RESUMO
The capsids of icosahedral viruses are closed shells assembled from a hexagonal lattice of proteins with fivefold angular defects located at the icosahedral vertices. Elasticity theory predicts that these disclinations are subject to an internal compressive prestress, which provides an explanation for the link between size and shape of capsids. Using a combination of experiment and elasticity theory we investigate the question of whether macromolecular assemblies are subject to residual prestress, due to basic geometric incompatibility of the subunits. Here we report the first direct experimental test of the theory: by controlled removal of protein pentamers from the icosahedral vertices, we measure the mechanical response of so-called "whiffle ball" capsids of herpes simplex virus, and demonstrate the signature of internal prestress locked into wild-type capsids during assembly.
Assuntos
Proteínas do Capsídeo/química , Capsídeo/química , Modelos Químicos , Complexos Multiproteicos/química , Nanoestruturas/química , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Elasticidade , Complexos Multiproteicos/metabolismo , Simplexvirus/química , Simplexvirus/metabolismoRESUMO
The current rapid growth in the use of nanosized particles is fueled in part by our increased understanding of their physical properties and ability to manipulate them, which is essential for achieving optimal functionality. Here we report detailed quantitative measurements of the mechanical response of nanosized protein shells (viral capsids) to large-scale physical deformations and compare them with theoretical descriptions from continuum elastic modeling and molecular dynamics (MD). Specifically, we used nanoindentation by atomic force microscopy to investigate the complex elastic behavior of Hepatitis B virus capsids. These capsids are hollow, approximately 30 nm in diameter, and conform to icosahedral (5-3-2) symmetry. First we show that their indentation behavior, which is symmetry-axis-dependent, cannot be reproduced by a simple model based on Föppl-von Kármán thin-shell elasticity with the fivefold vertices acting as prestressed disclinations. However, we can properly describe the measured nonlinear elastic and orientation-dependent force response with a three-dimensional, topographically detailed, finite-element model. Next, we show that coarse-grained MD simulations also yield good agreement with our nanoindentation measurements, even without any fitting of force-field parameters in the MD model. This study demonstrates that the material properties of viral nanoparticles can be correctly described by both modeling approaches. At the same time, we show that even for large deformations, it suffices to approximate the mechanical behavior of nanosized viral shells with a continuum approach, and ignore specific molecular interactions. This experimental validation of continuum elastic theory provides an example of a situation in which rules of macroscopic physics can apply to nanoscale molecular assemblies.
Assuntos
Elasticidade , Simulação de Dinâmica Molecular , Nanoestruturas/química , Tamanho da Partícula , Proteínas/química , Capsídeo/química , Capsídeo/ultraestrutura , Análise de Elementos Finitos , Vírus da Hepatite B/química , Microscopia de Força AtômicaRESUMO
Extracellular vesicles (EVs) have great potential as biomarkers since their composition and concentration in biofluids are disease state dependent and their cargo can contain disease-related information. Large tumor-derived EVs (tdEVs, >1 µm) in blood from cancer patients are associated with poor outcome, and changes in their number can be used to monitor therapy effectiveness. Whereas, small tumor-derived EVs (<1 µm) are likely to outnumber their larger counterparts, thereby offering better statistical significance, identification and quantification of small tdEVs are more challenging. In the blood of cancer patients, a subpopulation of EVs originate from tumor cells, but these EVs are outnumbered by non-EV particles and EVs from other origin. In the Dutch NWO Perspectief Cancer-ID program, we developed and evaluated detection and characterization techniques to distinguish EVs from non-EV particles and other EVs. Despite low signal amplitudes, we identified characteristics of these small tdEVs that may enable the enumeration of small tdEVs and extract relevant information. The insights obtained from Cancer-ID can help to explore the full potential of tdEVs in the clinic.
RESUMO
The ability of adenoviruses to infect a broad range of species has spurred a growing interest in nanomedicine to use adenovirus as a cargo delivery vehicle. While successful maturation of adenovirus and controlled disassembly are critical for efficient infection, the underlying mechanisms regulating these processes are not well understood. Here, we present Atomic Force Microscopy nanoindentation and fatigue studies of adenovirus capsids at different maturation stages to scrutinize their dynamic uncoating properties. Surprisingly, we find that the early intermediate immature (lacking DNA) capsid is mechanically indistinguishable in both break force and spring constant from the mature (containing DNA) capsid. However, mature and immature capsids do display distinct disassembly pathways, as revealed by our mechanically-induced fatigue analysis. The mature capsid first loses the pentons, followed by either long-term capsid stability or abrupt and complete disassembly. However, the immature capsid has a stable penton region and undergoes a stochastic disassembly mechanism, thought to be due to the absence of genomic pressure. Strikingly, the addition of the genome alone is not sufficient to achieve penton destabilization as indicated by the penton stability of the maturation-intermediate mutant, G33A. Full penton destabilization was achieved only when the genome was present in addition to the successful maturation-linked proteolytic cleavage of preprotein VI. Therefore these findings strongly indicate that maturation of adenovirus in concert with genomic pressure induces penton destabilization and thus, primes the capsid for controlled disassembly. This latter aspect is critical for efficient infection and successful cargo delivery.
