Eukaryotic cell locomotion depends on the propagation of self-organized reaction-diffusion waves and oscillations of actin filament assembly

Actin filament (F-actin) assembly kinetics determines the locomotion and shape of crawling eukaryotic cells, but the nature of these kinetics and their determining reactions are unclear. Live BHK21 fibroblasts, mouse melanoma cells, and Dictyostelium amoebae, locomoting on glass and expressing Green Fluorescent Protein-actin fusion proteins, were examined by confocal microscopy. The cells demonstrated three-dimensional bands of F-actin, which propagated throughout the cytoplasm at rates usually ranging between 2 and 5 microm/min in each cell type and produced lamellipodia or pseudopodia at the cell boundary. F-actin's dynamic behavior and supramolecular spatial patterns resembled in detail self-organized chemical waves in dissipative, physico-chemical systems. On this basis, the present observations provide the first evidence of self-organized, and probably autocatalytic, chemical reaction-diffusion waves of reversible actin filament assembly in vertebrate cells and a comprehensive record of wave and locomotory dynamics in vegetative-stage Dictyostelium cells. The intensity and frequency of F-actin wavefronts determine locomotory cell projections and the rotating oscillatory waves, which structure the cell surface. F-actin assembly waves thus provide a fundamental, deterministic, and nonlinear mechanism of cell locomotion and shape, which complements mechanisms based exclusively on stochastic molecular reaction kinetics.     

Architectural dynamics of F-actin in eupodia suggests their role in invasive locomotion in Dictyostelium.

Eupodia are F-actin-containing cortical structures similar to vertebrate podosomes or invadopodia found in metastatic cells. Eupodia are rich in alpha-actinin and myosin IB/D, but not a Dictyostelium homologue of talin. In the present study, we localized other actin-binding proteins, ABP120, cofilin, coronin, and fimbrin, in the eupodia and examined the three-dimensional organization of their F-actin system by confocal microscopy and transmission electron microscopy. To examine their function, we analyzed the assembly and disassembly dynamics of the F-actin system in eupodia and its relation to lamellipodial protrusion. Actin dynamics was examined by monitoring S65T-GFP-coronin and rhodamine-actin using a real-time confocal unit and a digital microscope system. Fluorescence morphometric analysis demonstrates the presence of a precise spatiotemporal coupling between F-actin assembly in eupodia and lamellipodial protrusion. When a lamellipodium advances to invade a tight space, additional rows of eupodia are sequentially formed at the base of that lamellipodium. These results indicate that mechanical stress at the leading edge modulates the structural integrity of actin and its binding proteins, such that eupodia are formed when anchorage is needed to boost for invasive protrusion of the leading edge.     

Filamin-regulated F-actin assembly is essential for morphogenesis and controls phototaxis in Dictyostelium

Dictyostelium strains lacking the F-actin cross-linking protein filamin (ddFLN) have a severe phototaxis defect at the multicellular slug stage. Filamins are rod-shaped homodimers that cross-link the actin cytoskeleton into highly viscous, orthogonal networks. Each monomer chain of filamin is comprised of an F-actin-binding domain and a rod domain. In rescue experiments only intact filamin re-established correct phototaxis in filamin minus mutants, whereas C-terminally truncated filamin proteins that had lost the dimerization domain and molecules lacking internal repeats but retaining the dimerization domain did not rescue the phototaxis defect. Deletion of individual rod repeats also changed their subcellular localization, and mutant filamins in general were less enriched at the cell cortex as compared with the full-length protein and were increasingly present in the cytoplasm. For correct phototaxis ddFLN is only required at the tip of the slug because expression under control of the cell type-specific extracellular-matrix protein A (ecmA) promoter and mixing experiments with wild type cells supported phototactic orientation. Likewise, in chimeric slugs wild type cells were primarily found at the tip of the slug, which acts as an organizer in Dictyostelium morphogenesis.     

