How T Cells Do the “Search for the Needle in the Haystack”

In the body, a T cell is confronted with millions of antigen-presenting cells (APCs) in the search for potentially harmful antigen. To elicit an appropriate immune response, this search has to be performed as fast and as precise as possible. These two requirements, however, are at odds with each other: fast searches lack accuracy, whereas high fidelity decisions are typically time-consuming. Here, we use the archetypical search for the needle in the haystack as an analogy for the T cell's search problem. We provide a statistical framework to quantitatively estimate the constraints of search strategies for rare instances. Particularly, we propose a solution for balancing the demand for high speed with low error rates. It takes advantage of a two-phase search process, which combines a first rapid scan with a second high-fidelity check. Finally, we provide arguments that support a two-phase search model for identification of antigen-positive APCs by T cells

Localization of fixed dipoles at high precision by accounting for sample drift during illumination

Single molecule localization microscopy relies on the precise quantification of the position of single dye emitters in a sample. This precision is improved by the number of photons that can be detected from each molecule. It is therefore recommendable to increase illumination times for the recording process. Particularly recording at cryogenic temperatures dramatically reduces photobleaching and thereby allows a massive increase in illumination times to several seconds. As a downside, microscope instabilities may well introduce jitter during such long illuminations, deteriorating the localization precision. In this paper, we theoretically demonstrate that a parallel recording of fiducial marker beads together with a novel fitting approach accounting for the full drift trajectory allows for largely eliminating drift effects for drift magnitudes of several hundred nanometers per frame.Comment: 12 pages, 7 figure

Cholesterol Slows down the Lateral Mobility of an Oxidized Phospholipid in a Supported Lipid Bilayer

We investigated the mobility and phase-partitioning of the fluorescent oxidized phospholipid analogue 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N-Alexa647-ethanolamine (PGPE-Alexa647) in supported lipid bilayers. Compared to the conventional phospholipid dihexadecanoylphosphoethanolamine (DHPE)-Bodipy we found consistently higher diffusion constants. The effect become dramatic when immobile obstacles were inserted into the bilayer. which essentially blocked the diffusion of DHPE-Bodipy but hardly influenced the movements of PGPE-Alexa647. In a supported lipid bilayer made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the differences in probe mobility leveled off with increasing cholesterol content. Using coarse-grained molecular dynamics simulations, we could ascribe this effect to increased interactions between the oxidized phospholipid and the membrane matrix, concomitant with a translation in the headgroup position of the oxidized phospholipid: at zero cholesterol content, its headgroup is shifted to the outside of the DOPC headgroup region, whereas increasing cholesterol concentrations pulls the headgroup into the bilayer plane

A Fast and Simple Contact Printing Approach to Generate 2D Protein Nanopatterns

Protein micropatterning has become an important tool for many biomedical applications as well as in academic research. Current techniques that allow to reduce the feature size of patterns below 1 μm are, however, often costly and require sophisticated equipment. We present here a straightforward and convenient method to generate highly condensed nanopatterns of proteins without the need for clean room facilities or expensive equipment. Our approach is based on nanocontact printing and allows for the fabrication of protein patterns with feature sizes of 80 nm and periodicities down to 140 nm. This was made possible by the use of the material X-poly(dimethylsiloxane) (X-PDMS) in a two-layer stamp layout for protein printing. In a proof of principle, different proteins at various scales were printed and the pattern quality was evaluated by atomic force microscopy (AFM) and super-resolution fluorescence microscopy

Oxidized Phospholipids Inhibit the Formation of Cholesterol-Dependent Plasma Membrane Nanoplatforms