Assuntos
Adenoviridae/metabolismo , Proteínas do Capsídeo/metabolismo , Endossomos/virologia , Proteínas do Capsídeo/química , Microscopia de Força Atômica , Nanoestruturas/química , Montagem de Vírus , Internalização do VírusRESUMO
Over the past two decades, single-molecule techniques have evolved into robust tools to study many fundamental biological processes. The combination of optical tweezers with fluorescence microscopy and microfluidics provides a powerful single-molecule manipulation and visualization technique that has found widespread application in biology. In this combined approach, the spatial (~nm) and temporal (~ms) resolution, as well as the force scale (~pN) accessible to optical tweezers is complemented with the power of fluorescence microscopy. Thereby, it provides information on the local presence, identity, spatial dynamics, and conformational dynamics of single biomolecules. Together, these techniques allow comprehensive studies of, among others, molecular motors, protein-protein and protein-DNA interactions, biomolecular conformational changes, and mechanotransduction pathways. In this chapter, recent applications of fluorescence microscopy in combination with optical trapping are discussed. After an introductory section, we provide a description of instrumentation together with the current capabilities and limitations of the approaches. Next we summarize recent studies that applied this combination of techniques in biological systems and highlight some representative biological assays to mark the exquisite opportunities that optical tweezers combined with fluorescence microscopy provide.
Assuntos
DNA/isolamento & purificação , Microscopia de Fluorescência/métodos , Pinças Ópticas , Proteínas/isolamento & purificação , Imagem Individual de Molécula/métodos , DNA/química , Mecanotransdução Celular , Microfluídica/métodos , Microscopia de Fluorescência/tendências , Nanotecnologia/tendências , Proteínas/química , Imagem Individual de Molécula/tendênciasRESUMO
Structure and function of viruses are intimately related, and one of the goals in virology is to elucidate the mechanisms behind this relation. A variety of research endeavours is focused on studying these mechanisms and a relatively new technique in this field is Atomic Force Microscopy (AFM). Using AFM virions and virus-like particles can be imaged and manipulated at the single particle level. Here we review recent AFM nano-indentations studies unveiling for instance the mechanics of capsid-genome interactions, morphological changes that drive viral maturation, capsid stabilizing factors and viral uncoating. We show that in an increasing amount of literature a clear link between mechanics and infectivity is observed, which not only provides us with new fundamental insights into virology, but also provides ways to improve virus-like particles for applications in nanomedicine and nanotechnology.
Assuntos
Microscopia de Força Atômica/métodos , Nanotecnologia/métodos , Vírion/química , Vírion/ultraestrutura , Vírus/química , Vírus/ultraestrutura , Animais , Capsídeo/química , Capsídeo/fisiologia , Proteínas do Capsídeo/química , Genoma Viral , Humanos , Camundongos , Vírion/fisiologia , Vírus/metabolismoRESUMO
Self-assembling protein nanocontainers are promising candidates for an increasingly wide scope of purposes. Their applications range from drug delivery vehicles and imaging agents to nanocompartments for controlled enzymatic activity. In order to exploit their full potential in these different fields, characterization of their properties is vital. For example, their mechanical properties give insight into the stability of a particle as a function of their internal content. The mechanics can be probed by atomic force microscopy nanoindentation, and while this single particle method is increasingly used to probe material properties of viral nanocages, it has hardly been used to characterize nonviral nanocages. Here we report nanoindentation studies on two types of nonviral nanocontainers: (i) lumazine synthase from Aquifex aeolicus (AaLS), which naturally self-assembles into icosahedral cages, and (ii) the artificial protein cage O3-33 originating from a computational design approach. In addition, we tested particles that had been engineered toward improved cargo loading capacity and compared these nanocages in empty and loaded states. We found that the thermostable AaLS cages are stiffer and resist higher forces before breaking than the O3-33 particles, but that mutations affecting the size of AaLS particles have a dramatic effect on their structural stability. Furthermore, we show that cargo packaging can occur while maintaining the cage's mechanical properties.
