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Utilizing cell membranes from diverse cell types for biointerfacing has demonstrated significant advantages in enhancing colloidal stability and incorporating biological properties, tailored specifically for various biomedical applications. However, the structures of these materials, particularly emulsions interfaced with red blood cell (RBC) or platelet (PLT) membranes, remain an underexplored area. This study systematically employs small- and ultra-small-angle neutron scattering (SANS and USANS) with contrast variation to investigate the structure of emulsions containing perfluorohexane within RBC (RBC/PFH) and PLT membranes (PLT/PFH). The findings reveal that the scattering length density of RBC and PLT membranes is 1.5 × 10-6 Å-2, similar to 30% (w/w) deuterium oxide. Using this solvent as a cell membrane-matching medium, estimated droplet diameters are 770 nm (RBC/PFH) and 1.5 µm (PLT/PFH), based on polydispersed sphere model fitting. Intriguingly, calculated patterns and invariant analysis reveal native droplet architectures featuring entirely liquid PFH cores, differing significantly from the observed bubble-droplet core system in electron microscopy. This highlights the advantage of SANS and USANS in differentiating genuine colloidal structures in complex dispersions. In summary, this work underscores the pivotal role of SANS and USANS in characterizing biointerfaced colloids and in uncovering novel colloidal structures with significant potential for biomedical applications and clinical translation.
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In this work, we present a pair of tools to improve the fiducial tracking and reconstruction quality of cryo-scanning transmission electron tomography (STET) datasets. We then demonstrate the effectiveness of these two tools on experimental cryo-STET data. The first tool, GoldDigger, improves the tracking of fiducials in cryo-STET by accommodating the changed appearance of highly defocussed fiducial markers. Since defocus effects are much stronger in scanning transmission electron microscopy than in conventional transmission electron microscopy, existing alignment tools do not perform well without manual intervention. The second tool, Checkers, combines image inpainting and unsupervised deep learning for denoising tomograms. Existing tools for denoising cryo-tomography often rely on paired noisy image frames, which are unavailable in cryo-STET datasets, necessitating a new approach. Finally, we make the two software tools freely available for the cryo-STET community.
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The compaction of chromatin is a prevalent paradigm in gene repression. Chromatin compaction is commonly thought to repress transcription by restricting chromatin accessibility. However, the spatial organisation and dynamics of chromatin compacted by gene-repressing factors are unknown. Using cryo-electron tomography, we solved the three-dimensional structure of chromatin condensed by the Polycomb Repressive Complex 1 (PRC1) in a complex with CBX8. PRC1-condensed chromatin is porous and stabilised through multivalent dynamic interactions of PRC1 with chromatin. Mechanistically, positively charged residues on the internally disordered regions (IDRs) of CBX8 mask negative charges on the DNA to stabilize the condensed state of chromatin. Within condensates, PRC1 remains dynamic while maintaining a static chromatin structure. In differentiated mouse embryonic stem cells, CBX8-bound chromatin remains accessible. These findings challenge the idea of rigidly compacted polycomb domains and instead provides a mechanistic framework for dynamic and accessible PRC1-chromatin condensates.
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Since its inception in the 1930s, transmission electron microscopy (TEM) has been a powerful method to explore the cellular structure of parasites. TEM usually requires samples of <100 nm thick and with protozoans being larger than 1 µm, their study requires resin embedding and ultrathin sectioning. During the past decade, several new methods have been developed to improve, facilitate, and speed up the structural characterisation of biological samples, offering new imaging modalities for the study of protozoans. In particular, scanning transmission electron microscopy (STEM) can be used to observe sample sections as thick as 1 µm thus becoming an alternative to conventional TEM. STEM can also be performed under cryogenic conditions in combination with cryo-electron tomography providing access to the study of thicker samples in their native hydrated states in 3D. This method, called cryo-scanning transmission electron tomography (cryo-STET), was first developed in 2014. This review presents the basic concepts and benefits of STEM methods and provides examples to illustrate the potential for new insights into the structure and ultrastructure of protozoans.