Subunits of the chaperonin CCT interact with F-actin and influence cell shape and cytoskeletal assembly

The integrity of the cytoskeleton is closely linked to the oligomeric chaperonin containing TCP-1 (CCT) via the folding requirements of actin and tubulin, but the role of CCT in cytoskeletal organization remains unclear. We address this issue by analyzing the effects of targeting CCT subunits via siRNA and assessing their location/assembly state in cultured mammalian cells. Reducing levels of individual CCT subunits implicates CCT? in influencing cell shape and reduced levels of this subunit limit the cells' ability to recover from microfilament depolymerization. Conversely, cells displayed enhanced microtubule regrowth when CCT subunit levels were altered by siRNA. Some CCT subunits co-localize with F-actin, whilst all are predominantly monomeric in extracts enriched for the cytoskeleton. This provides compelling evidence that some CCT subunits as monomers can influence cytoskeletal organization/polymerization. Therefore the activity of CCT may well extend beyond the folding of newly synthesized polypeptides, representing a novel function for CCT subunits distinct from their role in the CCT oligomer.     

Oscillations in cell shape and size during locomotion and in contractile activities of Physarum polycephalum, Dictyostelium discoideum, Amoeba proteus and macrophages

Changes in cell shape and size were measured during locomotion, together with the motive force of the protoplasmic streaming, in various amoeboid cells in different stages of their life cycle, and under various environmental conditions. The variations in these measurements with time were examined by Fourier spectral analysis. Notwithstanding a change in cell type in the life cycle of P. polycephalum, myxamoebae and tiny plasmodia showed a similar time pattern of locomotion, exhibiting oscillations having a mixture of several periods. A regular oscillation with protoplasmic streaming appeared in the plasmodium only above a critical cell size. D. discoideum amoebae oscillated with two periods of a few minutes in preaggregation stage, but with a period of 10 min in aggregation stage, the latter being induced by cAMP. Macrophages and A. proteus also oscillated with periods of a few minutes. Periods of all these oscillations were prolonged severalfold by respiratory inhibition with NaCN, but were unaffected by glycolytic inhibition with 2-deoxyglucose. Cell fragments of A. proteus containing fewer granules oscillated more slowly and with a larger amplitude than those containing more granules. Among the granules, the nucleus was excluded as a possible modifier of the oscillation. The oscillation in Physarum plasmodium was reversibly suppressed by combining respiratory and ATPase inhibitions in mitochondria with NaCN and oligomycin, intracellular ATP concentration being kept at an appropriate level. The present results show that amoeboid motility, as well as cell shape, is oscillatory and that mitochondria are involved in time keeping.     

Control of cell shape and locomotion by external calcium

Dependence of locomotion of Xenopus laevis epidermal cells on calcium influx from the external medium was investigated. Inhibition of Ca2+ influx by 2 mM La3+ or 4 mM Tb3+ in the culture medium causes an immediate stop to locomotion and a loss of motion at the outer margin of the lamella; microcolliculi disappear and the entire lamella becomes flat and very thin. The cell body region enlarges by spreading into the lamella to an extent approximately coincident with the distribution of myosin. The increase in thickness of this area is the result. The cytoskeletal elements actin, alpha-actinin and myosin become homogeneously distributed throughout the cell and a great number of straight microtubules extend to the margin after 20 min in La3+-containing media. Prekeratin distribution does not change. Reduction of calcium concentration in the external medium by EGTA leads to cessation of cell locomotion. Sr2+ (1-4 mM) is also able to replace calcium for triggering locomotion. These findings point to a control of Ca2+-activated contractions of actomyosin by influx of external Ca2+. According to our model of cell locomotion [14] the contractions generate a hydrostatic pressure extending the lamella by flow of hyaloplasm towards the margin. Small swellings (microcolliculi) appearing thereby will be dislocated by a calcium-dependent sol-gel transformation in this area, which contains actin but not myosin.     

