PeakForce Tapping resolves individual microvilli on living cells.

Microvilli are a common structure found on epithelial cells that increase the apical surface thus enhancing the transmembrane transport capacity and also serve as one of the cell's mechanosensors. These structures are composed of microfilaments and cytoplasm, covered by plasma membrane. Epithelial cell function is usually coupled to the density of microvilli and its individual size illustrated by diseases, in which microvilli degradation causes malabsorption and diarrhea. Atomic force microscopy (AFM) has been widely used to study the topography and morphology of living cells. Visualizing soft and flexible structures such as microvilli on the apical surface of a live cell has been very challenging because the native microvilli structures are displaced and deformed by the interaction with the probe. PeakForce Tapping® is an AFM imaging mode, which allows reducing tip-sample interactions in time (microseconds) and controlling force in the low pico-Newton range. Data acquisition of this mode was optimized by using a newly developed PeakForce QNM-Live Cell probe, having a short cantilever with a 17-µm-long tip that minimizes hydrodynamic effects between the cantilever and the sample surface. In this paper, we have demonstrated for the first time the visualization of the microvilli on living kidney cells with AFM using PeakForce Tapping. The structures observed display a force dependence representing either the whole microvilli or just the tips of the microvilli layer. Together, PeakForce Tapping allows force control in the low pico-Newton range and enables the visualization of very soft and flexible structures on living cells under physiological conditions.

Using bicellar mixtures to form supported and suspended lipid bilayers on silicon chips.

Bicellar mixtures, planar lipid bilayer assemblies comprising long- and short-chain phosphatidylcholine lipids in suspension, were used to form supported lipid bilayers on flat silicon substrate and on nanotextured silicon substrates containing arrays of parallel troughs (170 nm wide, 380 nm deep, and 300 nm apart). Confocal fluorescence and atomic force microscopies were used to characterize the resulting lipid bilayer. Formation of a continuous biphasic undulating lipid bilayer membrane, where the crests and troughs corresponded to supported and suspended lipid bilayer regions, is demonstrated. The use of interferometric lithography to fabricate nanotexured substrates provides an advantage over other nanotextured substrates such as nanoporous alumina by offering flexibility in designing different geometries for suspending lipid bilayers.

In situ scanning probe microscopy studies of tetanus toxin-membrane interactions.

Despite the considerable information available with regards to the structure of the clostridial neurotoxins, and their inherent threat as biological warfare agents, the mechanisms underpinning their interactions with and translocation through the cell membrane remain poorly understood. We report herein the results of an in situ scanning probe microscopy study of the interaction of tetanus toxin C-fragment (Tet C) with supported planar lipid bilayers containing the ganglioside receptor G(T1b). Our results show that Tet C preferentially binds to the surface of fluid phase domains within biphasic membranes containing G(T1b) and that with an extended incubation period these interactions lead to dramatic changes in the morphology of the lipid bilayer, including the formation of 40-80 nm diameter circular cavities. Combined atomic force microscopy/total internal reflection fluorescence microscopy experiments confirmed the presence of Tet C in the membrane after extended incubation. These morphological changes were found to be dependent upon the presence of G(T1b) and the solution pH.

Simultaneous in situ total internal reflectance fluorescence/atomic force microscopy studies of DPPC/dPOPC microdomains in supported planar lipid bilayers.

In situ simultaneous total internal reflectance fluorescence and in situ scanning probe microscopy performed on a phase-segregated supported planar lipid bilayer enabled direct in situ real-time correlated topographical and fluorescence images of nanometer-sized gel and fluid-phase lipid domains, presaging future in situ studies of membrane protein assemblies by single molecule imaging.

Properties of a novel magnetized alginate for magnetic resonance imaging.

Implanting recombinant cells encapsulated in alginate microcapsules to secrete therapeutic proteins has been proven clinically effective in treating several murine models of human diseases. However, once implanted, these microcapsules cannot be assessed without invasive surgery. We now report the preparation and characterization of a novel ferrofluid to render these microcapsules visible with magnetic resonance imaging (MRI). The ferrofluid was prepared as a colloidal iron oxide stabilized in water by alginate. The presence of iron particles in the ferrofluid was verified with chemical titration, dynamic light scattering, and magnetization measurement. The microcapsules fabricated with various concentrations of the ferrofluid in the core, or on the surface of alginate microcapsules, or both, all produced microcapsules with smooth surfaces as shown with light and scanning electron microscopy. However, at the nanoscale level, as revealed with atomic force microscopy, the ferrofluid-fabricated microcapsules demonstrated increased granularity, particularly when the ferrofluid was used to laminate the surface. From the force spectroscopy measurements, these modified microcapsules showed increasing surface rigidity in the following order: traditional alginate < ferrofluid in the core < ferrofluid on the surface. Although the mechanical stability of low-concentration ferrofluid (0.1%) microcapsules was reduced, increasing concentrations, up to 20%, were able to improve stability. When these ferrofluid microcapsules were examined with MRI, their T(2) relaxation time was reduced, thereby producing increased contrast readily detectable with MRI, whereas the traditional alginate microcapsules showed no difference when compared with water. In conclusion, such ferrofluid-enhanced alginate is suitable for fabricating microcapsules that offer the potential for in vivo tracking of implanted microcapsules without invasive surgery.

Elimination of host cell PtdIns(4,5)P(2) by bacterial SigD promotes membrane fission during invasion by Salmonella.

Salmonella invades mammalian cells by inducing membrane ruffling and macropinocytosis through actin remodelling. Because phosphoinositides are central to actin assembly, we have studied the dynamics of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) in HeLa cells during invasion by Salmonella typhimurium. Here we show that the outermost parts of the ruffles induced by invasion show a modest enrichment in PtdIns(4,5)P(2), but that PtdIns(4,5)P(2) is virtually absent from the invaginating regions. Rapid disappearance of PtdIns(4,5)P(2) requires the expression of the Salmonella phosphatase SigD (also known as SopB). Deletion of SigD markedly delays fission of the invaginating membranes, indicating that elimination of PtdIns(4,5)P(2) may be required for rapid formation of Salmonella-containing vacuoles. Heterologous expression of SigD is sufficient to promote the disappearance of PtdIns(4,5)P(2), to reduce the rigidity of the membrane skeleton, and to induce plasmalemmal invagination and fission. Hydrolysis of PtdIns(4,5)P(2) may be a common and essential feature of membrane fission during several internalization processes including invasion, phagocytosis and possibly endocytosis.

Single molecule imaging of supported planar lipid bilayer--reconstituted human insulin receptors by in situ scanning probe microscopy.

A 480-kDa disulfide-linked heterodimer single-pass transmembrane protein, the insulin receptor, is autophosphorylated upon insulin binding to its extracellular domain. Remarkably, the structural basis for this activation process remained largely unknown until the recent cryoelectron microscopy studies of the insulin-insulin receptor complex by Luo et al. [Science 285 (1999) 1077]. We report here the results of an in situ study by high-resolution scanning probe microscopy of the full-length insulin receptor reconstituted within supported planar lipid bilayers. Our preliminary studies confirm that (1) the intact receptor can be reconstituted constitutively within a lipid vesicle and (2) fusion of the receptor-containing vesicles to mica resulted in the formation of molecular flat 5.5-nm-thick supported planar bilayers populated by two populations of protrusions, the shape and size of which are consistent with those of the insulin receptor's intra- and extracellular domains as modeled by the cryo-EM data of Ottensmeyer et al. [Biochemistry 39 (2000) 12103]. These results establish a framework for real-time studies of insulin-insulin receptor binding by in situ SPM and single molecule force spectroscopy.