encodes a type B″ regulatory subunit of protein phosphatase 2A (PP2A), and transcriptomic analysis identified potential effector genes that may be involved in determining basal cell fate. The bas mutant phenotype is very similar to that observed in distag (dis) mutants, which lack a functional Tubulin-binding co-factor Cd1 (TBCCd1) protein, indicating that TBCCd1 and PP2A are two essential components of the cellular machinery that regulates the first cell division and mediates basal cell fate determination.

In-cell architecture of the nuclear pore and snapshots of its turnover.

Nuclear pore complexes (NPCs) fuse the inner and outer membranes of the nuclear envelope. They comprise hundreds of nucleoporins (Nups) that assemble into multiple subcomplexes and form large central channels for nucleocytoplasmic exchange1,2. How this architecture facilitates messenger RNA export, NPC biogenesis and turnover remains poorly understood. Here we combine in situ structural biology and integrative modelling with correlative light and electron microscopy and molecular perturbation to structurally analyse NPCs in intact Saccharomyces cerevisiae cells within the context of nuclear envelope remodelling. We find an in situ conformation and configuration of the Nup subcomplexes that was unexpected from the results of previous in vitro analyses. The configuration of the Nup159 complex appears critical to spatially accommodate its function as an mRNA export platform, and as a mediator of NPC turnover. The omega-shaped nuclear envelope herniae that accumulate in nup116Δ cells3 conceal partially assembled NPCs lacking multiple subcomplexes, including the Nup159 complex. Under conditions of starvation, herniae of a second type are formed that cytoplasmically expose NPCs. These results point to a model of NPC turnover in which NPC-containing vesicles bud off from the nuclear envelope before degradation by the autophagy machinery. Our study emphasizes the importance of investigating the structure-function relationship of macromolecular complexes in their cellular context.

Targeted volume correlative light and electron microscopy of an environmental marine microorganism.

Photosynthetic microalgae are responsible for an important fraction of CO2 fixation and O2 production on Earth. Three-dimensional (3D) ultrastructural characterization of these organisms in their natural environment can contribute to a deeper understanding of their cell biology. However, the low throughput of volume electron microscopy (vEM) methods along with the complexity and heterogeneity of environmental samples pose great technical challenges. In the present study, we used a workflow based on a specific electron microscopy sample preparation method compatible with both light and vEM imaging in order to target one cell among a complex natural community. This method revealed the 3D subcellular landscape of a photosynthetic dinoflagellate, which we identified as Ensiculifera tyrrhenica, with quantitative characterization of multiple organelles. We show that this cell contains a single convoluted chloroplast and show the arrangement of the flagellar apparatus with its associated photosensitive elements. Moreover, we observed partial chromatin unfolding, potentially associated with transcription activity in these organisms, in which chromosomes are permanently condensed. Together with providing insights in dinoflagellate biology, this proof-of-principle study illustrates an efficient tool for the targeted ultrastructural analysis of environmental microorganisms in heterogeneous mixes.

Spatial control of nucleoporin condensation by fragile X-related proteins.

Nucleoporins (Nups) build highly organized nuclear pore complexes (NPCs) at the nuclear envelope (NE). Several Nups assemble into a sieve-like hydrogel within the central channel of the NPCs. In the cytoplasm, the soluble Nups exist, but how their assembly is restricted to the NE is currently unknown. Here, we show that fragile X-related protein 1 (FXR1) can interact with several Nups and facilitate their localization to the NE during interphase through a microtubule-dependent mechanism. Downregulation of FXR1 or closely related orthologs FXR2 and fragile X mental retardation protein (FMRP) leads to the accumulation of cytoplasmic Nup condensates. Likewise, models of fragile X syndrome (FXS), characterized by a loss of FMRP, accumulate Nup granules. The Nup granule-containing cells show defects in protein export, nuclear morphology and cell cycle progression. Our results reveal an unexpected role for the FXR protein family in the spatial regulation of nucleoporin condensation.

Direct Decarboxylative C-2 Alkylation of Azoles through Minisci-Type Coupling.

This note discusses the application of a Minisci-type reaction for the direct alkylation of azoles with carboxylic acids as radical precursors. Different reaction conditions were investigated to achieve high yield of the desired products, focusing on acid strength and solvent screening. Moreover, the reactivity of imidazoles with various carboxylic acids was investigated, showing good yield for most cases. The study reveals the potential of this approach for late-stage functionalization in drug discovery.

GM130 and p115 play a key role in the organisation of the early secretory pathway during skeletal muscle differentiation.

Skeletal muscle (SKM) differentiation is a highly regulated process leading to the formation of specialised cells with reorganised compartments and organelles, such as those of the early secretory pathway. During SKM differentiation the Golgi complex (GC) redistributes close to the nuclear envelope and in small distinct peripheral structures distributed throughout the myotube. Concurrently, GC elements closely associate with endoplasmic reticulum-exit sites (ERES). The mechanisms underlying this reorganisation and its relevance for SKM differentiation are poorly understood. Here, we show, by time-lapse imaging studies, that the changes in GC organisation involve GC fragmentation and redistribution of ERES with the formation of tightly associated GC-ERES units. We show that knockdown of GM130 (also known as GOLGA2) or p115 (also known as USO1), two regulators of the early secretory pathway, impairs GC and ERES reorganisation. This in turn results in inhibition of myotube fusion and M-cadherin (also known as CDH15) transport to the sarcolemma. Taken together, our data suggest that the correct reorganisation of the early secretory pathway components plays an important role in SKM differentiation and, thus, associated pathologies.

