Victor Shahin

Human Endothelium: Target for Aldosterone

Abstract—Aldosterone has long been known to control water and electrolyte balance by acting on mineralocorticoid receptors in kidney. However, recent studies demonstrated the presence of these receptors in nonclassical locations, including the cardiovascular system. We tested the hypothesis whether endothelial cells respond to aldosterone with changes in cell volume, a measure for ion-mediated water movement across the cell membrane. By means of atomic force microscopy in fluid, we measured volume of adherent human umbilical venous endothelial cells exposed for 72 hours to 10 nmol/L aldosterone. Over this period of time, cells swell by ≈ 18%. Aldosterone-induced swelling is prevented by 100 nmol/L of the mineralocorticoid receptor antagonist spironolactone, added to the primary endothelial cell culture. Aldosterone-treated cells dramatically shrink when 1 μmol/L of the diuretic amiloride is applied. Cells deprived of aldosterone do not respond to amiloride. Our conclusions are: (1) aldosterone leads to sustained cell swelling inhibited by administration of spironolactone or the sodium channel blocker amiloride; (2) cells respond to amiloride after aldosterone exposure; (3) renal diuretics act on endothelial cells; and (4) both amiloride and spironolactone could be useful for medical applications to prevent aldosterone-mediated endothelial dysfunction.

Endothelial Cell Swelling by Aldosterone

There is accumulating evidence that mineralocorticoids not only act on kidney but also on the cardiovascular system. We investigated the response of human umbilical venous endothelial cells (HUVECs) to aldosterone at a time scale of 20 minutes in absence and presence of the aldosterone antagonist spironolactone or other transport inhibitors. We applied atomic force microscopy (AFM), which measures cell volume and volume shifts between cytosol and cell nucleus. We observed an immediate cell volume increase (about 10%) approximately 1 min after addition of aldosterone (0.1 µmol/l), approaching a maximum (about 18%) 10 min after aldosterone treatment. Cell volume returned to normal 20 min after hormone exposure. Spironolactone (1 µmol/l) or amiloride (1 µmol/l) prevented the late aldosterone-induced volume changes but not the immediate change observed 1 min after hormone exposure. AFM revealed nuclear swelling 5 min after aldosterone addition, followed by nuclear shrinkage 15 min later. The Na+/H+ exchange blocker cariporide (10 µmol/l) was ineffective. We conclude: (i) Aldosterone induces immediate (1 min) swelling independently of plasma membrane Na+ channels and intracellular mineralocorticoid receptors followed by late mineralocorticoid receptor- and Na+-channel-dependent swelling. (ii) Intracellular macromolecule shifts cause the changes in cell volume. (iii) Both amiloride and spironolactone may be useful for medical applications to prevent aldosterone-induced vasculopathies.

Steroids dilate nuclear pores imaged with atomic force microscopy

Macromolecules that act in the cell nucleus must overcome the nuclear envelope (NE). This barrier between cytosol and the nucleus is perforated by nuclear pore complexes (NPCs) that serve as translocation machineries. We visualized the translocation process at the NE surface, applying a nanotechnical approach using atomic force microscopy (AFM). In order to initiate protein targeting to NPCs, dexamethasone (dex) was injected into Xenopus laevis oocytes. Dex is a synthetic steroid of great therapeutic relevance that specifically binds to glucocorticoid receptors and thus triggers an intracellular signal cascade involving the cell nucleus. Ninety and 180 sec after dex injection cell nuclei were isolated, the NEs spread on glass and scanned with AFM. With single molecule resolution we observed that dex initiated proteins (DIPs) first bind to NPC‐free areas of the outer nuclear membrane. This causes NPCs to dilate. Then, in a second step, DIPs attach directly to NPCs and enter the dilated central channels. DIPs accumulation and NPC conformational changes were blocked by RU486, a specific glucocorticoid receptor antagonist. In conclusion, dex exposure induces NPC dilation. NPCs change conformation already prior to transport. The NPC dilation signal is most likely transmitted through NPC associated filaments or yet unknown structures in the NE outer membrane. NPC dilation could have significant impact on nuclear targeting of therapeutic macromolecules.

