Free-Standing DNA Origami Superlattice to Facilitate Cryo-EM Visualization of Membrane Vesicles.

Technological breakthroughs in cryo-electron microscopy (cryo-EM) methods open new perspectives for highly detailed structural characterizations of extracellular vesicles (EVs) and synthetic liposome-protein assemblies. Structural characterizations of these vesicles in solution under a nearly native hydrated state are of great importance to decipher cell-to-cell communication and to improve EVs' application as markers in diagnosis and as drug carriers in disease therapy. However, difficulties in preparing holey carbon cryo-EM grids with low vesicle heterogeneities, at low concentration and with kinetic control of the chemical reactions or assembly processes, have limited cryo-EM use in the EV study. We report a straightforward membrane vesicle cryo-EM sample preparation method that assists in circumventing these limitations by using a free-standing DNA-affinity superlattice for covering holey carbon cryo-EM grids. Our approach uses DNA origami to self-assemble to a solution-stable and micrometer-sized ordered molecular template in which structure and functional properties can be rationally controlled. We engineered the template with cholesterol-binding sites to specifically trap membrane vesicles. The advantages of this DNA-cholesterol-affinity lattice (DCAL) include (1) local enrichment of artificial and biological vesicles at low concentration and (2) isolation of heterogeneous cell-derived membrane vesicles (exosomes) from a prepurified pellet of cell culture conditioned medium on the grid.

The structure of pathogenic huntingtin exon 1 defines the bases of its aggregation propensity.

Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the first exon of the HTT gene, resulting in an extended polyglutamine (poly-Q) tract in huntingtin (httex1). The structural changes occurring to the poly-Q when increasing its length remain poorly understood due to its intrinsic flexibility and the strong compositional bias. The systematic application of site-specific isotopic labeling has enabled residue-specific NMR investigations of the poly-Q tract of pathogenic httex1 variants with 46 and 66 consecutive glutamines. Integrative data analysis reveals that the poly-Q tract adopts long α-helical conformations propagated and stabilized by glutamine side chain to backbone hydrogen bonds. We show that α-helical stability is a stronger signature in defining aggregation kinetics and the structure of the resulting fibrils than the number of glutamines. Our observations provide a structural perspective of the pathogenicity of expanded httex1 and pave the way to a deeper understanding of poly-Q-related diseases.

Structure and mechanics of the human nuclear pore complex basket using correlative AFM-fluorescence superresolution microscopy.

Nuclear pore complexes (NPCs) are the only gateways between the nucleus and cytoplasm in eukaryotic cells. They restrict free diffusion to molecules below 5 nm while facilitating the active transport of selected cargoes, sometimes as large as the pore itself. This versatility implies an important pore plasticity. Recently, cryo-EM and AI-based protein modeling of human NPC revealed with acute precision how most constituents are arranged. But the basket, a fish trap-like structure capping the nucleoplasmic side of the pore, remains poorly resolved. Here by atomic force microscopy (AFM) coupled to single molecule localization microscopy (SMLM) we revealed that the basket is very soft and explores a large conformational landscape: apart from its canonical basket shape, it dives into the central pore channel or opens, with filaments reaching to the pore sides. Our observations highlight how this structure can adapt and let morphologically diverse cargoes shuttle through NPCs.

Mechanical Control of Cell Migration by the Metastasis Suppressor Tetraspanin CD82/KAI1.

The plasma membrane is a key actor of cell migration. For instance, its tension controls persistent cell migration and cell surface caveolae integrity. Then, caveolae constituents such as caveolin-1 can initiate a mechanotransduction loop that involves actin- and focal adhesion-dependent control of the mechanosensor YAP to finely tune cell migration. Tetraspanin CD82 (also named KAI-1) is an integral membrane protein and a metastasis suppressor. Its expression is lost in many cancers including breast cancer. It is a strong inhibitor of cell migration by a little-known mechanism. We demonstrated here that CD82 controls persistent 2D migration of EGF-induced single cells, stress fibers and focal adhesion sizes and dynamics. Mechanistically, we found that CD82 regulates membrane tension, cell surface caveolae abundance and YAP nuclear translocation in a caveolin-1-dependent manner. Altogether, our data show that CD82 controls 2D cell migration using membrane-driven mechanics involving caveolin and the YAP pathway.