Alpha-synuclein fibrils recruit TBK1 and OPTN to lysosomal damage sites and induce autophagy in microglial cells.

Autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, but the exact mechanisms and causal connections are not clear and most previous work was done in neurons and not in microglial cells. Here, we report that exogenous fibrillary, but not monomeric, alpha-synuclein (AS, also known as SNCA) induces autophagy in microglial cells. We extensively studied the dynamics of this response using both live-cell imaging and correlative light-electron microscopy (CLEM), and found that it correlates with lysosomal damage and is characterised by the recruitment of the selective autophagy-associated proteins TANK-binding kinase 1 (TBK1) and optineurin (OPTN) to ubiquitylated lysosomes. In addition, we observed that LC3 (MAP1LC3B) recruitment to damaged lysosomes was dependent on TBK1 activity. In these fibrillar AS-treated cells, autophagy inhibition impairs mitochondrial function and leads to microglial cell death. Our results suggest that microglial autophagy is induced in response to lysosomal damage caused by persistent accumulation of AS fibrils. Importantly, triggering of the autophagic response appears to be an attempt at lysosomal quality control and not for engulfment of fibrillar AS.This article has an associated First Person interview with the first author of the paper.

Altered nuclear envelope structure and proteasome function of micronuclei.

Micronuclei are extra-nuclear bodies containing whole chromosomes that were not incorporated into the nucleus after cell division or damaged chromosome fragments. Even though the link between micronuclei and DNA damage is described for a long time, little is known about the functional organization of micronuclei and their contribution to tumorigenesis. We showed fusions between micronuclear membranes and lysosomes by electron microscopy and linked lysosome function to DNA damage levels in micronuclei. In addition, micronuclei drastically differ from primary nuclei in nuclear envelope composition, with a significant increase in the relative amount of nuclear envelope proteins LBR and emerin and a decrease in nuclear pore proteins. Strikingly, micronuclei lack active proteasomes, as the processing subunits and other factors of the ubiquitin proteasome system. Moreover, micronuclear chromatin shows a higher degree of compaction as compared to primary nuclei. The specific aberrations identified in micronuclei and the potential functional consequences of these defects may contribute to the role of micronuclei in catastrophic genomic rearrangements.

Positive feedback between Golgi membranes, microtubules and ER exit sites directs de novo biogenesis of the Golgi.

The Golgi complex is the central organelle of the secretory pathway. It undergoes dynamic changes during the cell cycle, but how it acquires and maintains its complex structure is unclear. To address this question, we have used laser nanosurgery to deplete BSC1 cells of the Golgi complex and have monitored its biogenesis by quantitative time-lapse microscopy and correlative electron microscopy. After Golgi depletion, endoplasmic reticulum (ER) export is inhibited and the number of ER exit sites (ERES) is reduced and does not increase for several hours. Occasional fusion of small post-ER carriers to form the first larger structures triggers a rapid and drastic growth of Golgi precursors, due to the capacity of these structures to attract more carriers by microtubule nucleation and to stimulate ERES biogenesis. Increasing the chances of post-ER carrier fusion close to ERES by depolymerizing microtubules results in the acceleration of Golgi and ERES biogenesis. Taken together, on the basis of our results, we propose a self-organizing principle of the early secretory pathway that integrates Golgi biogenesis, ERES biogenesis and the organization of the microtubule network by positive-feedback loops.

Metastatic liver disease from non-colorectal, non-neuroendocrine, non-sarcoma cancers: a systematic review.

BACKGROUND: Hepatic resection of liver metastases of non-colorectal, non-neuroendocrine, and non-sarcoma (NCNNNS) primary malignancies seems to improve survival in selected patients. The aims of the current review were to describe long-term results of surgery and to evaluate prognostic factors for survival in patients who underwent resection of NCNNNS liver metastases. METHODS: We identified 30 full texts (25 single-center and 5 multicenter studies) published after year 1995 and published in English with a total of 3849 patients. For NCNNNS liver metastases, 83.4 % of these subjects were resected. RESULTS: No prior systematic reviews or meta-analyses on this topic were identified. All studies were case series without matching control groups. The most common primary sites were breast (23.8 %), genito-urinary (21.8 %), and gastrointestinal tract (19.8 %). The median 5- and 10-year overall survival were 32.3 % (range 19-42 %) and 24 % (indicated only in two studies, range 23-25 %), respectively, with 71 % of R0 resections. CONCLUSIONS: There is evidence suggesting that surgery of NCNNNS metastases is safe, feasible, and effective if treatment is part of a multidisciplinary approach and if indication is based on the prognostic factors underlined in literature analysis.

