Signaling in chemotactic amoebae remains spatially confined to stimulated membrane regions.

Recent work has demonstrated that the receptor-mediated signaling system in chemotactic amoeboid cells shows typical properties of an excitable system. Here, we delivered spatially confined stimuli of the chemoattractant cAMP to the membrane of differentiated Dictyostelium discoideum cells to investigate whether localized receptor stimuli can induce the spreading of excitable waves in the G-protein-dependent signal transduction system. By imaging the spatiotemporal dynamics of fluorescent markers for phosphatidylinositol (3,4,5)-trisphosphate (PIP3), PTEN and filamentous actin, we observed that the activity of the signaling pathway remained spatially confined to the stimulated membrane region. Neighboring parts of the membrane were not excited and no receptor-initiated spatial spreading of excitation waves was observed. To generate localized cAMP stimuli, either particles that carried covalently bound cAMP molecules on their surface were brought into contact with the cell or a patch of the cell membrane was aspirated into a glass micropipette to shield this patch against freely diffusing cAMP molecules in the surrounding medium. Additionally, the binding site of the cAMP receptor was probed with different surface-immobilized cAMP molecules, confirming results from earlier ligand-binding studies.

Actin and PIP3 waves in giant cells reveal the inherent length scale of an excited state.

The membrane and actin cortex of a motile cell can autonomously differentiate into two states, one typical of the front, the other of the tail. On the substrate-attached surface of Dictyostelium discoideum cells, dynamic patterns of front-like and tail-like states are generated that are well suited to monitor transitions between these states. To image large-scale pattern dynamics independently of boundary effects, we produced giant cells by electric-pulse-induced cell fusion. In these cells, actin waves are coupled to the front and back of phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-rich bands that have a finite width. These composite waves propagate across the plasma membrane of the giant cells with undiminished velocity. After any disturbance, the bands of PIP3 return to their intrinsic width. Upon collision, the waves locally annihilate each other and change direction; at the cell border they are either extinguished or reflected. Accordingly, expanding areas of progressing PIP3 synthesis become unstable beyond a critical radius, their center switching from a front-like to a tail-like state. Our data suggest that PIP3 patterns in normal-sized cells are segments of the self-organizing patterns that evolve in giant cells.

High bacterial contamination of pig tonsils at slaughter.

Food-borne zoonoses have a major health impact in industrial countries. Campylobacter spp., Salmonella enterica, Yersinia enterocolitica and Listeria monocytogenes are high-risk food-borne zoonotic hazards in finishing pigs. The objectives of this work were (1) to study the isolation rate of pathogenic Y. enterocolitica, Salmonella spp., Campylobacter spp. and L. monocytogenes in the tonsils and feces and (2) to determine the number of mesophilic aerobic bacteria (MAB) and Escherichia coli in the tonsils of fattening pigs at slaughter. The samples, which were collected from one slaughterhouse on five occasions, originated from 50 pigs and 15 farms. The number of MAB varied from 6.40 to 7.82 log(10) CFU/g and E. coli from 4.38 to 6.53 log(10) CFU/g. Additionally, 31 (62%) of the tonsils were colonized with Y. enterocolitica and 16 (32%) with L. monocytogenes. Campylobacter spp. were more frequently excreted in feces and only 3 (6%) of the pigs carried Campylobacter spp. in the tonsils. No Salmonella spp. were isolated. The pig tonsils were shown to be colonized with a high number of bacteria including E. coli, which is the most important indicator for fecal contamination, and with Y. enterocolitica and L. monocytogenes, which are important food-borne pathogens. This study demonstrates that the tonsils are highly contaminated with micro-organisms and can be a very important source of contamination in the slaughterhouse.

Harnessing Motile Amoeboid Cells as Trucks for Microtransport and -Assembly.

Cell-driven microtransport is one of the most prominent applications in the emerging field of biohybrid systems. While bacterial cells have been successfully employed to drive the swimming motion of micrometer-sized cargo particles, the transport capacities of motile adherent cells remain largely unexplored. Here, it is demonstrated that motile amoeboid cells can act as efficient and versatile trucks to transport microcargo. When incubated together with microparticles, cells of the social amoeba Dictyostelium discoideum readily pick up and move the cargo particles. Relying on the unspecific adhesive properties of the amoeba, a wide range of different cargo materials can be used. The cell-driven transport can be directionally guided based on the chemotactic responses of amoeba to chemoattractant gradients. On the one hand, the cargo can be assembled into clusters in a self-organized fashion, relying on the developmentally induced chemotactic aggregation of cells. On the other hand, chemoattractant gradients can be externally imposed to guide the cellular microtrucks to a desired location. Finally, larger cargo particles of different shapes that exceed the size of a single cell by more than an order of magnitude, can also be transported by the collective effort of large numbers of motile cells.