Evidence for Ca2+- and ATP-sensitive peripheral channels in nuclear pore complexes

ABSTRACT In eukaryotic cells the nuclear envelope (NE) serves as a functional barrier between cytosol and nucleoplasm perforated by nuclear pore complexes (NPCs). Both active and passive transport of ions and macromolecules are thought to be mediated by the centrally located large NPC channel. However, 3‐di‐mensional imaging of NPCs based on electron microscopy indicates the existence of additional small channels of unknown function located in the NPC periphery. By means of the recently developed nuclear hourglass technique that measures NE electrical conductance, we evaluated passive electrically driven transport through NPCs. In isolated Xenopus laevis oocyte nuclei, we varied ambient Ca2+ and ATP in the cytoso‐lic solution and/or chelated Ca2+ in the perinuclear stores in order to assess the role of Ca2+ in regulating passive ion transport. We noticed that NE electrical conductance is large under conditions where macro‐molecule permeability is known to be low. In addition, atomic force microscopy applied to native NPCs detects multiple small pores in the NPC periphery consistent with channel openings. Peripheral pores were detectable only in the presence of ATP. We conclude that NPC transport of ions and macromolecules occurs through different routes. We present a model in which NE ion flux does not occur through the central NPC channel but rather through Ca2+‐ and ATP‐activated peripheral channels of individual NPCs.

The genome of HSV-1 translocates through the nuclear pore as a condensed rod-like structure

Incoming herpes simplex virus type-1 (HSV-1) capsids are known to dock to the nuclear pore complex (NPC) and release their genome. It has remained elusive, however, how the huge viral DNA translocates through the comparatively small NPC channel. In the present study, the interaction of HSV-1 with NPCs was analyzed by atomic force microscopy. In addition to capsids, smaller subviral structures - most with a diameter of 35-40 nm and a length of 130-160 nm - were visualized at the cytoplasmic side of the NPC. These components differed from capsids in their adhesion and stiffness properties, and were the sole subviral structures translocated through dilated NPCs towards the nucleus. It is presumed that they are the HSV-1 genome, and that a change in NPC conformation allows translocation of this genome as a densely packaged, rodlike structure.

Glucocorticoids remodel nuclear envelope structure and permeability

The present study describes glucocorticoid induced remodelling of nuclear envelope (NE) structure and permeability. A glucocorticoid analogue, triamcinolone acetonide (TA), is injected into Xenopus laevis oocytes that express an exogeneous glucocorticoid receptor (GR). Electrical, fluorescence and nano-imaging techniques are applied to study the permeability and the structure of the NE at 5 and 60 minutes after injection of TA. A remarkable dilation of nuclear pore complexes (NPCs), a rearrangement of NPC distribution and a significant increase of NE permeability for ions and fluorescent 20 kDa dextran are observed within 5 minutes of TA exposure. At regular distances on local NE patches, NPCs seem to adjoin forming clusters each consisting of several hundred NPCs. Interestingly, at the same time of exposure, hydrophobicity of NPC central channels and NPC-free NE surface increases. The changes in permeability and structure are transient as the NE permeability returns to its initial state within 60 minutes. In conclusion, the NE is a barrier of high plasticity sensitive to hydrophobic molecules. Remodelling of NE structure and permeability is a prerequisite for mediating physiological actions of glucocorticoids.

Imaging CFTR: A Tail to Tail Dimer with a Central Pore

The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein that belongs to the superfamily of ATP binding cassette (ABC) transporters. Mutations in the CFTR gene cause cystic fibrosis, an autosomal recessive disorder. The function of CFTR is versatile. It can serve as a regulatory protein, as a membrane transporter and as an ion channel. Dimerization of CFTR is necessary for full ion channel function although structural details of CFTR in native membrane are yet unknown. In order to identify CFTR in native plasma membrane we applied atomic force microscopy (AFM) to inside-out oriented membrane patches of CFTR-expressingXenopus laevis oocytes after cAMP stimulation. First, oocytes were injected with CFTR-cRNA and, three days later, voltage-clamped verifying successful CFTR expression and incorporation into the plasma membrane. Then, plasma membrane patches were isolated, placed inside-out on appropriate substrate and incubated with gold-labelled antibodies against the C-terminus of CFTR. Finally, the intracellular surface of the plasma membrane was scanned by AFM. In close vicinity to the immunogold labels we detected ring-like structures with bipartite symmetry. The substructure of the ring, formed by the extramembrane protein domains of CFTR, is consistent with the model of a CFTR dimer. Derived from AFM molecular mass analysis of the intramolecular domains we conclude that two CFTR molecules line up in parallel, tail by tail, forming a pore in its center. This molecular arrangement could represent the CFTR chloride channel configuration, operative in native plasma membrane.

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.

A Pathway Separate from the Central Channel through the Nuclear Pore Complex for Inorganic Ions and Small Macromolecules

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.

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