Author Correction: STEEP mediates STING ER exit and activation of signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

STEEP mediates STING ER exit and activation of signaling.

STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.

Nanoscale organization of tetraspanins during HIV-1 budding by correlative dSTORM/AFM.

Membrane partition and remodeling play a key role in numerous cell mechanisms, especially in viral replication cycles where viruses subvert the plasma membrane to enter and escape from the host cell. Specifically assembly and release of HIV-1 particles require specific cellular components, which are recruited to the egress site by the viral protein Gag. We previously demonstrated that HIV-1 assembly alters both partitioning and dynamics of the tetraspanins CD9 and CD81, which are key players in many infectious processes, forming enriched areas where the virus buds. In this study we correlated super resolution microscopy mapping of tetraspanins with membrane topography delineated by atomic force microscopy (AFM) in Gag-expressing cells. We revealed that CD9 is specifically trapped within the nascent viral particles, especially at buds tips, suggesting that Gag mediates CD9 and CD81 depletion from the plasma membrane. In addition, we showed that CD9 is organized as small membrane assemblies of few tens of nanometers that can coalesce upon Gag expression.

A Developmental Social Neuroscience Model for Understanding Pathways to Substance Use Disorders During Adolescence.

Adolescence is a transitional period of development characterized by critical changes in physical, neural, cognitive, affective, and social functions. Studies investigating the underlying mechanisms of substance use at levels of self-report, brain response, and behavioral data are generally consistent with suggestions from dual-process model that differential growth rates of frontally mediated control and striato-frontal reward processing are related to a heightened risk of substance use during adolescence. However, social theories highlight the important role of social context and environment in which adolescents grow up and suggest that growing up in an unfavorable environment and in particular exposure to adverse childhood experiences play a huge role in how this vulnerability is translated into actual risk. In this review, we provide a summary of recent theories that examine a number of key individual and social and environmental risk factors underlying risk for early initiation and escalation of substance misuse. We also present a model that expands the dual-process model to incorporate the role of negative self-concept and negative affect associated with growing up in an unfavorable environment and their interactions with cognitive control and inhibition to further explain vulnerability to early initiation and development of substance misuse in adolescents.

Membrane Curvature Sensing by Amphipathic Helices Is Modulated by the Surrounding Protein Backbone.

Membrane curvature is involved in numerous biological pathways like vesicle trafficking, endocytosis or nuclear pore complex assembly. In addition to its topological role, membrane curvature is sensed by specific proteins, enabling the coordination of biological processes in space and time. Amongst membrane curvature sensors are the ALPS (Amphipathic Lipid Packing Sensors). ALPS motifs are short peptides with peculiar amphipathic properties. They are found in proteins targeted to distinct curved membranes, mostly in the early secretory pathway. For instance, the ALPS motif of the golgin GMAP210 binds trafficking vesicles, while the ALPS motif of Nup133 targets nuclear pores. It is not clear if, besides curvature sensitivity, ALPS motifs also provide target specificity, or if other domains in the surrounding protein backbone are involved. To elucidate this aspect, we studied the subcellular localization of ALPS motifs outside their natural protein context. The ALPS motifs of GMAP210 or Nup133 were grafted on artificial fluorescent probes. Importantly, ALPS motifs are held in different positions and these contrasting architectures were mimicked by the fluorescent probes. The resulting chimeras recapitulated the original proteins localization, indicating that ALPS motifs are sufficient to specifically localize proteins. Modulating the electrostatic or hydrophobic content of Nup133 ALPS motif modified its avidity for cellular membranes but did not change its organelle targeting properties. In contrast, the structure of the backbone surrounding the helix strongly influenced targeting. In particular, introducing an artificial coiled-coil between ALPS and the fluorescent protein increased membrane curvature sensitivity. This coiled-coil domain also provided membrane curvature sensitivity to the amphipathic helix of Sar1. The degree of curvature sensitivity within the coiled-coil context remains correlated to the natural curvature sensitivity of the helices. This suggests that the chemistry of ALPS motifs is a key parameter for membrane curvature sensitivity, which can be further modulated by the surrounding protein backbone.