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Tomografia com Microscopia Eletrônica , Microscopia Eletrônica de Transmissão e Varredura/métodos , Tomografia com Microscopia Eletrônica/métodos , Microscopia Crioeletrônica/métodos , Microscopia Eletrônica de VarreduraRESUMO
Biocatalytic transformation has attracted increasing attention in the green synthesis of chemicals due to the diversity of enzymes, their high catalytic activities and specificities, and environmentally benign conditions. Most redox enzymes in nature are dependent on nicotinamide cofactors like ß-nicotinamide adenine dinucleotide (NAD+)/reduced nicotinamide adenine dinucleotide (NADH). The use of solar energy, especially visible light, in the regeneration of cofactors through the combination of photocatalysis and biocatalysis provides an extraordinary opportunity to make complete green processes. However, the combination of photocatalysts and enzymes has been challenged by the rapid degradation and deactivation of the enzymatic material by photogenerated reactive oxygen species (ROS). Here, we design core-shell structured polymer micelles and vesicles with aggregation-induced emission (AIE) as visible-light-mediated photocatalysts for highly stable and recyclable photobiocatalysis under aerobic conditions. NAD+ from NADH can be efficiently regenerated by the photoactive hydrophobic core of polymer micelles and the hydrophobic membrane of polymer vesicles, while the enzymatic material (glucose 1-dehydrogenase) is screened from the attack of photogenerated ROS by the hydrophilic surface layer of polymer colloids. After at least 10 regeneration cycles, the enzyme keeps its active state; meanwhile, polymer micelles and vesicles maintain their photocatalytic activity. These polymer colloids show the potential to be developed for the implementation of industrially relevant photobiocatalytic systems.
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Micelas , NAD , NAD/metabolismo , Oxirredução , Polímeros/metabolismo , Espécies Reativas de Oxigênio , BiocatáliseRESUMO
In photodynamic therapy (PDT), the uses of nanoparticles bearing photosensitizers (PSs) can overcome some of the drawbacks of using a PS alone (e.g., poor water solubility and low tumor selectivity). However, numerous nano-formulations are developed by physical encapsulation of PSs through Van der Waals interactions, which have not only a limited load efficiency but also some in vivo biodistribution problems caused by leakage or burst release. Herein, polymersomes made from an amphiphilic block copolymer, in which a PS with aggregation-induced emission (AIE-PS) is covalently attached to its hydrophobic poly(amino acid) block, are reported. These AIE-PS polymersomes dispersed in aqueous solution have a high AIE-PS load efficiency (up to 46% as a mass fraction), a hydrodynamic diameter of 86 nm that is suitable for in vivo applications, and an excellent colloidal stability for at least 1 month. They exhibit a red/near-infrared photoluminescence and ability to generate reactive oxygen species (ROS) under visible light. They are non-cytotoxic in the dark as tested on Hela cells up to concentration of 100 µm. Benefiting from colloidal stability, AIE property and ROS generation capability, such a family of polymersomes can be great candidates for image-guided PDT.
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Neoplasias , Fotoquimioterapia , Humanos , Espécies Reativas de Oxigênio , Células HeLa , Distribuição Tecidual , Fármacos Fotossensibilizantes/química , Neoplasias/tratamento farmacológicoRESUMO
The bacterial chromosomic DNA is packed within a membrane-less structure, the nucleoid, due to the association of DNA with proteins called Nucleoid Associated Proteins (NAPs). Among these NAPs, Hfq is one of the most intriguing as it plays both direct and indirect roles on DNA structure. Indeed, Hfq is best known to mediate post-transcriptional regulation by using small noncoding RNA (sRNA). Although Hfq presence in the nucleoid has been demonstrated for years, its precise role is still unclear. Recently, it has been shown in vitro that Hfq forms amyloid-like structures through its C-terminal region, hence belonging to the bridging family of NAPs. Here, using cryo soft X-ray tomography imaging of native unlabeled cells and using a semi-automatic analysis and segmentation procedure, we show that Hfq significantly remodels the Escherichia coli nucleoid. More specifically, Hfq influences nucleoid density especially during the stationary growth phase when it is more abundant. Our results indicate that Hfq could regulate nucleoid compaction directly via its interaction with DNA, but also at the post-transcriptional level via its interaction with RNAs. Taken together, our findings reveal a new role for this protein in nucleoid remodeling in vivo, that may serve in response to stress conditions and in adapting to changing environments.
