Ivan Liashkovich

Membrane potential depolarization decreases the stiffness of vascular endothelial cells.

The stiffness of vascular endothelial cells is crucial to mechanically withstand blood flow and, at the same time, to control deformation-dependent nitric oxide release. However, the regulation of mechanical stiffness is not yet understood. There is evidence that a possible regulator is the electrical plasma membrane potential difference. Using a novel technique that combines fluorescence-based membrane potential recordings with atomic force microscopy (AFM)-based stiffness measurements, the present study shows that membrane depolarization is associated with a decrease in the stiffness of endothelial cells. Three different depolarization protocols were applied, all of which led to a similar and significant decrease in cell stiffness, independently of changes in cell volume. Moreover, experiments using the actin-destabilizing agent cytochalasin D indicated that depolarization acts by affecting the cortical actin cytoskeleton. A model is proposed whereby a change of the electrical field across the plasma membrane is directly sensed by the submembranous actin network, regulating the actin polymerization:depolymerization ratio and thus cell stiffness. This depolarization-induced decrease in the stiffness of endothelial cells could play a role in flow-mediated nitric-oxide-dependent vasodilation.

Nanomechanics of vascular endothelium

The mechanical characteristics of endothelial cells reveal four distinct compartments, namely glycocalyx, cell cortex, cytoplasm and nucleus. There is accumulating evidence that endothelial nanomechanics of these individual compartments control vascular physiology. Depending on protein composition, filament formation and interaction with cross-linker proteins, these four compartments determine endothelial stiffness. Structural organization and mechanical properties directly influence physiological processes such as endothelial barrier function, nitric oxide release and gene expression. This review will focus on endothelial nanomechanics and its impact on vascular function.

Nanoscale stiffness topography reveals structure and mechanics of the transport barrier in intact nuclear pore complexes

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 ~5 nm must bind to nuclear transport receptors to overcome a selective barrier within the NPC1. Whilst the structure and shape of the cytoplasmic ring of the NPC are relatively well characterized2-5, the selective barrier is situated deep within the central channel of the NPC and depends critically on unstructured nuclear pore proteins5,6, and is therefore not well understood. Here, we show that stiffness topography7 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 microscopy2-5. 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.

AFM imaging of functionalized carbon nanotubes on biological membranes

Multifunctional carbon nanotubes are promising for biomedical applications as their nano-size, together with their physical stability, gives access into the cell and various cellular compartments including the nucleus. However, the direct and label-free detection of carbon nanotube uptake into cells is a challenging task. The atomic force microscope (AFM) is capable of resolving details of cellular surfaces at the nanometer scale and thus allows following of the docking of carbon nanotubes to biological membranes. Here we present topographical AFM images of non-covalently functionalized single walled (SWNT) and double walled carbon nanotubes (DWNT) immobilized on different biological membranes, such as plasma membranes and nuclear envelopes, as well as on a monolayer of avidin molecules. We were able to visualize DWNT on the nuclear membrane while at the same time resolving individual nuclear pore complexes. Furthermore, we succeeded in localizing individual SWNT at the border of incubated cells and in identifying bundles of DWNT on cell surfaces by AFM imaging.

Exceptional mechanical and structural stability of HSV-1 unveiled with fluid atomic force microscopy

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 ∼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.

Nuclear delivery mechanism of herpes simplex virus type 1 genome

Herpes simplex virus type 1 (HSV‐1) is a widespread human pathogen infecting more than 80% of the population worldwide. Its replication involves an essential, poorly understood multistep process, referred to as uncoating. Uncoating steps are as follows: (1) The incoming capsid pinpoints the nuclear pore complex (NPC). (2) It opens up at the NPC and releases the highly pressurized viral genome. (3) The viral genome translocates through the NPC. In the present review, we highlight recent advances in this field and propose mechanisms underlying the individual steps of uncoating. We presume that the incoming HSV‐1 capsid pinpoints the NPC by hydrophobic interactions and opens up upon binding to NPC proteins. Genome translocation is initially pressure‐driven. Copyright

Simultaneous mechanical stiffness and electrical potential measurements of living vascular endothelial cells using combined atomic force and epifluorescence microscopy

The degree of mechanical stiffness of vascular endothelial cells determines the endogenous production of the vasodilating gas nitric oxide (NO). However, the underlying mechanisms are not yet understood. Experiments on vascular endothelial cells suggest that the electrical plasma membrane potential is involved in this regulatory process. To test this hypothesis we developed a technique that simultaneously measures the electrical membrane potential and stiffness of vascular endothelial cells (GM7373 cell line derived from bovine aortic endothelium) under continuous perfusion with physiological electrolyte solution. The cellular stiffness was determined by nano-indentation using an atomic force microscope (AFM) while the electrical membrane potential was measured with bis-oxonol, a voltage-reporting fluorescent dye. These two methods were combined using an AFM attached to an epifluorescence microscope. The electrical membrane potential and mechanical stiffness of the same cell were continuously recorded for a time span of 5 min. Fast fluctuations (in the range of seconds) of both the electrical membrane potential and mechanical stiffness could be observed that were not related to each other. In contrast, slow cell depolarizations (in the range of minutes) were paralleled by significant increases in mechanical stiffness. In conclusion, using the combined AFM-fluorescence technique we monitored for the first time simultaneously the electrical plasma membrane potential and mechanical stiffness in a living cell. Vascular endothelial cells exhibit oscillatory non-synchronized waves of electrical potential and mechanical stiffness. The sustained membrane depolarization, however, is paralleled by a concomitant increase of cell stiffness. The described method is applicable for any fluorophore, which opens new perspectives in biomedical research.

Atomic force microscopy visualises a hydrophobic meshwork in the central channel of the nuclear pore

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 β, 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.

Exceptional structural and mechanical flexibility of the nuclear pore complex

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. J. Cell. Physiol. 226: 675–682, 2011.

Structural organization of the nuclear pore permeability barrier.

The efficiency of gene therapy in non-dividing cells is particularly poor due to restricted nuclear delivery rates of exogenously applied macromolecules across the nuclear pore complexes (NPCs). Therefore, improved intranuclear delivery of transgenes requires an ability to modulate the barrier function of the NPC. Despite a large body of experimental evidence accumulated to date, the contribution of individual NPC proteins (nucleoporins) to the formation of the NPC permeability barrier as well as their structural organization within the NPC remains under debate. In the present study, we revisit the view on the spatial arrangement of the Phe-Gly rich domains (FG-domains) of a subset of nucleoporins known as FG-nucleoporins. They are generally believed to be the key constituents of the NPC permeability barrier. Comparison of the binding pattern of a transport receptor importin β fragment, that binds specifically to FG-domains, with the binding pattern of wheat germ agglutinin that binds elsewhere in the NPC, reveals that FG-domains tend to cluster in the very center of the NPC. Furthermore, a controlled sequential release of the barrier-forming nucleoporins results in a gradual breakdown of the NPC permeability barrier. The breakdown is initiated by a dissociation of Nup62 from the NPC. This is accompanied by an increased passive diffusion of small molecules across the NPC. Subsequent dissociation of Nup98 and possibly other nucleoporins results in a collapse of the barrier for larger molecules. We therefore conclude that FG-nucleoporins do not contribute equally to the maintenance of the NPC permeability barrier exclusion limit. This implies that a controlled release of nucleoporins that contribute most to the formation and maintenance of the NPC barrier can facilitate access of therapeutic macromolecules into the nucleus.