Nicoletta Kahya

Fluorescence correlation studies of lipid domains in model membranes (Review)

Advances in optical microscopy techniques and single-molecule detection have paved the way to exploring new approaches for investigating membrane dynamics and organization, thereby revealing details on the processing of signals, complex association/dissociation, chemical reactions and transport at and around the membrane. These events rely on a tight regulation of lipid-protein and protein-protein interactions in space and time. Fluorescence Correlation Spectroscopy (FCS) provides exquisite sensitivity in measuring local concentrations, association/dissociation constants, chemical rate constants and, in general, in probing the chemical environment of the species of interest and its interactions with potential partners. Here, we review some applications of FCS to lipid and protein organization in biomimetic membranes with lateral heterogeneities, which share some physico-chemical properties with cellular rafts. What we learn from investigations of lipid-lipid and lipid-protein interactions in simple model membranes can be regarded as an essential basic lecture for studies in more complex cellular membranes.

Reconstitution and Anchoring of Cytoskeleton inside Giant Unilamellar Vesicles

Among the requirements for all life forms is the ability to self‐replicate. In eukaryotic cellular systems, this division is achieved through cytokinesis, and is facilitated by the (re)arrangement and interaction of cytoskeletal proteins with lipids and other proteins localized to the plasma membrane. A fascinating challenge of modern synthetic biology is the bottom‐up reconstitution of such processes for the generation of an artificial cell. One crucial step towards this goal is the functional reconstitution of the protein‐anchoring machinery to facilitate cytokinesis into lipid vesicles. True to the ideal of a minimal cell‐like system, we here describe the formation of an actin‐based cytoskeleton within giant unilamellar vesicles (GUVs) made from porcine brain lipid extracts. We demonstrate that the actin filaments are localised and anchored to the interior walls of the GUVs through the spectrin/ankyrin proteins, and produce tightly packed actin bundles. These studies allow for the examination of cytoskeletal rearrangements within a cell‐like model membrane system and represent important first steps in reconstituting the minimal machinery required for the division of an artificial cell. In addition, the study of such minimal systems can shed light on protein functions that are commonly unobservable or hidden within the overwhelming complexity of cells.

Mobility of Min-proteins in Escherichia coli measured by fluorescence correlation spectroscopy

In the bacterium Escherichia coli, selection of the division site involves pole-to-pole oscillations of the proteins MinD and MinE. Different oscillation mechanisms based on cooperative effects between Min-proteins and on the exchange of Min-proteins between the cytoplasm and the cytoplasmic membrane have been proposed. The parameters characterizing the dynamics of the Min-proteins in vivo are not known. It has therefore been difficult to compare the models quantitatively with experiments. Here, we present in vivo measurements of the mobility of MinD and MinE using fluorescence correlation spectroscopy. Two distinct timescales are clearly visible in the correlation curves. While the faster timescale can be attributed to cytoplasmic diffusion, the slower timescale could result from diffusion of membrane-bound proteins or from protein exchange between the cytoplasm and the membrane. We determine the diffusion constant of cytoplasmic MinD to be approximately 16 microm(2) s(-1), while for MinE we find about 10 microm(2) s(-1), independently of the processes responsible for the slower time-scale. The implications of the measured values for the oscillation mechanism are discussed.

How Phospholipid-Cholesterol Interactions Modulate Lipid Lateral Diffusion, as Revealed by Fluorescence Correlation Spectroscopy

Cholesterol is a key player in regulating physico-chemical properties of cellular membranes and, thereby, ensuring cell viability. In particular, lipid-cholesterol interactions may provide important information on the spatio-temporal organization of membrane components. Here, we apply confocal imaging and Fluorescence Correlation Spectroscopy (FCS) to Giant Unilamellar Vesicles (GUVs) composed of binary mixtures of lipids and cholesterol.The effect of cholesterol on lipid dynamics and molecular packing order of unsaturated, monounsaturated, fully saturated (with both low and high phase transition temperatures, Tm) glycero-phospholipids and sphingomyelin has been investigated. We show that, for unsaturated glycerophospholipids, the decrease of the lipid diffusion coefficient as a result of the interaction with cholesterol does not depend on the fatty acid chain length. However, the values of the diffusion coefficient change as a function of chain length. The monounsaturated phospholipid palmitoyl-oleoyl-phosphatidylcholine (POPC) exhibits a dynamic behavior very similar to the unsaturated dioleoyl-phosphatidylcholine (DOPC). By contrast, for saturated (low Tm) glycero-phospholipids, cholesterol causes a decrease of lipid mobility in a chain length-dependent manner.FCS can be employed as a valuable tool to study lipid-sterol interactions and their effect on lipid dynamics, molecular packing and degree of conformational order.