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Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Tomografia por Raios X , DNA , Proteínas de Escherichia coli/genética , Fator Proteico 1 do Hospedeiro/genéticaRESUMO
Crystallization from solution often occurs via "nonclassical" routes; that is, it involves transient, non-crystalline states like reactant-rich liquid droplets and amorphous particles. However, in mineral crystals, the well-defined thermodynamic character of liquid droplets and whether they convertâor notâinto amorphous phases have remained unassessed. Here, by combining cryo-transmission electron microscopy and X-ray scattering down to a 250 ms reaction time, we unveil that crystallization of cerium oxalate involves a metastable chemical equilibrium between transient liquid droplets and solid amorphous particles: contrary to the usual expectation, reactant-rich droplets do not evolve into amorphous solids. Instead, at concentrations above 2.5 to 10 mmol L-1, both amorphous and reactant-rich liquid phases coexist for several tens of seconds and their molar fractions remain constant and follow the lever rule in a multicomponent phase diagram. Such a metastable chemical equilibrium between solid and liquid precursors has been so far overlooked in multistep nucleation theories and highlights the interest of rationalizing phase transformations using multicomponent phase diagrams not only when designing and recycling rare earths materials but also more generally when describing nonclassical crystallization.
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Amyloid fibrils are aggregates of proteins or peptides. In humans, they are associated with various pathologies ranging from neurodegenerative diseases such as Alzheimer's and Parkinson's to systemic diseases like type 2 diabetes. In bacteria, amyloids can exert functional roles such as biofilm formation or gene regulation. Up to now, the aggregation mechanism leading to amyloid fibril formation is poorly understood as proteins with different amino acid sequences can fold into similar 3D structures. Understanding the formation of amyloid fibrils constitutes a central challenge for fighting major human health issues such as neurodegenerative diseases and biofilm formation in ports (implantable chambers). Since the dogma linking protein sequence, 3D structure, and function is increasingly disrupted by the growing understanding of the importance of disordered domains in proteins, it is crucial to possess a method capable of building accurate atomic models of amyloids. Aided by the leap forward of cryo-electron microscopy (cryo-EM), which can now routinely achieve sub-nanometric resolutions, it has become the method of choice for studying amyloids. In this chapter, we use the Hfq protein from Escherichia coli as an example to present general protocols in cryo-EM to unveil the structure of bacterial amyloids and improve our knowledge of their aggregation mechanism.
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Amiloide , Diabetes Mellitus Tipo 2 , Sequência de Aminoácidos , Amiloide/química , Bactérias/metabolismo , Microscopia Crioeletrônica/métodos , Escherichia coli/metabolismo , HumanosRESUMO
Bacterial chromosomal DNA is packed within a non-membranous structure, the nucleoid, thanks to nucleoid associated proteins (NAPs). The role of bacterial amyloid has recently emerged among these NAPs, particularly with the nucleoid-associated protein Hfq that plays a direct role in DNA compaction. In this chapter, we present a 3D imaging technique, cryo-soft X-ray tomography (cryo-SXT) to obtain a detailed 3D visualization of subcellular bacterial structures, especially the nucleoid. Cryo-SXT imaging of native unlabeled cells enables observation of the nucleoid in 3D with a high resolution, allowing to evidence in vivo the role of amyloids on DNA compaction. The precise experimental methods to obtain 3D tomograms will be presented.