First total synthesis of caerulomycin K: a case study on selective, multiple C-H functionalizations of pyridines.

Caerulomycins, natural alkaloids with antimicrobial properties, have been previously synthesized starting with highly pre-functionalized building blocks or requiring many functional group manipulations. In this work, we report the first total synthesis of caerulomycin K, a diversely trifunctionalized pyridine readily assembled in three steps exploiting the recent advancements in the C-H activation of N-heterocycles.

Electrochemical Synthesis of Unnatural Amino Acids Embedding 5- and 6-Membered Heteroaromatics.

Using a commercially available potentiostat, the electrochemical synthesis of unnatural amino acids bearing heteroaromatics on the lateral chain has been accomplished. This strategy exploits the side-chain decarboxylative arylation of aspartic/glutamic acid, a reaction that becomes challenging with electron-rich coupling partners such as 5- and 6-membered heteroaromatics. These rings are underrepresented in unnatural amino acids, therefore allowing a wider exploration of the chemical space, given the abundance of the aryl bromides employable in this reaction.

Discovery and Optimization of Pyridazinones as PI3Kδ Selective Inhibitors for Administration by Inhalation.

A hit-to-lead campaign pursuing the identification of novel inhalant small-molecule phosphatidylinositol 3-kinase (PI3K) inhibitors for the treatment of inflammatory respiratory diseases is disclosed. A synthetically versatile pyridazin-3(2H)-one scaffold was designed, and three exit vectors on the core moiety were used to explore chemical diversity and optimize pharmacological and absorption, distribution, metabolism, and excretion (ADME) properties. Desired modulation of PI3Kδ selectivity and cellular potency as well as ADME properties in view of administration by inhalation was achieved. Intratracheal administration of lead compound 26 resulted in a promising pharmacokinetic profile, thus demonstrating that the optimization strategy of in vitro profiles successfully translated to an in vivo setting.

Adhesion energy controls lipid binding-mediated endocytosis.

Several bacterial toxins and viruses can deform membranes through multivalent binding to lipids for clathrin-independent endocytosis. However, it remains unclear, how membrane deformation and endocytic internalization are mechanistically linked. Here we show that many lipid-binding virions induce membrane deformation and clathrin-independent endocytosis, suggesting a common mechanism based on multivalent lipid binding by globular particles. We create a synthetic cellular system consisting of a lipid-anchored receptor in the form of GPI-anchored anti-GFP nanobodies and a multivalent globular binder exposing 180 regularly-spaced GFP molecules on its surface. We show that these globular, 40 nm diameter, particles bind to cells expressing the receptor, deform the plasma membrane upon adhesion and become endocytosed in a clathrin-independent manner. We explore the role of the membrane adhesion energy in endocytosis by using receptors with affinities varying over 7 orders of magnitude. Using this system, we find that once a threshold in adhesion energy is overcome to allow for membrane deformation, endocytosis occurs reliably. Multivalent, binding-induced membrane deformation by globular binders is thus sufficient for internalization to occur and we suggest it is the common, purely biophysical mechanism for lipid-binding mediated endocytosis of toxins and pathogens.

A novel laser nanosurgery approach supports de novo Golgi biogenesis in mammalian cells.

The Golgi complex has a central role in the secretory pathway of all higher organisms. To explain the synthesis of its unique stacked structure in mammalian cells, two major models have been proposed. One suggests that it is synthesized de novo from the endoplasmic reticulum. The second model postulates a pre-existing Golgi template that serves as a scaffold for its biogenesis. To test these hypotheses directly, we have developed an approach in which we deplete the Golgi complex from living cells by laser nanosurgery, and subsequently analyze the 'Golgi-depleted' karyoplast using time-lapse and electron microscopy. We show that biosynthetic transport is blocked after Golgi depletion, but is restored 12 hours later. This recovery of secretory transport coincides with an ordered assembly of stacked Golgi structures, and we also observe the appearance of matrix proteins before that of Golgi enzymes. Functional experiments using RNA interference-mediated knockdown of GM130 further demonstrate the importance of the matrix during Golgi biogenesis. By contrast, the centrosome, which can also be removed by laser nanosurgery and is not reformed within the considered time frame, is not required for this process. Altogether, our data provide evidence that de novo Golgi biogenesis can occur in mammalian cells.

Synthesis of a structural analogue of the repeating unit from Streptococcus pneumoniae 19F capsular polysaccharide based on the cross-metathesis-selenocyclization reaction sequence.

Pseudo-oligosaccharides have attracted much interest as scaffolds for the synthesis of sugar mimics endowed with very similar biological properties but structurally and synthetically simpler than their natural counterparts. Herein, the synthesis of pseudo-oligosaccharides using the cross-metathesis reaction between distinct sugar-olefins followed by intramolecular selenocyclization of the obtained heterodimer as key steps is first investigated. This methodology has been then applied to the preparation of structural analogues of the trisaccharide repeating unit from Streptococcus pneumoniae 19F. The inhibition abilities of the synthetic molecules were evaluated by a competitive ELISA assay using a rabbit polyclonal anti-19F serum.