Christian Westendorf

Cilia-based flow network in the brain ventricles.

Going with the flow The interstitial spaces of the brain are filled with cerebrospinal fluid (CSF). Faubel et al. studied fluid transport in the third ventricle of the brain of mice, rats, and pigs. Sophisticated, state-of-the-art fluid dynamic studies revealed a complex pattern of cilia beating that leads to an intricate network of “highways” of CSF flow. This flow rapidly and efficiently transports and partitions CSF. Science, this issue p. 176 A cilia-based transport network that suggests how cerebrospinal fluid constituents are actively distributed is revealed in the brain. Cerebrospinal fluid conveys many physiologically important signaling factors through the ventricular cavities of the brain. We investigated the transport of cerebrospinal fluid in the third ventricle of the mouse brain and discovered a highly organized pattern of cilia modules, which collectively give rise to a network of fluid flows that allows for precise transport within this ventricle. We also discovered a cilia-based switch that reliably and periodically alters the flow pattern so as to create a dynamic subdivision that may control substance distribution in the third ventricle. Complex flow patterns were also present in the third ventricles of rats and pigs. Our work suggests that ciliated epithelia can generate and maintain complex, spatiotemporally regulated flow networks.

Actin cytoskeleton of chemotactic amoebae operates close to the onset of oscillations

The rapid reorganization of the actin cytoskeleton in response to external stimuli is an essential property of many motile eukaryotic cells. Here, we report evidence that the actin machinery of chemotactic Dictyostelium cells operates close to an oscillatory instability. When averaging the actin response of many cells to a short pulse of the chemoattractant cAMP, we observed a transient accumulation of cortical actin reminiscent of a damped oscillation. At the single-cell level, however, the response dynamics ranged from short, strongly damped responses to slowly decaying, weakly damped oscillations. Furthermore, in a small subpopulation, we observed self-sustained oscillations in the cortical F-actin concentration. To substantiate that an oscillatory mechanism governs the actin dynamics in these cells, we systematically exposed a large number of cells to periodic pulse trains of different frequencies. Our results indicate a resonance peak at a stimulation period of around 20 s. We propose a delayed feedback model that explains our experimental findings based on a time-delay in the regulatory network of the actin system. To test the model, we performed stimulation experiments with cells that express GFP-tagged fusion proteins of Coronin and actin-interacting protein 1, as well as knockout mutants that lack Coronin and actin-interacting protein 1. These actin-binding proteins enhance the disassembly of actin filaments and thus allow us to estimate the delay time in the regulatory feedback loop. Based on this independent estimate, our model predicts an intrinsic period of 20 s, which agrees with the resonance observed in our periodic stimulation experiments.

Chemotaxis of Dictyostelium discoideum: Collective Oscillation of Cellular Contacts

Chemotactic responses of Dictyostelium discoideum cells to periodic self-generated signals of extracellular cAMP comprise a large number of intricate morphological changes on different length scales. Here, we scrutinized chemotaxis of single Dictyostelium discoideum cells under conditions of starvation using a variety of optical, electrical and acoustic methods. Amebas were seeded on gold electrodes displaying impedance oscillations that were simultaneously analyzed by optical video microscopy to relate synchronous changes in cell density, morphology, and distance from the surface to the transient impedance signal. We found that starved amebas periodically reduce their overall distance from the surface producing a larger impedance and higher total fluorescence intensity in total internal reflection fluorescence microscopy. Therefore, we propose that the dominant sources of the observed impedance oscillations observed on electric cell-substrate impedance sensing electrodes are periodic changes of the overall cell-substrate distance of a cell. These synchronous changes of the cell-electrode distance were also observed in the oscillating signal of acoustic resonators covered with amebas. We also found that periodic cell-cell aggregation into transient clusters correlates with changes in the cell-substrate distance and might also contribute to the impedance signal. It turned out that cell-cell contacts as well as cell-substrate contacts form synchronously during chemotaxis of Dictyostelium discoideum cells.

Live cell flattening — traditional and novel approaches

Eukaryotic cell flattening is valuable for improving microscopic observations, ranging from bright field (BF) to total internal reflection fluorescence (TIRF) microscopy. Fundamental processes, such as mitosis and in vivo actin polymerization, have been investigated using these techniques. Here, we review the well known agar overlayer protocol and the oil overlay method. In addition, we present more elaborate microfluidics-based techniques that provide us with a greater level of control. We demonstrate these techniques on the social amoebae Dictyostelium discoideum, comparing the advantages and disadvantages of each method. PACS Codes: 87.64.-t, 47.61.-k, 87.80.Ek

Shape oscillations of Dictyostelium discoideum cells on ultramicroelectrodes monitored by impedance analysis.

Motile cells change their shape and crawl in response to external stimuli by the formation and retraction of lamellipodia, requiring remodeling of cytoskeletal actin. [ 1 ] Some migrating cells display cyclic periodicities in their cellular shape changes such as neutrophils, [ 2 ] Dictyostelium discoideum , [ 3 ] Physarum polycephalum , fi broblasts, and Amoeba proteus . [ 4 ] The social amoebae D. discoideum proliferate as single cells but aggregate under starvation conditions into clusters of about 10 5 cells that eventually turn into a migrating “slug”, which further differentiates into a fruiting body to facilitate spore dispersal. [ 3 c, 5 ] It is assumed that cells from the aggregation center periodically produce and secrete the chemoattractant cyclic adenosine 3′,5′-monophosphate (cAMP) into the extracellular medium, where it is detected by adjacent cells via cell-surface receptors. The neighboring cells become stimulated and in turn produce and secrete more cAMP, resulting in spatiotemporal wave patterns of cAMP propagating from the aggregation center. [ 5 d, 6 ] Stimulated cells crawl in the direction of increasing cAMP concentration, producing waves. The cells are detectable by light scattering since optical density waves originate from cell shape changes that occur during the chemotactic cycle. Resting cells scatter less light than elongated moving ones, resulting in an alternating spatial pattern of bright and dark bands in dark-fi eld microscopy. [ 5 d, 6 ] So far, most of our knowledge is based on population-level optical density observations or isotope dilution assays of cAMP. [ 3 a, 5 a, 5 d, 6 b, 7 ] Individual cells or small ensembles are rarely investigated with respect to

Variability and order in cytoskeletal dynamics of motile amoeboid cells

The chemotactic motion of eukaryotic cells such as leukocytes or metastatic cancer cells relies on membrane protrusions driven by the polymerization and depolymerization of actin. Here we show that the response of the actin system to a receptor stimulus is subject to a threshold value that varies strongly from cell to cell. Above the threshold, we observe pronounced cell-to-cell variability in the response amplitude. The polymerization time, however, is almost constant over the entire range of response amplitudes, while the depolymerization time increases with increasing amplitude. We show that cell-to-cell variability in the response amplitude correlates with the amount of Arp2/3, a protein that enhances actin polymerization. A time-delayed feedback model for the cortical actin concentration is consistent with all our observations and confirms the role of Arp2/3 in the observed cell-to-cell variability. Taken together, our observations highlight robust regulation of the actin response that enables a reliable timing of cell movement.

Collective behavior of Dictyostelium discoideum monitored by impedance analysis

Dictyostelium discoideum cells respond to periodic signals of extracellular cAMP by collective changes of cell-cell and cell-substrate contacts. This was confirmed by dielectric analysis employing electric cell-substrate impedance sensing (ECIS) and impedance measurements involving cell-filled micro channels in conjunction with optical microscopy providing a comprehensive picture of chemotaxis under conditions of starvation.