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Organelas , Tomografia por Raios X , Proteínas Amiloidogênicas , Proteínas de Bactérias , DNA , DNA Bacteriano , Imageamento Tridimensional/métodos , Organelas/ultraestrutura , Tomografia por Raios X/métodosRESUMO
Electromagnetic radiation-triggered therapeutic effect has attracted a great interest over the last 50 years. However, translation to clinical applications of photoactive molecular systems developed to date is dramatically limited, mainly because their activation requires excitation by low-energy photons from the ultraviolet to near infra-red range, preventing any activation deeper than few millimetres under the skin. Herein we conceive a strategy for photosensitive-system activation potentially adapted to biological tissues without any restriction in depth. High-energy stimuli, such as those employed for radiotherapy, are used to carry energy while molecular activation is provided by local energy conversion. This concept is applied to azobenzene, one of the most established photoswitches, to build a radioswitch. The radiation-responsive molecular system developed is used to trigger cytotoxic effect on cancer cells upon gamma-ray irradiation. This breakthrough activation concept is expected to expand the scope of applications of photosensitive systems and paves the way towards the development of original therapeutic approaches.
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Fótons , Radiação Ionizante , Fótons/uso terapêuticoRESUMO
Pigment organelles of vertebrates belong to the lysosome-related organelle (LRO) family, of which melanin-producing melanosomes are the prototypes. While their anabolism has been extensively unraveled through the study of melanosomes in skin melanocytes, their catabolism remains poorly known. Here, we tap into the unique ability of crab spiders to reversibly change body coloration to examine the catabolism of their pigment organelles. By combining ultrastructural and metal analyses on high-pressure frozen integuments, we first assess whether pigment organelles of crab spiders belong to the LRO family and second, how their catabolism is intracellularly processed. Using scanning transmission electron microscopy, electron tomography, and nanoscale Synchrotron-based scanning X-ray fluorescence, we show that pigment organelles possess ultrastructural and chemical hallmarks of LROs, including intraluminal vesicles and metal deposits, similar to melanosomes. Monitoring ultrastructural changes during bleaching suggests that the catabolism of pigment organelles involves the degradation and removal of their intraluminal content, possibly through lysosomal mechanisms. In contrast to skin melanosomes, anabolism and catabolism of pigments proceed within the same cell without requiring either cell death or secretion/phagocytosis. Our work hence provides support for the hypothesis that the endolysosomal system is fully functionalized for within-cell turnover of pigments, leading to functional maintenance under adverse conditions and phenotypic plasticity. First formulated for eye melanosomes in the context of human vision, the hypothesis of intracellular turnover of pigments gets unprecedented strong support from pigment organelles of spiders.
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Cor , Lisossomos/metabolismo , Melanossomas/fisiologia , Organelas/fisiologia , Pigmentos Biológicos/fisiologia , Pele/metabolismo , Aranhas/fisiologia , Animais , Endossomos/metabolismoRESUMO
Fluorescent polymer cubosomes and hexosomes with aggregation-induced emission (AIE) were prepared from amphiphilic block copolymers PEG-b-PTPEMA where the hydrophobic block PTPEMA was a polymethacrylate with tetraphenylethene (TPE) as the AIE side group. Four highly asymmetric block copolymers with hydrophilic block weight ratio f PEG ≤ 20% were synthesized. Cubosomes and hexosomes with strong fluorescence emission were obtained by nanoprecipitation of polymers with f PEG < 9% in dioxane/water and THF/water systems. Their ordered internal structures were studied by electron microscopy (cryo-EM, SEM and TEM) and the X-ray scattering technique (SAXS). To elucidate the formation mechanisms of these inverted colloids, other parameters influencing the morphologies, like the water content during self-assembly and the organic solvent composition, were also investigated. This study not only inspires people to design novel building blocks for the preparation of functional cubosomes and hexosomes, but also presents the first AIE fluorescent polymer cubosome and hexosome with potential applications in bio-related fields.
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Hfq is a bacterial regulator with key roles in gene expression. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, thanks to its binding to small regulatory noncoding RNAs. This property is of primary importance for bacterial adaptation and survival in hosts. Small RNAs and Hfq are, for instance, involved in the response to antibiotics. Previous work has shown that the E. coli Hfq C-terminal region (Hfq-CTR) self-assembles into an amyloid structure. It was also demonstrated that the green tea compound EpiGallo Catechin Gallate (EGCG) binds to Hfq-CTR amyloid fibrils and remodels them into nonamyloid structures. Thus, compounds that target the amyloid region of Hfq may be used as antibacterial agents. Here, we show that another compound that inhibits amyloid formation, apomorphine, may also serve as a new antibacterial. Our results provide an alternative in order to repurpose apomorphine, commonly used in the treatment of Parkinson's disease, as an antibiotic to block bacterial adaptation to treat infections.
