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.

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. © 2015 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.

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.

Molecular level associations between components within the lipid bilayer as proteins complex to the membrane surface are fundamental to the complete understanding of protein-membrane interactions.To characterize these protein-induced membrane-organizational processes, we have prepared a new fluorescently labeled glycolipid (PSMU) that enables monitoring of glycolipid aggregation within the lipid membrane. The glycolipid's mannosamine headgroup was specifically recognized by the lectin concanavalin A (Con A). Fluorescence studies with liposomes composed of 5 mol % PSMU/distearylphosphatidylcholine found that the membrane reorganized in response to Con A adsorption. Initially aggregated structures of glycolipid were dispersed as a consequence of specific affinity with the lectin through steric restrictions imposed by other bound Con A, the distance between mannosyl receptor sites, and possible protein insertion events. The protein binding and membrane reorganizational process was slow (ca. days). An association constant for Con A with the glycolipid membrane was estimated to be 3 × 10 6 M -1 , around 2 orders of magnitude higher than that for methyl-D-R-mannopyranoside. Nanoscale imaging with the atomic force microscope found that the glycolipid formed 10 nm wide dendrite structures throughout the membrane and that the bound Con A was associated with those nanoscale features.