RESUMO
Symmetry is rarely found on cellular surfaces. An exception is the brush border of microvilli, which are essential for the proper function of transport epithelia. In a healthy intestine, they appear densely packed as a 2D-hexagonal lattice. For in vitro testing of intestinal transport the cell line Caco-2 has been established. As reported by electron microscopy, their microvilli arrange primarily in clusters developing secondly into a 2D-hexagonal lattice. Here, atomic force microscopy (AFM) was employed under aqueous buffer conditions on Caco-2 cells, which were cultivated on permeable filter membranes for optimum differentiation. For analysis, the exact position of each microvillus was detected by computer vision; subsequent Fourier transformation yielded the type of 2D-lattice. It was confirmed, that Caco-2 cells can build a hexagonal lattice of microvilli and form clusters. Moreover, a second type of arrangement was discovered, namely a rhombic lattice, which appeared at sub-maximal densities of microvilli with (29 ± 4) microvilli / µm2. Altogether, the findings indicate the existence of a yet undescribed pattern in cellular organization.
Assuntos
Enterócitos/ultraestrutura , Microvilosidades/ultraestrutura , Adenocarcinoma/patologia , Técnicas de Cultura de Células/instrumentação , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Análise de Fourier , Humanos , Microscopia de Força Atômica , Microscopia Eletrônica de VarreduraRESUMO
The nuclear pore complex (NPC) is the gate for transport between the cell nucleus and the cytoplasm. Small molecules cross the NPC by passive diffusion, but molecules larger than â¼5â nm must bind to nuclear transport receptors to overcome a selective barrier within the NPC. Although the structure and shape of the cytoplasmic ring of the NPC are relatively well characterized, the selective barrier is situated deep within the central channel of the NPC and depends critically on unstructured nuclear pore proteins, and is therefore not well understood. Here, we show that stiffness topography with sharp atomic force microscopy tips can generate nanoscale cross-sections of the NPC. The cross-sections reveal two distinct structures, a cytoplasmic ring and a central plug structure, which are consistent with the three-dimensional NPC structure derived from electron microscopy. The central plug persists after reactivation of the transport cycle and resultant cargo release, indicating that the plug is an intrinsic part of the NPC barrier. Added nuclear transport receptors accumulate on the intact transport barrier and lead to a homogenization of the barrier stiffness. The observed nanomechanical properties in the NPC indicate the presence of a cohesive barrier to transport and are quantitatively consistent with the presence of a central condensate of nuclear pore proteins in the NPC channel.
Assuntos
Nanopartículas/química , Nanopartículas/ultraestrutura , Poro Nuclear/química , Poro Nuclear/ultraestrutura , Transporte Ativo do Núcleo Celular , Adesividade , Adsorção , Módulo de Elasticidade , Fricção , Teste de Materiais , Microscopia de Força Atômica/métodos , Estresse Mecânico , Propriedades de SuperfícieRESUMO
Nuclear pore complexes (NPCs) mediate all transport between the cytosol and the nucleus and therefore take centre stage in physiology. While transport through NPCs has been extensively investigated little is known about their structural and barley anything about their mechanical flexibility. Structural and mechanical flexibility of NPCs, however, are presumably of key importance. Like the cell and the cell nucleus, NPCs themselves are regularly exposed to physiological mechanical forces. Besides, NPCs reveal striking transport properties which are likely to require fairly high structural flexibility. The NPC transports up to 1,000 molecules per second through a physically 9 nm wide channel which repeatedly opens to accommodate macromolecules significantly larger than its physical diameter. We hypothesised that NPCs possess remarkable structural and mechanical stability. Here, we tested this hypothesis at the single NPC level using the nano-imaging and probing approach atomic force microscopy (AFM). AFM presents the NPC as a highly flexible structure. The NPC channel dilates by striking 35% on exposure to trans-cyclohexane-1,2-diol (TCHD), which is known to transiently collapse the hydrophobic phase in the NPC channel like receptor-cargo complexes do in transit. It constricts again to its initial size after TCHD removal. AFM-based nano-indentation measurements show that the 50 nm long NPC basket can astonishingly be squeezed completely into the NPC channel on exposure to incremental mechanical loads but recovers its original vertical position within the nuclear envelope plane when relieved. We conclude that the NPC possesses exceptional structural and mechanical flexibility which is important to fulfilling its functions.