Theta oscillations as a substrate for medial prefrontal-hippocampal assembly interactions

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Correlation among endothelial cell shape, F-actin arrangement, and prostacyclin synthesis

Though many factors have been identified which modulate prostacyclin (PGI2) synthesis, there is little information on cellular mechanisms whereby endothelial cells (EC) regulate their basal eicosanoid metabolism. Using substrates of various adhesive capacities, bovine and porcine aortic EC shape and cytoskeletal F-actin arrangement could be modulated. Staining with rhodamine-phalloidin (R-P) permitted analysis of F-actin arrangement, while differences in cell shape were determined by measurement of cell perimeter surface area (CPSA). Spectrophotoflurometric measurements were used to quantitate the R-P binding capacity of the cultures. Cultures of reduced CPSA (225.2 +/- 13.5 mu2) generated the highest levels of basal PGl2 (6.14 +/- 0.51 pg/ug cell protein); had a diffuse arrangement of F-actin and an increased binding capacity for R-P (463.55 +/- 50.58 nmoles/ug cell protein). Cultures of enlarged CPSA (1399.3 +/- 148.3 mu2), with many actin cables and a significantly reduced (p less than 0.001) R-P binding capacity (74.941 +/- 11.79 nmoles/ug of cell protein) produced significantly smaller (p less than 0.001) basal quantities of PGl2 (1.33 +/- 0.14 pg/ug cell protein). Similarly, arachidonic acid stimulation of cultures of reduced CPSA resulted in an increased synthesis of PGl2 when compared to stimulated cultures of enlarged cells. These findings suggest a role for cell shape and the cytoskeleton in the mechanism controlling PGl2 production and indicate that alteration of the arrangement of F-actin may be of importance in regulation of EC eicosanoid metabolism.     

A quantitative method for the analysis of cell shape and locomotion

Summary A rapid, semiautomated system to quantitate and analyze leukocyte shape and locomotion was developed. Video images of moving leukocytes were obtained using a Vidicon camera mounted on a Nikon phase microscope. The video signal was either inputted directly, or indirectly via a video cassette recorder, to a Datacube video analog-digital, digital-analog converter. A Digital Equipment Corporation LSI 11/23 computer using the RT-11/TSX-Plus operating system and computer programs written in FORTRAN and MARCO assembly language permitted image segmentation, image display, and calculation of position, speed, direction of movement and orientation of each leukocyte at 10 s intervals. These data were stored on a winchester disk for subsequent evaluation of the leukocyte orientation, speed and direction of movement using statistical and graphical methods. The reproducibility of measurements made with the video system was tested by comparison with manual measurements; a correlation coefficient of 0.998 was obtained for the two methods. Rates of chemokinesis were then determined for unstimulated and chemokinetically stimulated polymorphonuclear leukocytes (PMNs) and found to average 12.8 m/min and 18.1 m/min, respectively. The high speed, ease of data analysis, and potential for multiparameter evaluation makes this system useful for directly evaluating leukocyte locomotion.In honour of Prof. P. van Duijn     

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. Actin's properties of oscillation and self-organization might also fundamentally determine the nature of the eukaryotic cell's reactions of adaptation, timing and signal response.     

A quantitative method for the analysis of cell shape and locomotion

A rapid, semiautomated system to quantitate and analyze leukocyte shape and locomotion was developed. Video images of moving leukocytes were obtained using a Vidicon camera mounted on a Nikon phase microscope. The video signal was either inputted directly, or indirectly via a video cassette recorder, to a Datacube video analog-digital, digital-analog converter. A Digital Equipment Corporation LSI 11/23 computer using the RT-11/TSX-Plus operating system and computer programs written in FORTRAN and MARCO assembly language permitted image segmentation, image display, and calculation of position, speed, direction of movement and orientation of each leukocyte at 10 s intervals. These data were stored on a winchester disk for subsequent evaluation of the leukocyte orientation, speed and direction of movement using statistical and graphical methods. The reproducibility of measurements made with the video system was tested by comparison with manual measurements; a correlation coefficient of 0.998 was obtained for the two methods. Rates of chemokinesis were then determined for unstimulated and chemokinetically stimulated polymorphonuclear leukocytes (PMNs) and found to average 12.8 micron/min and 18.1 micron/min, respectively. The high speed, ease of data analysis, and potential for multiparameter evaluation makes this system useful for directly evaluating leukocyte locomotion.     