We previously developed a single-molecule microscopy method termed TOCCSL (thinning out clusters while conserving stoichiometry of labeling), which allows for direct imaging of stable nanoscopic platforms with raft-like properties diffusing in the plasma membrane. As a consensus raft marker, we chose monomeric GFP linked via a glycosylphosphatidylinositol (GPI) anchor to the cell membrane (mGFP-GPI). With this probe, we previously observed cholesterol-dependent homo-association to nanoplatforms diffusing in the plasma membrane of live CHO cells. Here, we report the release of this homo-association upon addition of 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) or 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine, two oxidized phospholipids (oxPLs) that are typically present in oxidatively modified low-density lipoprotein. We found a dose-response relationship for mGFP-GPI nanoplatform disintegration upon addition of POVPC, correlating with the signal of the apoptosis marker Annexin V-Cy3. Similar concentrations of lysolipid showed no effect, indicating that the observed phenomena were not linked to properties of the lipid bilayer itself. Inhibition of acid sphingomyelinase by NB-19 before addition of POVPC completely abolished nanoplatform disintegration by oxPLs. In conclusion, we were able to determine how oxidized lipid species disrupt mGFP-GPI nanoplatforms in the plasma membrane. Our results favor an indirect mechanism involving acid sphingomyelinase activity rather than a direct interaction of oxPLs with nanoplatform constituents. © 2016 Biophysical Society

Temporal resolution of protein–protein interactions in the live-cell plasma membrane

We have recently devised a method to quantify interactions between a membrane protein (“bait”) and a fluorophore-labeled protein (“prey”) directly in the live-cell plasma membrane (Schwarzenbacher et al. Nature Methods 5:1053–1060 2008). The idea is to seed cells on surfaces containing micro-patterned antibodies against the exoplasmic domain of the bait, and monitor the co-patterning of the fluorescent prey via fluorescence microscopy. Here, we characterized the time course of bait and prey micropattern formation upon seeding the cells onto the micro-biochip. Patterns were formed immediately after contact of the cells with the surface. Cells were able to migrate over the chip surface without affecting the micropattern contrast, which remained constant over hours. On single cells, bait contrast may be subject to fluctuations, indicating that the bait can be released from and recaptured on the micropatterns. We conclude that interaction studies can be performed at any time-point ranging from 5 min to several hours post seeding. Monitoring interactions with time opens up the possibility for new assays, which are briefly sketched in the discussion section

Membrane-Lipid Therapy in Operation: The HSP Co-Inducer BGP-15 Activates Stress Signal Transduction Pathways by Remodeling Plasma Membrane Rafts

Aging and pathophysiological conditions are linked to membrane changes which modulate membrane-controlled molecular switches, causing dysregulated heat shock protein (HSP) expression. HSP co-inducer hydroxylamines such as BGP-15 provide advanced therapeutic candidates for many diseases since they preferentially affect stressed cells and are unlikely have major side effects. In the present study in vitro molecular dynamic simulation, experiments with lipid monolayers and in vivo ultrasensitive fluorescence microscopy showed that BGP-15 alters the organization of cholesterol-rich membrane domains. Imaging of nanoscopic long-lived platforms using the raft marker glycosylphosphatidylinositol-anchored monomeric green fluorescent protein diffusing in the live Chinese hamster ovary (CHO) cell plasma membrane demonstrated that BGP-15 prevents the transient structural disintegration of rafts induced by fever-type heat stress. Moreover, BGP-15 was able to remodel cholesterol-enriched lipid platforms reminiscent of those observed earlier following non-lethal heat priming or membrane stress, and were shown to be obligate for the generation and transmission of stress signals. BGP-15 activation of HSP expression in B16-F10 mouse melanoma cells involves the Rac1 signaling cascade in accordance with the previous observation that cholesterol affects the targeting of Rac1 to membranes. Finally, in a human embryonic kidney cell line we demonstrate that BGP-15 is able to inhibit the rapid heat shock factor 1 (HSF1) acetylation monitored during the early phase of heat stress, thereby promoting a prolonged duration of HSF1 binding to heat shock elements. Taken together, our results indicate that BGP-15 has the potential to become a new class of pharmaceuticals for use in ‘membrane-lipid therapy’ to combat many various protein-misfolding diseases associated with aging