Fluorescence Readout of a Patch Clamped Membrane by Laser Scanning Microscopy.

In this chapter, we describe how to shield a patch of a cell membrane against extracellularly applied chemoattractant stimuli. Classical patch clamp methodology is applied to allow for controlled shielding of a membrane patch by measuring the seal resistivity. In Dictyostelium cells, a seal resistivity of 50 MΩ proved to be tight enough to exclude molecules from diffusing into the shielded membrane region. This allowed for separating a shielded and a non-shielded region of a cell membrane to study the spatiotemporal dynamics of intracellular chemotactic signaling events at the interface between shielded and non-shielded areas. The spatiotemporal dynamics of signaling events in the membrane was read out by means of appropriate fluorescent markers using laser scanning confocal microscopy.

Cell-substrate impedance fluctuations of single amoeboid cells encode cell-shape and adhesion dynamics.

We show systematic electrical impedance measurements of single motile cells on microelectrodes. Wild-type cells and mutant strains were studied that differ in their cell-substrate adhesion strength. We recorded the projected cell area by time-lapse microscopy and observed irregular oscillations of the cell shape. These oscillations were correlated with long-term variations in the impedance signal. Superposed to these long-term trends, we observed fluctuations in the impedance signal. Their magnitude clearly correlated with the adhesion strength, suggesting that strongly adherent cells display more dynamic cell-substrate interactions.

Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells.

Cyclic GMP (cGMP) is a ubiquitous second messenger in eukaryotic cells. It is assumed to regulate the association of myosin II with the cytoskeleton of motile cells. When cells of the social amoeba Dictyostelium discoideum are exposed to chemoattractants or to increased osmotic stress, intracellular cGMP levels rise, preceding the accumulation of myosin II in the cell cortex. To directly investigate the impact of intracellular cGMP on cytoskeletal dynamics in a living cell, we released cGMP inside the cell by laser-induced photo-cleavage of a caged precursor. With this approach, we could directly show in a live cell experiment that an increase in intracellular cGMP indeed induces myosin II to accumulate in the cortex. Unexpectedly, we observed for the first time that also the amount of filamentous actin in the cell cortex increases upon a rise in the cGMP concentration, independently of cAMP receptor activation and signaling. We discuss our results in the light of recent work on the cGMP signaling pathway and suggest possible links between cGMP signaling and the actin system.

Monopolar vs. bipolar subretinal stimulation-an in vitro study.

This study uses an in vitro rd10 mouse model to quantify and compare the ability of the monopolar and the (concentric) bipolar electrode configurations for subretinal stimulation. To allow for results which can be directly compared an identical region of the retina was stimulated due to the circumstance that the bipolar electrode configuration allows also for monopolar stimulation, if the concentric counter-electrode is set potential-free (floating). A ganglion cell, located centrally over the bipolar electrode configuration was selected to extracellularly record action potentials during stimulation. To analyse the recorded action potentials, we introduce a new method which combines the advantages of (a) singular value decomposition (SVD) for weighting similar modulation patterns with which the recorded action potentials are characterized and (b) multi curve fitting to identify a common threshold level, required to finally assemble a strength-duration relationship (SDR). By directly comparing the obtained SDR curves, we found that the efficiency of stimulation with the monopolar electrode configuration is significantly higher than with the bipolar electrode configuration. All obtained SDR curves were fitted using the Lapicque model to estimate the chronaxie times and the rheobase currents. Liquid inclusions, eventually separating the retina from the electrodes are discussed to be a major cause for low ganglion cell responses during stimulation with the bipolar electrode configuration.

Electric field stimulation of bipolar cells in a degenerated retina--a theoretical study.

Subretinal implants are the subject of clinical investigation for their ability to evoke useful visual sensations in blind individuals via electrical stimulation of the diseased retina. We investigated the spatial characteristic of the retinal polarization obtained by electric field stimulation through a subretinally located monopolar electrode array and bipolar electrode array. By combining electric potential simulation through a boundary element method with a segmented cell model, we computed the membrane voltage at the axon terminal of the bipolar cells as a function of the axon length (50-110 microm) and the electrode diameter. We found that short OFF bipolar cells are predominantly addressed by small bipolar electrodes (diameter between 60 and 100 microm) and by using a short duration ( < 150 micros) of the stimulating voltage pulse. Long ON cells are best addressed by large monopolar electrodes (diameter > 100 microm) and a long pulse duration ( > 150 micros). However, the low selectivity of the electric field stimulation with regard to the cell length does not enable the individual depolarization of long OFF cells and short ON cells. When the stimulation must take place at multiple retinal sites simultaneously, the bipolar electrode arrays allow for higher spatial modulation of the polarization of the axon terminal than the monopolar arrays.