A novel nano-photonics biosensor concept for rapid molecular diagnostics

We present a novel nano-photonics biosensor concept that offers an ultra-high surface specificity and excellent suppression of background signals due to the sample fluid on top of the biosensor. In our contribution, we will briefly discuss the operation principle and fabrication of the biosensor, followed by a more detailed discussion on the experimentally determined performance parameters. Recent results on detection of fluorescently labeled molecules in a highly fluorescent background will be shown, and we will give an outlook on real-time detection of bio-molecules such as proteins and nucleic acids.

Pushing the Complexity of Model Bilayers: Novel Prospects for Membrane Biophysics

As an interface between different biological compartments, membranes guarantee an efficient exchangeof matter, energy and/or signals. For this purpose, such an interface has to be designed as a verydynamic system, yet with a non-random distribution of its components, lipids and proteins. A delicatebalance of lipid and protein interactions is the basis of tightly regulated mechanisms to concentrate moleculesat the site of interest at a specific time and, thereby, exclude unwanted components. In order toelucidate this highly intricate architecture, the top-down approach—by looking at the intact cell—isbest complemented by a bottom-up strategy, by building the whole complexity starting from a minimalnumber of components. Within this framework, model membranes are key systems to isolate the biologicalmachinery and identify its function. In this paper, we review research on biomimetic membranes for opticalmicrospectroscopy. In particular, we focus on giant unilamellar vesicles (GUVs), and their applicationto studies on domain assembly and on membrane curvature and deformations. In order to build complexity,efforts must be made towards mimicking cellular compositions, by using GUVs with native lipid compositions,to reconstitute (complexes of) membrane proteins and to include components of an artificial cytoskeletonunderneath the bilayer. Novel exciting avenues lie ahead in the arena of membrane biophysics, many of whichare strongly coupled to the promising developments of optical technologies.

Procedure and step-based analysis of the occupational radiation dose during endovascular aneurysm repair in the hybrid operating room

Objective: This study measured the cumulative occupational X‐ray radiation dose received by support staff during endovascular aortic procedures and during additional intraoperative steps in the hybrid operating room. Methods: Radiation dose measurements were performed during interventions on 65 patients receiving 90 stent grafts during endovascular aneurysm repair (EVAR), bifurcated EVAR, thoracic EVAR, iliac branched device deployment, aortouni‐iliac stenting, and fenestrated/branched EVAR (F/BrEVAR). X‐ray imaging was acquired using the Philips Allura FD20 Clarity System (Philips Medical Systems, Best, The Netherlands). The occupational radiation dose (also referred to as the estimated effective dose, E, measured in millisieverts) was measured with the DoseAware Xtend system (Philips Medical Systems) personal dosimeters. E was reported per staff member (ESTAFF), where “staff” was a generic term for each staff member included in the study: the first operator (FO), the second operator (ESO), a virtual maximum operator (MO), and all additional supporting staff, including the sterile nurse, nonsterile nurse, anaesthesiologist, and radiation technician. The primary outcome was the median cumulative ESTAFF (or EFO, EMO, and so on), which was presented as the median cumulative dose per intervention and stratified for several within‐interventional EVAR and F/BrEVAR steps or stents. The second outcome was the percentage of the absorbed E by a supporting staff member in relation to the E measured by the reference badge attached on the C‐arm (ESTAFF% or EFO%, EMO%, and so on). All outcomes are presented as median with interquartile range, unless stated differently. Results: The occupational effective dose in millisieverts of the MO (EMO) was 0.055 (0.029–0.082) for aortouni‐iliac stenting (n = 6), 0.084 (0.054–0.141) during thoracic EVAR (n = 14), 0.036 (0.026–0.068) during bifurcated EVAR (n = 38), 0.054 (0.035–0.126) during iliac branched device deployment (n = 8), and 0.345 (0.235–0.757) during F/BrEVAR (n = 24). The median EMO in millisieverts was 0.025 (0.012–0.062) per renal target vessel (TV) and 0.146 (0.07–0.315) for a nonrenal visceral TV. During all noncomplex interventions, the EMO% was 4.4% (2.7%‐7.3%), with the lowest median rate at 3.5% (2.5%‐5%) for EVAR. The highest median rate EMO% was found for F/BrEVAR procedures: 8.2% (5.0%‐14.4%). Conclusions: With maximum operator shielding during femoral access, relative occupational radiation risk can be minimized. However, digital subtraction angiography image acquisition, recanalization of TVs, recanalization of superior mesenteric artery or celiac artery, and recanalization of branched TVs are predictors for increased occupational radiation dose risks caused by increased radiation doses to the patient and reduced options for shielding of the operator.