Microfluidic array cytometer based on refractive optical tweezers for parallel trapping, imaging and sorting of individual cells

Analysis of genetic and functional variability in populations of living cells requires experimental techniques capable of monitoring cellular processes such as cell signaling of many single cells in parallel while offering the possibility to sort interesting cell phenotypes for further investigations. Although flow cytometry is able to sequentially probe and sort thousands of cells per second, dynamic processes cannot be experimentally accessed on single cells due to the sub-second sampling time. Cellular dynamics can be measured by image cytometry of surface-immobilized cells, however, cell sorting is complicated under these conditions due to cell attachment. We here developed a cytometric tool based on refractive multiple optical tweezers combined with microfluidics and optical microscopy. We demonstrate contact-free immobilization of more than 200 yeast cells into a high-density array of optical traps in a microfluidic chip. The cell array could be moved to specific locations of the chip enabling us to expose in a controlled manner the cells to reagents and to analyze the responses of individual cells in a highly parallel format using fluorescence microscopy. We further established a method to sort single cells within the microfluidic device using an additional steerable optical trap. Ratiometric fluorescence imaging of intracellular pH of trapped yeast cells allowed us on the one hand to measure the effect of the trapping laser on the cells' viability and on the other hand to probe the dynamic response of the cells upon glucose sensing

Phosphatidylserine positive microparticles improve hemostasis in in-vitro hemophilia A plasma models

Circulating microparticles (MPs) are procoagulant due to the surface containing phosphatidylserine (PS), which facilitates coagulation. We investigated if MPs improve hemostasis in HA plasma models. MPs isolated from pooled normal human plasma were added to severe, moderate and mild HA plasma models (0%, 2.5%, 20% FVIII). The MPs' effect on hemostasis was evaluated by calibrated automated thrombogram (CAT) and overall hemostasis potential (OHP) assays, while fibrin structure was imaged by standard confocal, stimulated emission depletion (STED) microscopy and scanning electron microscopy (SEM). MPs partially restored thrombin generation and fibrin formation in all HA plasma models. The procoagulant effect of MPs requires PS exposure, to a less extent of contact pathway activation, but not tissue factor exposure or in vitro stimulation of MPs. MPs partially normalized the fibrin structure, and using super-resolution STED, MPs attached to fibrin were clearly resolved. In summary, our results demonstrate that PS positive MPs could improve hemostasis in HA plasma models

Cell-derived plasma membrane vesicles as minimal cells for analyzing transmembrane signaling

Cellular signalling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show how cell-derived vesicles can be used for anlaysing transmembrane signalling. The vesicles are derived from live mammalian cells by using either chemicals, or by optical tweezers and they comprise parts of the plasma membrane and cytosol of the mother cell. We measured in vesicles derived from individual cells with single molecule sensitivity the different steps of G protein-coupled receptor mediated signalling like ligand binding to receptors, subsequent G protein activation and finally receptor deactivation by interaction with arrestin. Cell-derived plasma membrane vesicles represent the smallest autonomous containers capable of performing cellular signaling reactions thus functioning like minimal cells. Observing cellular signalling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis and investigating receptor mediated signalling in multiarray format

Association of Alleles Carried at TNFA -850 and BAT1 -22 with Alzheimer\u27s Disease

Background: Inflammatory changes are a prominent feature of brains affected by Alzheimer\u27s disease (AD). Activated glial cells release inflammatory cytokines which modulate the neurodegenerative process. These cytokines are encoded by genes representing several interleukins and TNFA, which are associated with AD. The gene coding for HLA-B associated transcript 1 (BAT1) lies adjacent to TNFA in the central major histocompatibility complex (MHC). BAT1, a member of the DEAD-box family of RNA helicases, appears to regulate the production of inflammatory cytokines associated with AD pathology. In the current study TNFA and BAT1 promoter polymorphisms were analysed in AD and control cases and BAT1 mRNA levels were investigated in brain tissue from AD and control cases. Methods: Genotyping was performed for polymorphisms at positions -850 and -308 in the proximal promoter of TNFA and position -22 in the promoter of BAT1. These were investigated singly or in haplotypic association in a cohort of Australian AD patients with AD stratified on the basis of their APOE ε4 genotype. Semi-quantitative RT-PCR was also performed for BAT1 from RNA isolated from brain tissue from AD and control cases. Results: APOE ε4 was associated with an independent increase in risk for AD in individuals with TNFA -850*2, while carriage of BAT1 -22*2 reduced the risk for AD, independent of APOE ε4 genotype. Semi-quantitative mRNA analysis in human brain tissue showed elevated levels of BAT1 mRNA in frontal cortex of AD cases. Conclusion: These findings lend support to the application of TNFA and BAT1 polymorphisms in early diagnosis or risk assessment strategies for AD and suggest a potential role for BAT1 in the regulation of inflammatory reactions in AD pathology