Assuntos
Fenômenos Mecânicos , Poro Nuclear/química , Poro Nuclear/metabolismo , Xenopus laevis/metabolismo , Animais , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força Atômica , Poro Nuclear/ultraestrutura , Oócitos/citologia , Oócitos/metabolismo , Permeabilidade , MaleabilidadeRESUMO
Advances in nanomedicine require conceptual understanding of physiological processes. Apoptosis is a fundamental physiological process that is characterized, among other things, by an increased permeability of the nuclear envelope (NE). The latter is a tight transport barrier, known to restrict nuclear delivery rate of therapeutic nanoparticles. Therefore, an understanding of the underlying mechanism that leads to the breakdown of the barrier during apoptosis could stimulate the development of new approaches in gene therapy. We set out to elucidate this mechanism following induction of apoptosis on isolated cell nuclei. We tested the hypothesis whether caspases, mediators of apoptosis, trigger the NE leakiness at the level of the nuclear pore complexes (NPCs) using fluorescence techniques. As the permeability barrier inside the NPC channel is thought to be based on hydrophobic-hydrophobic protein interactions we further investigated the NPC channel hydrophobicity using atomic force microscopy. Caspase-9 was found to induce NE leakiness to large macromolecules. Leakiness was prevented by pretreatment of NPCs with an importin-ß mutant, which irreversibly binds and thereby obstructs the NPC channel. Utilizing an ultra-sharp, hydrophobic atomic force microscope tip as a chemical nanosensor that reaches deep into the apoptotic NPC channel, a remarkable decrease of hydrophobic binding sites was detected therein. We conclude that caspase 9 gives rise to NE leakiness by perturbing the hydrophobicity-based barrier inside the NPC channel. This explains the high passive NE permeability in early apoptosis. FROM THE CLINICAL EDITOR: In this study, biological processes taking place in the nucleus during the course of apoptosis have been monitored using atomic force microscopy-based nanosensors. The conclusion was that one of the caspases, caspase 9 perturbs the hydrophobicity-based barrier inside the nuclear pore complex channel causing nuclear envelope leakiness.
Assuntos
Caspase 9/metabolismo , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Animais , Apoptose/efeitos dos fármacos , Citocromos c/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força Atômica , Membrana Nuclear/efeitos dos fármacos , Poro Nuclear/efeitos dos fármacos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Xenopus laevisRESUMO
Oily core nanocapsules were prepared by sequential addition of positively and negatively charged polyelectrolytes based on a nanoemulsion and transformation thereof into a core-shell structure. The capsules were well characterized by photon correlation spectroscopy, laser diffraction, zeta-potential and transmission electron microscopy and feature an average size of 150nm and a negative surface charge. The aim of the current study was to improve the dispersion stability and mechanic rigidity of the capsule wall by depositing an increasing number of up to five layers. Therefore, atomic force microscopy (AFM) and ultrasonic resonator technology (URT) were applied to investigate the shell of the nanoemulsion, the intermediate and final nanocapsules in more detail. AFM was performed to investigate the shape, morphology and mechanic properties of the emulsion and capsule shell. It proved to be a feasible technique to distinguish nanoemulsions from nanocapsules by stiffness analysis. URT was utilized in order to observe the ultrasound velocity and could confirm the AFM results. Both techniques demonstrated that the shell around an oil droplet solidified with increasing number of polyelectrolyte layers. Since a solid wall might have the potential of a strong diffusion barrier, nanocapsules might present a feasible prolonged release drug delivery system in contrast to nanoemulsions.