Can cannabis use be prevented by targeting personality risk in schools? Twenty-four-month outcome of the adventure trial on cannabis use: a cluster-randomized controlled trial.

AIMS: To examine the effectiveness of a personality-targeted intervention program (Adventure trial) delivered by trained teachers to high-risk (HR) high-school students on reducing marijuana use and frequency of use. DESIGN: A cluster-randomized controlled trial. SETTING: Secondary schools in London, UK. PARTICIPANTS: Twenty-one secondary schools were randomized to intervention (n = 12) or control (n = 9) conditions, encompassing a total of 1038 HR students in the ninth grade [mean (standard deviation) age = 13.7 (0.33) years]. INTERVENTIONS: Brief personality-targeted interventions to students with one of four HR profiles: anxiety sensitivity, hopelessness, impulsivity and sensation-seeking. PRIMARY OUTCOME: marijuana use. Secondary outcome: frequency of use. Assessed using the Reckless Behaviour Questionnaire at intervals of 6 months for 2 years. Personality risk was measured with the Substance Use Risk Profile Scale. FINDINGS: Logistic regression analysis revealed significant intervention effects on cannabis use rates at the 6-month follow-up in the intent-to-treat sample [odds ratio (OR) = 0.67, P = 0.05, 95% confidence interval (CI) = 0.45-1.0] and significant reductions in frequency of use at 12- and 18-month follow-up (ß = -0.14, P ≤ 0.05, 95% CI = -0.6 to -0.01; ß = -0.12, P ≤ 0.05, 95% CI = -0.54 to 0.0), but this was not supported in two-part latent growth models. Subgroup analyses (both logistic and two-part models) reveal that the sensation-seeking intervention delayed the onset of cannabis use among sensation seekers (OR = 0.25, ß = -0.833, standard error = 0.342, P = 0.015). CONCLUSIONS: Personality-targeted interventions can be delivered effectively by trained school staff to delay marijuana use onset among a subset of high-risk teenagers: sensation-seekers.

Nuclear pore biogenesis into an intact nuclear envelope.

Nuclear pore complexes (NPCs) serve as transport channels across the nuclear membrane, a double lipid bilayer that physically separates the nucleoplasm and cytoplasm of eukaryotic cells. New evidence suggests that the multiprotein nuclear pores also play a role in chromatin organization and gene expression. Given the importance of NPC function, it is not surprising that a growing list of human diseases and developmental defects have been linked to its malfunction. In order to fully understand the functional repertoire of NPCs and their essential role for nuclear organization, it is critical to determine the sequence of events that lead to the formation of nuclear pores. This is particularly relevant since NPC number, and possibly composition, are tightly linked to metabolic activity. Most of our knowledge is derived from NPC formation that occurs in dividing cells at the end of mitosis when the nuclear envelope (NE) and NPCs reform from disassembled precursors. However, NPC assembly also takes place during interphase into an intact NE. Importantly, this process is not restricted to dividing cells but also occurs during cell differentiation. Here, we will review aspects unique to this process, namely the regulation of nuclear expansion and the mechanisms of fusion between the outer and inner nuclear membranes. We will then discuss conserved and diverging mechanisms between post-mitotic and interphase assembly of the proteinaceous structure in light of recently published data.

Cell cycle-dependent differences in nuclear pore complex assembly in metazoa.

In metazoa, nuclear pore complexes (NPCs) assemble from disassembled precursors into a reforming nuclear envelope (NE) at the end of mitosis and into growing intact NEs during interphase. Here, we show via RNAi-mediated knockdown that ELYS, a nucleoporin critical for the recruitment of the essential Nup107/160 complex to chromatin, is required for NPC assembly at the end of mitosis but not during interphase. Conversely, the transmembrane nucleoporin POM121 is critical for the incorporation of the Nup107/160 complex into new assembly sites specifically during interphase. Strikingly, recruitment of the Nup107/160 complex to an intact NE involves a membrane curvature-sensing domain of its constituent Nup133, which is not required for postmitotic NPC formation. Our results suggest that in organisms with open mitosis, NPCs assemble via two distinct mechanisms to accommodate cell cycle-dependent differences in NE topology.