Cortical actomyosin breakage triggers shape oscillations in cells and cell fragments

Cell shape and movements rely on complex biochemical pathways that regulate actin, microtubules, and substrate adhesions. Some of these pathways act through altering the cortex contractility. Here we examined cellular systems where contractility is enhanced by disassembly of the microtubules. We found that adherent cells, when detached from their substrate, developed a membrane bulge devoid of detectable actin and myosin. A constriction ring at the base of the bulge oscillated from one side of the cell to the other. The movement was accompanied by sequential redistribution of actin and myosin to the membrane. We observed this oscillatory behavior also in cell fragments of various sizes, providing a simplified, nucleus-free system for biophysical studies. Our observations suggest a mechanism based on active gel dynamics and inspired by symmetry breaking of actin gels growing around beads. The proposed mechanism for breakage of the actomyosin cortex may be used for cell polarization.     

Molecular genetics of cell migration: Dictyostelium as a model system

A central unresolved issue in modern cell biology concerns how eukaryotic cell migration is achieved. Although the underlying mechanics of cell locomotion appear similar in cells ranging from amoebae to leukocytes, the organisms that have been historically studied have not been amenable to the techniques of modern molecular genetics. The recent development of high-efficiency gene targeting technology for Dictyostelium discoideum, coupled with the classic cell migration behavior of this organism, offers an opportunity to resolve many of the controversial issues concerning cell locomotion.     

Pelvic girdle shape predicts locomotion and phylogeny in batoids

In terrestrial vertebrates, the pelvic girdle can reliably predict locomotor mode. Because of the diminished gravitational effects on positively buoyant bony fish, the same relationship does not appear to exist. However, within the negatively buoyant elasmobranch fishes, benthic batoids employ pelvic fin bottom‐walking and punting as primary or supplementary forms of locomotion. Therefore, in this study, we employed geometric and linear morphometrics to investigate if their pelvic girdles exhibit shape characteristics similar to those of sprawling terrestrial vertebrates. We tested for correlates of pelvic girdle shape with 1) Order, 2) Family, 3) Swim Mode, and/or 4) Punt Mode. Landmarks and semilandmarks were placed along outlines of dorsal views of 61 batoid pelvic girdles (3/3 orders, 10/13 families, 35/72 genera). The first three relative warps explained 88.45% of the variation among individuals (P < 0.01%). Only Order and Punt Mode contained groups that were all significantly different from each other (P < 0.01%). Discriminant function analyses indicated that the majority of variation within each category was due to differences in extension of lateral and prepelvic processes and puboischiac bar angle. Over 60% of the original specimens and 55% of the cross‐validated specimens were correctly classified. The neutral angle of the propterygium, which articulates with the pelvic girdle, was significantly different among punt modes, whereas only pectoral fin oscillators had differently shaped pelvic girdles when compared with batoids that perform other swimming modes (P < 0.01). Pelvic girdles of batoids vary greatly, and therefore, likely function in ways not previously described in teleost fishes. This study illustrates that pelvic girdle shape is a good predictor of punt mode, some forms of swimming mode, and a species' Order. Such correlation between locomotor style and pelvic girdle shape provides evidence for the convergent evolution of morphological features that support both sprawled‐gait terrestrial walking and aquatic bottom‐walking. J. Morphol. 275:100–110, 2014. © 2013 Wiley Periodicals, Inc.     

Biochemical characterization of a Dictyostelium myosin II heavy-chain phosphatase that promotes filament assembly.