Assuntos
Emulsões/química , Microscopia de Força Atômica/métodos , Nanocápsulas/química , Nanoestruturas/química , Nanotecnologia/métodos , Ultrassom , Fenômenos Químicos , Portadores de Fármacos/síntese química , Portadores de Fármacos/química , Emulsões/síntese química , Nanocápsulas/ultraestrutura , Nanoestruturas/ultraestrutura , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Evidence is emerging that changes in the structural and mechanical properties of viral particles are closely linked and that such changes are essential to infectivity. Here, applying the nanostructural and nanomechanical approach of atomic force microscopy, we visualised capsids of the ubiquitous human pathogen herpes simplex virus type 1 (HSV-1) at nano-scale resolution in physiologically relevant conditions. Simultaneously performed nano-indentation measurements on genome-containing and genome-free capsids revealed that genome-containing HSV-1 capsids withstand an exceptionally large mechanical force of approximately 6 nN, which is three times larger than the highest values previously reported for other viruses. Greater mechanical forces, however, led to a release of the viral genome. The resulting genome-free capsids, which largely retained their overall structure, were found to be utterly elastic. HSV-1 capsids thus exhibit an exceptional structural and mechanical stability, which is largely provided by the densely packaged genome. This stability might be the key determinant for capsid survival over long distances in the axonal cytoplasm where it is exposed to mechanical forces by molecular motors before it reaches the nuclear pore for crucial genome uncoating.
Assuntos
Herpesvirus Humano 1/química , Herpesvirus Humano 1/ultraestrutura , Microscopia de Força Atômica , Fenômenos Biomecânicos , Capsídeo/química , Capsídeo/ultraestrutura , DNA Viral/metabolismo , Microscopia de FluorescênciaRESUMO
Nuclear pore complexes (NPCs) mediate and control the transport of virtually all material between the cytosol and the nucleus. It is, therefore, unsurprising that they have long taken centre stage in physiology. A precise understanding of the NPC structure and function that remain to be thoroughly investigated yet is, thus, of crucial importance. The NPC can mediate transport both actively and passively. It remains to be clarified, however, whether transport of small molecules and macromolecules proceeds through the same route in the NPC. Furthermore, it has been shown that surface hydrophobicity represents a major sorting criterion for the active transport through NPCs. Transport factors like importin beta, which exhibit a rather large surface hydrophobicity, bind to their cargo and are believed to interact with a supposedly hydrophobic meshwork that is assumed to reside in the central channel of the NPC but has not yet been visualised. This interaction is presumed to lead to a partial breakdown of the meshwork, thereby, permitting the transport-cargo complexes to pass through. In this study, by using the nano-imaging approach, atomic force microscopy, we visualised under near-physiological conditions, for the first time, the presence of a hydrophobic meshwork in the NPC central channel. Furthermore, our data lend strong support for the existence of two segregated transport routes in the NPC.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força Atômica/métodos , Poro Nuclear/ultraestrutura , Animais , Anticorpos/imunologia , Feminino , Poro Nuclear/imunologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Oócitos/citologia , Oócitos/metabolismo , Oócitos/ultraestrutura , Xenopus laevis , beta Carioferinas/metabolismo , beta Carioferinas/ultraestruturaRESUMO
Nuclear pore complexes (NPCs) are supramolecular nanomachines that mediate the exchange of macromolecules and inorganic ions between the nucleus and the cytosol. Although there is no doubt that large cargo is transported through the centrally located channel, the route of ions and small molecules remains debatable. We thus tested the hypothesis that there are two separate pathways by imaging NPCs using atomic force microscopy, NPC electrical conductivity measurements, and macromolecule permeability assays. Our data indicate a spatial separation between the active transport of macromolecules through the central channel and the passive transport of ions and small macromolecules through the pore periphery.
