Michael G. Vicker

Reaction–diffusion waves of actin filament polymerization/depolymerization in Dictyostelium pseudopodium extension and cell locomotion

Cell surface movements and the intracellular spatial patterns and dynamics of actin filament (F-actin) were investigated in living and formalin-fixed cells of Dictyostelium discoideum by confocal microscopy. Excitation waves of F-actin assembly developed and propagated several micrometers at up to 26 microm/min in cells which had been intracellularly loaded with fluorescently labeled actin monomer. Wave propagation and extinction corresponded with the initiation and attenuation of pseudopodium extension and cell advance, respectively. The identification of chemical waves was supported by the ring, sphere, spiral and scroll wave patterns, which were observed in the extensions of fixed cells stained with phalloidin-rhodamine, and by the similar, asymmetrical [F-actin] distribution in wavefronts in living and fixed cells. These F-actin patterns and dynamics in Dictyostelium provide evidence for a new supramolecular state of actin, which propagates as a self-organized, reaction-diffusion wave of reversible F-actin assembly and affects pseudopodium extension. Actins properties of oscillation and self-organization might also fundamentally determine the nature of the eukaryotic cells reactions of adaptation, timing and signal response.

F-actin assembly in Dictyostelium cell locomotion and shape oscillations propagates as a self-organized reaction–diffusion wave

The crawling locomotion and shape of eukaryotic cells have been associated with the stochastic molecular dynamics of actin and its protein regulators, chiefly Arp2/3 and Rho family GTPases, in making a cytoskeleton meshwork within cell extensions. However, the cells actin‐dependent oscillatory shape and extension dynamics may also yield insights into locomotory mechanisms. Confocal observations of live Dictyostelium cells, expressing a green fluorescent protein–actin fusion protein, demonstrate oscillating supramolecular patterns of filamentous actin throughout the cell, which generate pseudopodia at the cell edge. The distinctively dissipative spatio‐temporal behavior of these structures provides strong evidence that reversible actin filament assembly propagates as a self‐organized, chemical reaction–diffusion wave.

Cell movement and shape are non-random and determined by intracellular, oscillatory rotating waves in Dictyostelium amoebae

We present evidence for a mechanism of eukaryotic cell movement. The pseudopodial dynamics and shape of Dictyostelium discoideum amoebae were investigated using computer-supported video microscopy. An examination of the cell periphery by the novel method of serial circular maps revealed explicit, classical wave patterns, which indicate the existence of intrinsic intracellular oscillations. The patterns are generated by the transit of self-organized, super-positioned, harmonic modes of rotating oscillatory waves (ROWS). These waves are probably associated with the dynamics of intracellular actin polymerisation and depolymerisation. A Karhunen-Loève expansion was conducted on one cell during 10 min of locomotion using points each 10 degrees around the cells boundary. The results show that only 2-3 modes are necessary to describe the most essential features of cell movement and shape. Based on this analysis, a wave model was developed, which accurately simulates the dynamics of cell movement and shape during this time. The model was tested by reconstructing the cells dynamical form by means of the Karhunen-Loève transform. No difference was detected between this reconstruction and the actual cell outline. Although cell movement and shape have hitherto been viewed as random, our results demonstrate that ROWS determine the spatio-temporal expression of pseudopodia, and consequently govern cell shape and movement, non-randomly.

Ideal and non-ideal concentration gradient propagation in chemotaxis studies

Abstract The shape and spatio-temporal development of a concentration gradient depend on the method and medium of its generation. Model gradients of low molecular weight (MW) dyes develop with predictable characteristics in 1% agarose. In contrast, any method of delivering dyes into free solution, e.g., via capillary pipets, produces anomalous gradients, unless the chambers are extremely shallow (less than 1 mm deep). Thus, most methods of chemo-attractant delivery which are popular in chemotaxis studies are incapable of propagating gradients by molecular diffusion; rather, they tend to be dominated by the effects of wholesale flow of the chemo-attractant and convection and turbulence of the free solution. This may have serious consequences for the interpretation of some chemotaxis experiments, since several gradient qualities of chemo-attractants (and probably morphogens) could influence cell behaviour. The possible effects on cells of the motion and development of gradients are also discussed.

Case study: visualization of laser confocal microscopy datasets

The paper presents an example of how existing visualization methods can be successfully applied-after minor modifications-for allowing new, sometimes unexpected insight into scientific questions, in this case for better understanding of unknown, microscopic biological structures. The authors present a volume rendering system supporting the visualization of LCM datasets, a new microscopic tomographic method allowing for the first time accurate and fast in-vivo inspection of the spatial structure of microscopic structures, especially important in (but not restricted to) biology. The speed, flexibility and versatility of the system allows fast, convenient, interactive operation with large datasets on small computers (workstation or PC). By testing different datasets, they have been able to significantly improve the performance of understanding the internal structure of LCM data. Most important, they have been able to show static and dynamic structures of cells never seen before and allowing significant insight in the cell movement process. Therefore they regard the system as a universal tool for the visualization of such data.