Single bead affinity detection (SINBAD) for the analysis of protein-protein interactions.

We present a miniaturized pull-down method for the detection of protein-protein interactions using standard affinity chromatography reagents. Binding events between different proteins, which are color-coded with quantum dots (QDs), are visualized on single affinity chromatography beads by fluorescence microscopy. The use of QDs for single molecule detection allows the simultaneous analysis of multiple protein-protein binding events and reduces the amount of time and material needed to perform a pull-down experiment.

A novel role for PA28gamma-proteasome in nuclear speckle organization and SR protein trafficking.

In eukaryotic cells, proteasomes play an essential role in intracellular proteolysis and are involved in the control of most biological processes through regulated degradation of key proteins. Analysis of 20S proteasome localization in human cell lines, using ectopic expression of its CFP-tagged alpha7 subunit, revealed the presence in nuclear foci of a specific and proteolytically active complex made by association of the 20S proteasome with its PA28gamma regulator. Identification of these foci as the nuclear speckles (NS), which are dynamic subnuclear structures enriched in splicing factors (including the SR protein family), prompted us to analyze the role(s) of proteasome-PA28gamma complexes in the NS. Here, we show that knockdown of these complexes by small interfering RNAs directed against PA28gamma strongly impacts the organization of the NS. Further analysis of PA28gamma-depleted cells demonstrated an alteration of intranuclear trafficking of SR proteins. Thus, our data identify proteasome-PA28gamma complexes as a novel regulator of NS organization and function, acting most likely through selective proteolysis. These results constitute the first demonstration of a role of a specific proteasome complex in a defined subnuclear compartment and suggest that proteolysis plays important functions in the precise control of splicing factors trafficking within the nucleus.

Roles and potential therapeutic targets of the ubiquitin proteasome system in muscle wasting.

Muscle wasting, characterized by the loss of protein mass in myofibers, is in most cases largely due to the activation of intracellular protein degradation by the ubiquitin proteasome system (UPS). During the last decade, mechanisms contributing to this activation have been unraveled and key mediators of this process identified. Even though much remains to be understood, the available information already suggests screens for new compounds inhibiting these mechanisms and highlights the potential for pharmaceutical drugs able to treat muscle wasting when it becomes deleterious. This review presents an overview of the main pathways contributing to UPS activation in muscle and describes the present state of efforts made to develop new strategies aimed at blocking or slowing muscle wasting. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

Multiple phosphorylation events control mitotic degradation of the muscle transcription factor Myf5.

BACKGROUND: The two myogenic regulatory factors Myf5 and MyoD are basic helix-loop-helix muscle transcription factors undergoing differential cell cycle dependent proteolysis in proliferating myoblasts. This regulated degradation results in the striking expression of these two factors at distinct phases of the cell cycle, and suggests that their precise and alternated disappearance is an important feature of myoblasts, maybe connected to the maintenance of the proliferative status and/or commitment to the myogenic lineage of these cells. One way to understand the biological function(s) of the cyclic expression of these proteins is to specifically alter their degradation, and to analyze the effects of their stabilization on cells. To this aim, we undertook the biochemical analysis of the mechanisms governing Myf5 mitotic degradation, using heterologous systems. RESULTS: We show here that mitotic degradation of Myf5 is conserved in non-myogenic cells, and is thus strictly under the control of the cell cycle apparatus. Using Xenopus egg extracts as an in vitro system to dissect the main steps of Myf5 mitotic proteolysis, we show that (1) Myf5 stability is regulated by a complex interplay of phosphorylation/dephosphorylation, probably involving various kinases and phosphatases, (2) Myf5 is ubiquitylated in mitotic extracts, and this is a prerequisite to its degradation by the proteasome and (3) at least in the Xenopus system, the E3 responsible for its mitotic degradation is not the APC/C (the major E3 during mitosis). CONCLUSION: Altogether, our data strongly suggest that the mitotic degradation of Myf5 by the ubiquitin-proteasome system is precisely controlled by multiple phosphorylation of the protein, and that the APC/C is not involved in this process.