It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell receptor (TCR) triggering, but how segregation would occur and whether it can initiate signaling is unclear. Using structural and biophysical analysis we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of new structures characterized by spontaneous sub-micron scale CD45 and kinase segregation, called ‘close-contacts’, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the unexpectedly potent signaling effects of local CD45 and kinase segregation. TCR ligands could heighten signaling simply by holding receptors in close-contacts.

Fluorescence recovery after photobleaching has been an established technique of quantifying the mobility of molecular species in cells and cell membranes for more than 30 years. However, under nonideal experimental conditions, the current methods of analysis still suffer from occasional problems; for example, when the signal/noise ratio is low, when there are temporal fluctuations in the illumination, or when there is bleaching during the recovery process. We here present a method of analysis that overcomes these problems, yielding accurate results even under nonideal experimental conditions. The method is based on circular averaging of each image, followed by spatial frequency analysis of the averaged radial data, and requires no prior knowledge of the shape of the bleached area. The method was validated using both simulated and experimental fluorescence recovery after photobleaching data, illustrating that the diffusion coefficient of a single diffusing component can be determined to within approximately 1%, even for small signal levels (100 photon counts), and that at typical signal levels (5000 photon counts) a system with two diffusion coefficients can be analyzed with <10% error.

This paper presents the use of the localized surface plasmon resonance (LSPR) sensor concept to probe the formation of macroscopic and laterally mobile supported lipid bilayers (SLBs) on SiOx-encapsulated nanohole-containing Au and Ag films. A comparison between Au- and Ag-based sensor templates demonstrates a higher sensitivity for Au-based templates with respect to both bulk and interfacial refractive index (RI) changes in aqueous solution. The lateral mobility of SLBs formed on the SiOx-encapsulated nanohole templates was analyzed using fluorescence recovery after photobleaching (FRAP), demonstrating essentially complete (>96%) recovery, but a reduction in diffusivity of about 35% compared with SLBs formed on flat SiOx substrates. Furthermore, upon SLB formation, the temporal variation in extinction peak position of the LSPR active templates display a characteristic shape, illustrating what, to the best of our knowledge, is the first example where the nanoplasmonic concept is shown capable of probing biomacromolecular structural changes without the introduction of labels. With a signal-to-noise ratio better than 5 x 10(2) upon protein binding to the cell-membrane mimics, the sensor concept is also proven competitive with state-of-the-art label-free sensors.

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We report on a single-molecule readout scheme on total internal reflection fluorescence microscopy (TIRFM) demonstrating a detection limit in the low fM regime for short (30-mer) unlabeled DNA strands. Detection of unlabeled DNA targets is accomplished by letting them mediate the binding of suspended fluorescently labeled DNA-modified small unilamellar vesicles (Ø approximately 100 nm) to a DNA-modified substrate. On top of rapid and sensitive detection, the technique is also shown capable of extracting kinetics data from statistics of the residence time of the binding reaction in equilibrium, that is, without following neither the rate of binding upon injection nor release upon rinsing. The potential of this feature is demonstrated by discriminating a single mismatch from a fully complementary sequence. The success of the method is critically dependent on a surface modification that provides sufficiently low background. This was achieved through self-assembly of a biotinylated copolymer, Poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) on a silicon dioxide surface, followed by subsequent addition of streptavidin and biotinylated DNA. The proposed detection scheme is particularly appealing due to the simplicity of the sensor, which relies on self-assembly principles and conventional TIRFM. Therefore, we foresee a great potential of the concept to serve as an important component in future multiplexed sensing schemes. This holds in particular true in cases when information about binding kinetics is valuable, such as in single nucleotide polymorphism diagnostics.

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In this work, we demonstrate how a lateral motion of a supported lipid bilayer (SLB) and its constituents can be created without relying on self-spreading forces. The force driving the SLB is instead a viscous shear force arising from a pressure-driven bulk flow acting on the SLB that is formed on a glass wall inside a microfluidic channel. In contrast to self-spreading bilayers, this method allows for accurate control of the bilayer motion by altering the bulk flow in the channel. Experiments showed that an egg yolk phosphatidylcholine SLB formed on a glass support moved in a rolling motion under these shear forces, with the lipids in the upper leaflet of the bilayer moving at twice the velocity of the bilayer front. The drift velocity of different lipid probes in the SLB was observed to be sensitive to the interactions between the lipid probe and the surrounding molecules, resulting in drift velocities that varied by up to 1 order of magnitude for the different lipid probes in our experiments. Since the method provides a so far unattainable control of the motion of all molecules in an SLB, we foresee great potential for this technique, alone or in combination with other methods, for studies of lipid bilayers and different membrane-associated molecules.

This paper presents a study of supported lipid bilayer (SLB) formation and subsequent protein binding using a sensor that combines localized surface plasmon resonance (LSPR) and quartz crystal microbalance with dissipation (QCM-D) monitoring. The LSPR activity arises from silicon oxide (SiO x ) coated nanometric apertures in a thin gold film, which also serves as the active electrode of a QCM-D crystal. Both transducer principles provide signatures for the formation of a SLB upon adsorption and subsequent rupture of adsorbed lipid vesicles. However, the two techniques are sensitive over different regions of the sample: LSPR primarily inside and on the rim of the holes and QCM-D primarily on the planar areas between the holes. Although the dimension of the lipid vesicles is on the same order as the dimension of the nanoholes, it is concluded from the response of the combined system that vesicle rupture in the nanoholes and on the planar region between the holes is synchronized. Furthermore, by determining the thickness of the SLB from the QCM-D response, the characteristic decay length of the LSPR field intensity could be determined. This made it possible not only to determine the mass and refractive index of the homogeneous SLB but also to postulate a generic means to quantify the LSPR response in terms of mass-uptake also for nonhomogeneous films. This is exemplified by measuring the adsorbed lipid mass during vesicle adsorption, yielding the critical lipid vesicle coverage at which spontaneous rupture into a planar bilayer occurs. The generic applicability and versatility of the method is demonstrated from specific protein binding to a functionalized SLB. From the absolute refractive index of the protein, provided from the LSPR data alone, it was possible to determine both the effective thickness of the protein film and the molecular mass (or number) of bound protein.