Assuntos
Compostos Inorgânicos/química , Íons/química , Substâncias Macromoleculares/química , Poro Nuclear/metabolismo , Animais , Transporte Biológico , Bovinos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Citosol/metabolismo , Citosol/ultraestrutura , Condutividade Elétrica , Feminino , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Microscopia de Força Atômica , Mutação , Membrana Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Oócitos/citologia , Oócitos/metabolismo , Plasmídeos , Soroalbumina Bovina/metabolismo , Xenopus laevis/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismo , beta Carioferinas/ultraestruturaRESUMO
Ethanol is the most frequently used drug among humans. We tested the hypothesis whether ethanol, at clinically relevant concentrations modifies, signaling across the nuclear envelope (NE). In cell nuclei isolated from Xenopus oocytes, we measured NE electrical resistance and NE macromolecule permeability 1 to 20 h after addition of ethanol (0.05 to 0.2%). Furthermore, with atomic force microscopy, nuclear pores of the NE were imaged after exposure to ethanol. We found that NE permeability decreased within hours of ethanol exposure. In parallel, nuclei swell and nuclear pores form clusters in the NE. Force measurements on individual nuclear pores indicate that pores found in clusters are stiffer than those found randomly distributed in the NE. Application of a transcription blocker (actinomycin D) or RNase treatment of isolated nuclei in vitro after ethanol exposure prevents the permeability changes. In conclusion, ethanol, at commonly used concentrations, changes NE structure by transcriptional processes in the cell nucleus. Within hours, the NE becomes less permeable for diffusible ions and macromolecules. This could explain altered signaling to and communication with the cell nucleus in the pathophysiology of alcohol abuse.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Poro Nuclear/metabolismo , Oócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Feminino , Microscopia de Força Atômica , Poro Nuclear/ultraestrutura , Oócitos/citologia , Permeabilidade/efeitos dos fármacos , Xenopus laevisRESUMO
A Glucocorticoid-sensitive cell rapidly responds to hormone stimulation with bidirectional exchange of specific macromolecules between cytosol and nucleus. Glucocorticoid-initiated macromolecules (GIMs) must overcome the nuclear envelope (NE) to enter or leave the nucleus. GIM translocation occurs through nuclear pore complexes (NPCs) that span the NE. We investigated the question whether transport of GIMs through NPCs occurs random or involves selected groups of NPCs (hot spots). Glucocorticoid receptors were expressed in Xenopus laevis oocytes and GIM transport was activated by triamcinolone acetonide, a potent synthetic glucocorticoid analogon. Glucocorticoid receptors associated with the NE and the chromatin were identified using western blot analysis and, at single molecule level, atomic force microscopy. Fluorescence-labeled dextran was used to describe passive NE permeability. We observed that after hormone injection (i) small GIMs, most likely GRs, localize within seconds on both sides of the NE. (ii) large GIMs, most likely ribonucleoproteins, localize within minutes on NPCs at the nucleoplasmic side (iii) both small and large GIMs accumulate on selected NPC clusters (iv) NE permeability transiently decreases when GIMs attach to NPCs. We conclude that GIM transport across the nuclear barrier does not randomly take place but is carried out by a selected population of NPCs.
Assuntos
Membrana Nuclear/efeitos dos fármacos , Poro Nuclear/metabolismo , Oócitos/efeitos dos fármacos , Esteroides/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Western Blotting , Células Cultivadas , Feminino , Microscopia de Força Atômica , Membrana Nuclear/metabolismo , Poro Nuclear/efeitos dos fármacos , Oócitos/citologia , Permeabilidade , Receptores de Glucocorticoides/metabolismo , Triancinolona Acetonida/farmacologia , Xenopus laevisRESUMO
We have successfully established a novel protein microarray-based kinase assay, which we applied to identify target proteins of the barley protein kinase CK2alpha. As a source of recombinant barley proteins we cloned cDNAs specific for filial tissues of developing barley seeds into an E. coli expression vector. By using robot technology, 21,500 library clones were arrayed in microtiter plates and gridded onto high-density filters. Protein expressing clones were detected using an anti-RGS-His6 antibody and rearrayed into a sublibrary of 4100 clones. All of these clones were sequenced from the 5'-end and the sequences were analysed by homology searches against protein databases. Based on these results we selected 768 clones expressing different barley proteins for protein purification. The purified proteins were robotically arrayed onto FAST slides. The generated protein microarrays were incubated with an expression library-derived barley CK2alpha in the presence of [gamma-33P]ATP, and signals were detected by X-ray film or phosphor imager. We were able to demonstrate the power of the protein microarray technology by identification of 21 potential targets out of 768 proteins including such well-known substrates of CK2alpha as high mobility group proteins and calreticulin.