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Structuring pores into stable membrane and controlling their opening is extremely useful for applications that require nanopores as channels for material exchange and transportation. In this work, nanoporous vesicles with aggregation-induced emission (AIE) properties were developed from the amphiphilic polymer PEG550-TPE-Chol, in which the hydrophobic part is composed of a tetraphenylethene (TPE) group and a cholesterol moiety and the hydrophilic block is a poly(ethylene glycol) (PEG, Mn = 550 Da). Two stereoisomers, trans-PEG550-TPE-Chol and cis-PEG550-TPE-Chol, were successfully synthesized. These thermally stable stereoisomers showed distinct self-assembly behavior in water: trans-PEG550-TPE-Chol formed classical vesicles, while cis-PEG550-TPE-Chol self-assembled into cylindrical micelles. Interestingly, trans/cis mixtures of PEG550-TPE-Chol (trans/cis = 60/40), either naturally synthesized without isomers' separation during the synthesis or intentionally mixed using trans- and cis-isomers, constructed perforated vesicles with nanopores. Moreover, under the illumination of high intensity UV light (365 nm, 15 mW/cm2), the classical vesicles of trans-PEG550-TPE-Chol were perforated by its cis counterparts generated from the trans-cis photoisomerization, while the cylindrical micelles of cis-PEG550-TPE-Chol interweaved to form meshes and nanoporous membranes due to the trans-isomers produced by cis-trans photoisomerization. All of these assemblies in water emitted bright cyan fluorescence under UV light, while their constituent molecules were not fluorescent when solubilized in organic solvent. The AIE fluorescent normal vesicles and nanoporous vesicles may find potential applications in biotechnology as light-gated delivery vehicles and capsules with nanochannels for material exchange.
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Allergen immunotherapy (AIT) is the only disease-modifying treatment with evidence for sustained efficacy. However, it is poorly developed compared to symptomatic drugs. The main reasons come from treatment duration implying monthly injections during 3 to 5 years or daily sublingual use, and the risk of allergic side-effects. To become a more attractive alternative to lifelong symptomatic drug use, improvements to AIT are needed. Among the most promising new immunotherapy strategies is the use of bioparticles for the presentation of target antigen to the immune system as they can elicit strong T cell and B cell immune responses. Virus-like particles (VLPs) are a specific class of bioparticles in which the structural and immunogenic constituents are from viral origin. However, VLPs are ill-suited for use in AIT as their antigenicity is linked to structure. Recently, synthetic biology has been used to produce artificial modular bioparticles, in which supramolecular assemblies are made of elements from heterogeneous biological sources promoting the design and use of in vivo-assembling enveloped bioparticles for viral and non-viral antigens presentation. We have used a coiled-coil hybrid assembly for the design of an enveloped bioparticle (eBP) that present trimers of the Der p 2 allergen at its surface, This bioparticle was produced as recombinant and in vivo assembled eBPs in plant. This allergen biotherapeutic was used to demonstrate i) the capacity of plants to produce synthetic supramolecular allergen bioparticles, and ii) the immunomodulatory potential of naturally-assembled allergen bioparticles. Our results show that allergens exposed on eBPs induced a very strong IgG response consisting predominantly of IgG2a in favor of the TH1 response. Finally, our results demonstrate that rDer p 2 present on the surface of BPs show a very limited potential to stimulate the basophil degranulation of patient allergic to this allergen which is predictive of a high safety potential.