Pseudopodium extension and amoeboid locomotion in Dictyostelium discoideum : possible autowave behaviour of F-actin

Abstract Supramolecular patterns of filamentous (F-)actin up to several micrometres across were visualized within projections of locomotory amoebae after cell fixation and staining with phalloidin-rhodamine. The patterns included rings, single and double spirals, some apparently colliding and disintegrating. Cell stimulation with a pulse of the chemoattractant cyclic AMP induced damping oscillations in F-actin ring frequency with a period of 6–7 s. Ring front propagation after stimulation was modelled by Markov and Fourier methods at 3.1–17.5 μm/min, similar to actual cell speed. We argue that the dynamics and detailed morphological correspondence of these F-actin structures to wave patterns in chemical reaction-diffusion systems strongly supports the interpretation that Dictyostelium cytoplasm behaves as an unstable, excitable medium enabling the propagation of self-organized, physico-chemical relaxation oscillations, i.e. autowaves, of reversible F-actin assembly or aggregation — a new state of actin - fundamental to pseudopodium extension, cell locomotion, chemotaxis and other cell functions.

Dual chemotaxis signalling regulates Dictyostelium development : Intercellular cyclic AMP pulses and intracellular F-actin disassembly waves induce each other

Aggregating Dictyostelium discoideum amoebae periodically emit and relay cAMP, which regulates their chemotaxis and morphogenesis into a multicellular, differentiated organism. Cyclic AMP also stimulates F-actin assembly and chemotactic pseudopodium extension. We used actin-GFP expression to visualise for the first time intracellular F-actin assembly as a spatio-temporal indicator of cell reactions to cAMP, and thus the kinematics of cell communication, in aggregating streams. Every natural cAMP signal pulse induces an autowave of F-actin disassembly, which propagates from each cells leading end to its trailing end at a linear rate, much slower than the calculated and measured velocities of cAMP diffusion in aggregating Dictyostelium. A sequence of transient reactions follows behind the wave, including anterior F-actin assembly, chemotactic pseudopodium extension and cell advance at the cell front and, at the back, F-actin assembly, extension of a small retrograde pseudopodium (forcing a brief cell retreat) and chemotactic stimulation of the following cell, yielding a 20s cAMP relay delay. These dynamics indicate that stream cell behaviour is mediated by a dual signalling system: a short-range cAMP pulse directed from one cell tail to an immediately following cell front and a slower, long-range wave of intracellular F-actin disassembly, each inducing the other.

SIGNALS FOR CHEMOTAXIS AND CHEMOKINESIS IN CELLS OF DICTYOSTELIUM DISCOIDEUM

Tactic behaviour and its external signal requirements were examined in two ways. 1) The development of a stimulus spatial gradient was simulated in order to predict how cells might behave if able to read either the spatial concentration or relative spatial gradients. 2) Cells of Dictyostelium discoi-deum were exposed to two types of cyclic AMP signal fields while migrating in a micropore filter: a directed pulse lasting 40 s or a stable spatial gradient (which developed from an initially isotropic concentration after removal of the cAMP from one side of the filter). The cell population shifted up-field after a pulse, but down-field in a spatial gradient with no pulse. The optimum for cell motility is 30–50 nM cAMP in a gradient, but ≤ 1 nM as a pulse. The results demonstrate that taxis requires a directed, temporal stimulus and is not induced by cAMP spatial gradients.

Mathematical Analysis of Cell Shape

Cell motility involves translocation of the cell’s centroid as well as changes or distortions in the cell’s shape. Clues about the mechanism of cell movement may be obtained from information about its shape changes in time. The changes occur in multiple dimensions and can be highly periodic, however they may elude superficial observation. The techniques outlined in this contribution might help to reveal otherwise undetectable periodic shape changes.

The Biological Mechanism of Low Dose Ionizing Radiation: Induction of Inflammatory Reactions in Human Blood

Niedrige Dosen ionisierender γ-Strahlung von 137-Casium wirken im menschlichen Blut als unphysiologisches Stimulanz von Entzundungsreaktionen. Strahlung erhoht die Aktivierung des “oxidative burst” nach Behandlung des Blutes mit Aktivatoren, wie Ca2+ Ionophor A23187 oder Phorbolester. Diese Reaktion wird durch Amplifizierung der zellularen Chemilumineszenz mit Luminol gemessen. Die erhohte Lumineszenz kann einige Minuten nach der Bestrahlung beobachtet werden, dauert mindestens 1 h, ist durch die interzellulare Verbreitung eines Vermittlermolekuls gekennzeichnet und ist γ-dosisabhangig (5–50 μSv, in vitro). Eine ahnliche Reaktion konnte bei Patienten nach einer routinemasigen Rontgenaufnahme der Lunge festgestellt werden. Die Reaktion ist gehemmt durch EGTA und Adenosin (zwei Zeichen ihrer Ca2+-Abhangigkeit) und durch den Phospho-lipase A2-Blocker p-Bromphenacylbromid. Der Cyclo-oxygenase-Blocker Aspirin ist dagegen nur teilweise effektiv. Diese Reaktionen auf niedrige Dosen implizieren die Wirkung von zweiten Boten-Systemen, insbesondere Metaboliten von Arachidonsaure und sind von molekularer „Beschadigung- & Reparatur-“ Reaktionen unabhangig. Die Ergebnisse zeigen einen Mechanismus der niedrigen Strahlendosen mit Konsequenzen fur die Homoeostasis von Entzundungs- und zweiten Boten-Reaktionen.