Murine malaria is associated with significant hearing impairment

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum </it>malaria has been suspected to cause hearing loss. Developmental, cognitive and language disorders have been observed in children, surviving cerebral malaria. This prospective study aims to evaluate whether malaria influences hearing in mice.</p> <p>Methods</p> <p>Twenty mice were included in a standardized murine cerebral malaria model. Auditory evoked brainstem responses were assessed before infection and at the peak of the illness.</p> <p>Results</p> <p>A significant hearing impairment could be demonstrated in mice with malaria, especially the cerebral form. The control group did not show any alterations. No therapy was used.</p> <p>Conclusion</p> <p>This suggests that malaria itself leads to a hearing impairment in mice.</p

Advanced Fluorescence Microscopy to Study Plasma Membrane Protein Dynamics

Membrane protein dynamics is of great importance for living organisms. The precise localization of proteins composing a synapse on the membrane facing a nerve terminus is essential for proper functioning of the nervous system. In muscle fibers, the nicotinic acetylcholine is densely packed under the motor nerve termini. A receptor associated protein, rapsyn, acts as a linker between the receptor and the other components of the synaptic suramolecular assembly. Advances in fluorescence microscopy have allowed to measure the behavior of a single receptor in the cell membrane. In this work single-molecule microscopy was used to track the motion of ionotropic acetylcholine (nAChR) and serotonin (5HT3R) receptors in the plasma membrane of cells. We present methods for measuring single-molecule diffusion and their analysis. Single molecule tracking has shown a high dependence of acetylcholine receptors diffusion on its associated protein rapsyn. Comparing muscle cells that either express rapsyn or are devoid of it, we found that rapsyn plays an important role on receptor immobilization. A three-fold increase of receptor mobility was observed in muscle cells devoid of rapsyn. However, in these cells, a certain fraction of immobilized receptors was also found immobile. Furthermore, nAChR were strongly confined in membrane domains of few tens of nanometers. This showed that membrane composition and membrane associated proteins influence on receptor localization. During muscle cell differentiation, the fraction of immobile nAChR diminished along with the decreasing nAChR and stable rapsyn expression levels. The importance of rapsyn in nAChR immobilization has been further confirmed by measurements in HEK 293 cells, where co-expression of rapsyn increased immobilization of the receptor. nAChR is a ligand-gated ion-channel of the Cys-loop family. In mammals, members of this receptor family share general structural and functional features. They are homo- or hetero-pentamers and form a membrane-spanning ion channel. Subunits have three major regions, an extracellular ligand binding domain, a transmembrane channel and a large intracellular loop. 5HT3R was used as a model to study the effect of this loop on receptor mobility. Single-molecule tracking experiments on receptors with progressively larger deletions in the intracellular loop did not show a dependence of the size of the loop on the diffusion coefficient of mobile receptors. However, two regions were identified to play a role in receptor mobility by changing the fractions of immobile and directed receptors. Interestingly, a prokaryotic homologue of cys-loop receptors, ELIC, devoid of a large cytoplasmic loop was found to be immobile or to show directed diffusion similar as the wild-type 5HT3R. The scaffolding protein rapsyn stabilizes nAChR clusters in a concentration dependent manner. We have measured the density and self-interactions of rapsyn using FRET microscopy. Point-mutations of rapsyn, known to provoke myopathies, destabilized rapsyn self-interactions. Rapsyn-N88K, and R91L were found at high concentration in the cytoplasm suggesting that this modification disturbs membrane association of rapsyn. A25V was found to accumulate in the endoplasmic reticulum. Fluorescent tools to measure intracellular concentration of calcium ions are of great value to study the function of neurons. Rapsyn is highly abundant at the neuromuscular junction and thus is a genuine synaptic marker. A fusion protein of rapsyn with a genetically encoded ratiometric calcium sensor has been made to probe synapse activity. This thesis has shown that the combined use of biologically relevant system and modern fluorescence microscopy techniques deliver important information on pLGIC behaviour in the cell membrane.QC 20151217</p

Single-Molecule Imaging Deciphers the Relation between Mobility and Signaling of a Prototypical G Protein-Coupled Receptor in Living Cells

Lateral diffusion enables efficient interactions between membrane proteins leading to signal transmission across the plasma membrane. An open question is how the spatio-temporal distribution of cell surface receptors influences the transmembrane signaling network. Here we addressed this issue studying the mobility of a prototypical G protein-coupled receptor, the neurokinin-1 receptor (NK1R) during its different phases of cellular signaling. Attaching a single quantum dot to individual NK1Rs enabled us to follow with high spatial and temporal resolution over long time regimes the fate of individual receptors at the plasma membrane. Single receptor trajectories revealed a very heterogeneous mobility distribution pattern with diffusion constants ranging from 0.0005 to 0.1 μm2/s comprising receptors freely diffusing, confined in 100-600 nm sized membrane domains, as well as immobile ones. A two-dimensional representation of mobility and confinement resolved two major, broadly distributed receptor populations, one showing high mobility and low lateral restriction, the other low mobility and high restriction. We found that about 40% of the receptors in the basal state are already confined in membrane domains and are associated with clathrin. After stimulation with an agonist, additional 30% of receptors became further confined. Using inhibitors of clathrin-mediated endocytosis, we showed that the fraction of confined receptors at the basal state depends on the quantity of membrane-associated clathtrin and is correlated to a significant decrease of the receptors' canonical pathway activity. This shows that the high plasticity of receptor mobility is of central importance for receptor homeostasis and fine regulation of receptor activity