RESUMEN
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.
Asunto(s)
Nanopartículas/química , Nanopartículas/ultraestructura , Poro Nuclear/química , Poro Nuclear/ultraestructura , Transporte Activo de Núcleo Celular , Adhesividad , Adsorción , Módulo de Elasticidad , Fricción , Ensayo de Materiales , Microscopía de Fuerza Atómica/métodos , Estrés Mecánico , Propiedades de SuperficieRESUMEN
Structural variability and flexibility are crucial factors for biomolecular function. Here we have reduced the invasiness and enhanced the spatial resolution of atomic force microscopy (AFM) to visualize, for the first time, different structural conformations of the two polynucleotide strands in the DNA double helix, for single molecules under near-physiological conditions. This is achieved by identifying and tracking the anomalous resonance behavior of nanoscale AFM cantilevers in the immediate vicinity of the sample.