In Dictyostelium cells, myosin II is found as cytosolic nonassembled monomers and cytoskeletal bipolar filaments. It is thought that the phosphorylation state of three threonine residues in the tail of myosin II heavy chain regulates the molecular motor's assembly state and localization. Phosphorylation of the myosin heavy chain at threonine residues 1823, 1833 and 2029 is responsible for maintaining myosin in the nonassembled state, and subsequent dephosphorylation of these residues is a prerequisite for assembly into the cytoskeleton. We report here the characterization of myosin heavy-chain phosphatase activities in Dictyostelium utilizing myosin II phosphorylated by myosin heavy-chain kinase A as a substrate. One of the myosin heavy-chain phosphatase activities was identified as protein phosphatase 2A and the purified holoenzyme was composed of a 37-kDa catalytic subunit, a 65-kDa A subunit and a 55-kDa B subunit. The protein phosphatase 2A holoenzyme displays two orders of magnitude higher activity towards myosin phosphorylated on the heavy chains than it does towards myosin phosphorylated on the regulatory light chains, consistent with a role in the control of filament assembly. The purified myosin heavy-chain phosphatase activity promotes bipolar filament assembly in vitro via dephosphorylation of the myosin heavy chain. This system should provide a valuable model for studying the regulation and localization of protein phosphatase 2A in the context of cytoskeletal reorganization.     

Wave-pinning and cell polarity from a bistable reaction-diffusion system

Motile eukaryotic cells polarize in response to external signals. Numerous mechanisms have been suggested to account for this symmetry breaking and for the ensuing robust polarization. Implicated in this process are various proteins that are recruited to the plasma membrane and segregate at an emergent front or back of the polarizing cell. Among these are PI3K, PTEN, and members of the Rho family GTPases such as Cdc42, Rac, and Rho. Many such proteins, including the Rho GTPases, cycle between active membrane-bound forms and inactive cytosolic forms. In previous work, we have shown that this property, together with appropriate crosstalk, endows a biochemical circuit (Cdc42, Rac, and Rho) with the property of inherent polarizability. Here we show that this property is present in an even simpler system comprised of a single active/inactive protein pair with positive feedback to its own activation. The simplicity of this minimal system also allows us to explain the mechanism using insights from mathematical analysis. The basic idea resides in a well-known property of reaction-diffusion systems with bistable kinetics, namely, propagation of fronts. However, it crucially depends on exchange between active and inactive forms of the chemicals with unequal rates of diffusion, and overall conservation to pin the waves into a stable polar distribution. We refer to these dynamics as wave-pinning and we show that this phenomenon is distinct from Turing-instability-generated pattern formation that occurs in reaction-diffusion systems that appear to be very similar. We explain the mathematical basis of the phenomenon, relate it to spatial segregation of Rho GTPases, and show how it can account for spatial amplification and maintenance of polarity, as well as sensitivity to new stimuli typical in polarization of eukaryotic cells.     

Respiratory burst oscillations in human neutrophils and their correlation with fluctuations in apparent cell shape

Neutrophils pretreated with phorbol 12-myristate 13-acetate (1-10 nM) and stimulated with low concentrations of chemotactic agonists (1-10nM) exhibited a marked increase in respiratory burst activity that was characterized by regular oscillations. These were accompanied by parallel oscillations in turbidity having the same phase and period. Four different agonists, f-Met-Leu-Phe, complement fragment C5a, platelet-activating factor, and leukotriene B4, induced virtually identical oscillations, with mean periods of 7.9 +/- 0.6 s (respiratory burst) and 7.9 +/- 0.8 s (turbidity) at 37 degrees C. No burst oscillations were observed at high agonist concentrations (50-100 nM) unless the fungal metabolite 17-hydroxywortmannin was added prior to stimulation. In the absence of phorbol 12-myristate 13-acetate, the respiratory burst activity was inhibited by 17-hydroxywortmannin, the protein kinase C inhibitor staurosporine, and calcium depletion, while agonist-dependent turbidity changes including the oscillations were unaffected. Turbidity changes reflect corresponding changes in cell size and/or shape, suggesting that cyclic alterations in morphology such as lamellipod extension and retraction physically affect the catalytic efficiency of the membrane-bound burst enzyme NADPH-oxidase. The oscillations appear to be controlled via receptor-dependent activation mechanisms which do not involve PKC activation or the rise in internal calcium presumably derived from phospholipase C activation.