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Alérgenos/imunologia , Imunomodulação/imunologia , Alérgenos/biossíntese , Alérgenos/química , Sequência de Aminoácidos , Animais , Antígenos de Dermatophagoides/imunologia , Basófilos/imunologia , Hiper-Reatividade Brônquica/imunologia , Líquido da Lavagem Broncoalveolar , DNA/metabolismo , Feminino , Humanos , Hipersensibilidade/imunologia , Imunização , Camundongos Endogâmicos BALB C , Nanopartículas/química , Nanopartículas/ultraestrutura , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/químicaRESUMO
The flagellum of Trypanosoma brucei is a 20 µm-long organelle responsible for locomotion and cell morphogenesis. The flagellum attachment zone (FAZ) is a multi-protein complex whose function is to attach the flagellum to the cell body but also to guide cytokinesis. Cryo-transmission electron microscopy is a tool of choice to access the structure of the FAZ in a close-to-native state. However, because of the large dimension of the cell body, the whole FAZ cannot be structurally studied in situ at the nanometre scale in 3D using classical transmission electron microscopy approaches. In the present work, cryo-scanning transmission electron tomography, a new method capable of investigating cryo-fixed thick biological samples, has been used to study whole T. brucei cells at the bloodstream stage. The method has been used to visualise and characterise the structure and organisation of the FAZ filament. It is composed of an array of cytoplasmic stick-like structures. These sticks are heterogeneously distributed between the posterior part and the anterior tip of the cell. This cryo-STET investigation provides new insights into the structure of the FAZ filament. In combination with protein structure predictions, this work proposes a new model for the elongation of the FAZ.
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The mechanisms of Z-ring assembly and regulation in bacteria are poorly understood, particularly in non-model organisms. Actinobacteria, a large bacterial phylum that includes the pathogen Mycobacterium tuberculosis, lack the canonical FtsZ-membrane anchors and Z-ring regulators described for E. coli. Here we investigate the physiological function of Corynebacterium glutamicum SepF, the only cell division-associated protein from Actinobacteria known to interact with the conserved C-terminal tail of FtsZ. We show an essential interdependence of FtsZ and SepF for formation of a functional Z-ring in C. glutamicum. The crystal structure of the SepF-FtsZ complex reveals a hydrophobic FtsZ-binding pocket, which defines the SepF homodimer as the functional unit, and suggests a reversible oligomerization interface. FtsZ filaments and lipid membranes have opposing effects on SepF polymerization, indicating that SepF has multiple roles at the cell division site, involving FtsZ bundling, Z-ring tethering and membrane reshaping activities that are needed for proper Z-ring assembly and function.
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Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/citologia , Corynebacterium glutamicum/metabolismo , Proteínas do Citoesqueleto/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Divisão Celular , Corynebacterium glutamicum/química , Corynebacterium glutamicum/genética , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Dimerização , Regulação Bacteriana da Expressão Gênica , Ligação Proteica , Alinhamento de SequênciaRESUMO
A superoxide dismutase mimic (Mn1) was functionalized with three positively charged-peptides: RRRRRRRRR (Mn1-R9), RRWWWRRWRR (Mn1-RW9) or Fx-r-Fx-K (Mn1-MPP). Characterization of the physico-chemical properties of the complexes show that they share similar binding affinity for Mn2+, apparent reduction potential and intrinsic superoxide dismutase activity. However, their accumulation in cells is different (Mn1-R9 < Mn1-MPP < Mn1-RW9 < Mn1), as well as their subcellular distribution. In addition, the three functionalized-complexes display a better anti-inflammatory activity than Mn1 when assayed at 10 µM. This improvement is due to a combination of an anti-inflammatory effect of the peptidyl moiety itself, and of the SOD mimic for Mn1-RW9 and Mn1-MPP. In contrast, the enhanced anti-inflammatory activity of Mn1-R9 is solely due to the SOD mimic.
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Anti-Inflamatórios não Esteroides/farmacologia , Peptídeos Penetradores de Células/farmacologia , Superóxido Dismutase/metabolismo , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/metabolismo , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Células HT29 , Humanos , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Estrutura Molecular , Superóxido Dismutase/química , TermodinâmicaRESUMO
Biocompatible polymersomes are prepared from amphiphilic block copolypeptoids with aggregation-induced emission, where the hydrophobic block P(TPE-NAG) is a tetraphenylethylene (TPE)-modified poly(N-allylglycine) and the hydrophilic block is polysarcosine. These nanoparticles are non-cytotoxic and show strong fluorescence emission in aqueous solution.