Assuntos
Proteínas de Bactérias/química , Complexos Multienzimáticos/química , Nanoestruturas/química , Bactérias/química , Proteínas de Bactérias/genética , Fenômenos Biomecânicos , Clonagem Molecular , Expressão Gênica , Microscopia de Força Atômica , Complexos Multienzimáticos/genética , Nanoestruturas/ultraestrutura , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
Eukaryotic cells obtain their morphology and mechanical strength from the cytoskeleton and in particular from the cross-linked actin network that branches throughout the whole cell. This actin cortex lies like a quasi-two-dimensional (2D) biopolymer network just below the cell membrane, to which it is attached. In the quest for building an artificial cell, one needs to make a biomimetic model of the actin cortex and combine this in a bottom-up approach with other "synthetic" components. Here, we describe a reconstitution method for such an artificial actin cortex, which is freely suspended on top of a regular array of pillars. By this immobilization method, the actin network is only attached to a surface at discrete points and can fluctuate freely in between. By discussing the method to make the micropillars and the way to reconstitute a quasi-2D actin network on top, we show how one can study an isolated, reconstituted part of a cell. This allows the study of fundamental interaction mechanisms of actin networks, providing handles to design a functional actin cortex in an artificial cell.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Biopolímeros/metabolismo , Adesão Celular/fisiologia , Animais , Microtúbulos/metabolismo , CoelhosRESUMO
The interaction between a viral capsid and its genome governs crucial steps in the life cycle of a virus, such as assembly and genome uncoating. Tuning cargo-capsid interactions is also essential for successful design and cargo delivery in engineered viral systems. Here we investigate the interplay between cargo and capsid for the picorna-like Triatoma virus using a combined native mass spectrometry and atomic force microscopy approach. We propose a topology and assembly model in which heterotrimeric pentons that consist of five copies of structural proteins VP1, VP2 and VP3 are the free principal units of assembly. The interpenton contacts are established primarily by VP2. The dual role of the genome is first to stabilize the densely packed virion and, second, on an increase in pH to trigger uncoating by relaxing the stabilizing interactions with the capsid. Uncoating occurs through a labile intermediate state of the virion that reversibly disassembles into pentons with the concomitant release of protein VP4.
Assuntos
Fenômenos Biofísicos , Capsídeo/metabolismo , Genoma Viral , Vírus de Insetos/genética , Vírus de Insetos/fisiologia , Animais , Fenômenos Biomecânicos , Capsídeo/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Proteica , Triatoma/virologia , Desenvelopamento do VírusRESUMO
The effects of changes in the loading rate during the forced dissociation of single bonds have been studied for a wide variety of interactions. Less is known on the loading rate dependent behaviour of more complex systems that consist of multiple bonds. Here we focus on viral nanoparticles, in particular the protein shell (capsid) that protects the viral genome. As model systems we use the well-studied capsids of the plant virus Cowpea Chlorotic Mottle Virus (CCMV) and of the bacteriophages φ29 and HK97. By applying an atomic force microscopy (AFM) nanoindentation approach we study the loading rate dependency of their mechanical properties. Our AFM results show very diverse behaviour for the different systems. In particular, we find that not only the breaking force, but also the spring constant of some capsids depend on the loading rate. We describe and compare the measured data with simulation results from the literature. The unexpected complex loading rate dependencies that we report present a challenge for the current theoretical considerations aimed at understanding the molecular level interactions of highly ordered protein assemblies.
Assuntos
Bacteriófagos/fisiologia , Bacteriófagos/ultraestrutura , Bromovirus/fisiologia , Bromovirus/ultraestrutura , Capsídeo/fisiologia , Capsídeo/ultraestrutura , Fenômenos Mecânicos , Fenômenos Biomecânicos , Microscopia de Força Atômica , Nanopartículas/ultraestruturaRESUMO
Bioabsorbable polymers are increasingly being used in tissue engineering strategies. Despite the knowledge that some sterilization techniques may affect the physical properties of these polymers, this aspect is often overlooked. We speculate that the type of sterilization method used may influence cellular responses by altering the surface characteristics. We cultured adipose stem cells on bioabsorbable poly(l-lactide-co-caprolactone) (PLCL) sheets, sterilized using either ethylene oxide (EO), argon glow discharge (aGD) or electron beam (e-beam). Significantly higher values for surface roughness in the order EO>aGD>e-beam and significant differences in contact angles (EO>e-beam>aGD) and surface energies (aGD>e-beam>EO) were observed. Increased cell attachment and proliferation rates were observed with lower contact angles. The alkaline phosphatase activity was significantly higher for the ethylene oxide sterilized PLCL sheet. In conclusion, the type of sterilization for bioabsorbable polymers should be considered in the design of new scaffolds, since it might affect, or can be used to enhance, the outcome of the tissue engineered construct.
Assuntos
Adipócitos/citologia , Substitutos Ósseos/síntese química , Óxido de Etileno/química , Osteoblastos/citologia , Poliésteres/química , Células-Tronco/citologia , Engenharia Tecidual/métodos , Adipócitos/fisiologia , Diferenciação Celular , Células Cultivadas , Elétrons , Gases/química , Temperatura Alta , Humanos , Teste de Materiais , Osteoblastos/fisiologia , Células-Tronco/fisiologia , Propriedades de SuperfícieRESUMO
The main functions of viral capsids are to protect, transport and deliver their genome. The mechanical properties of capsids are supposed to be adapted to these tasks. Bacteriophage capsids also need to withstand the high pressures the DNA is exerting onto it as a result of the DNA packaging and its consequent confinement within the capsid. It is proposed that this pressure helps driving the genome into the host, but other mechanisms also seem to play an important role in ejection. DNA packaging and ejection strategies are obviously dependent on the mechanical properties of the capsid. This review focuses on the mechanical properties of viral capsids in general and the elucidation of the biophysical aspects of genome packaging mechanisms and genome delivery processes of double-stranded DNA bacteriophages in particular.