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.     

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.     

Bleb-driven chemotaxis of Dictyostelium cells

Blebs and F-actin–driven pseudopods are alternative ways of extending the leading edge of migrating cells. We show that Dictyostelium cells switch from using predominantly pseudopods to blebs when migrating under agarose overlays of increasing stiffness. Blebs expand faster than pseudopods leaving behind F-actin scars, but are less persistent. Blebbing cells are strongly chemotactic to cyclic-AMP, producing nearly all of their blebs up-gradient. When cells re-orientate to a needle releasing cyclic-AMP, they stereotypically produce first microspikes, then blebs and pseudopods only later. Genetically, blebbing requires myosin-II and increases when actin polymerization or cortical function is impaired. Cyclic-AMP induces transient blebbing independently of much of the known chemotactic signal transduction machinery, but involving PI3-kinase and downstream PH domain proteins, CRAC and PhdA. Impairment of this PI3-kinase pathway results in slow movement under agarose and cells that produce few blebs, though actin polymerization appears unaffected. We propose that mechanical resistance induces bleb-driven movement in Dictyostelium, which is chemotactic and controlled through PI3-kinase.     

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 cell's 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.     

Protein phosphatases in developing Dictyostelium discoideum cells

Protein phosphatase activities in developing Dictyostelium discoideum cells were investigated. Substrates were prepared by phosphorylation of histone H2b and kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) using cAMP-dependent protein kinase. Two histone phosphatase activities (M_r 170 000 and 520 000) and one kemptide phosphatase activity (M_r 230 000) were found in the cytosolic cell fraction. Histone phosphatase was also present in the particulate fraction, kemptide phosphatase was not. All phosphatase activities were present throughout development. No differences in protein phosphatase activities were found in prespore and prestalk cells. A heat-stable factor which inhibits the particulate and both soluble histone phosphatase activities was isolated.     

Location of actin, myosin, and microtubular structures during directed locomotion of Dictyostelium amebae

During their life cycle, amebae of the cellular slime mould Dictyostelium discoideum aggregate to form multicellular structures in which differentiation takes place. Aggregation depends upon the release of chemotactic signals of 3',5'-cAMP from aggregation centers. In response to the signals, aggregating amebae elongate, actively more toward the attractive source, and may be easily identified from the other cells because of their polarized appearance. To examine the role of cytoskeletal components during ameboid locomotion, immunofluorescence microscopy with antibodies to actin, myosin, and to a microtubule-associated component was used. In addition, rhodamine-labeled phallotoxin was employed. Actin and myosin display a rather uniform distribution in rounded unstretched cells. In polarized locomoting cells, actin fluorescence (due to both labeled phallotoxin and specific antibody) is prevalently concentrated in the anterior pseudopod while myosin fluorescence appears to be excluded from the pseudopod. Similarly, microtubules in locomoting cells are excluded from the leading pseudopod. The cell nucleus is attached to the microtubule network by way of a nucleus-associated organelle serving as a microtubule-organizing center and seems to be maintained in a rather fixed position by the microtubules. These findings, together with available morphological and biochemical evidences, are consistent with a mechanism in which polymerized actin is moved into the pseudopod through its interaction with myosin at the base of the pseudopod. Microtubules, apparently, do not actively participate in movement but seem to behave as anchorage structures for the nucleus and possibly other cytoplasmic organelles.     

Group locomotion of PtK1 cells

PtK1 cells were found to form groups of variable size which locomoted in unison. A continuous spectrum of net displacements during two days of observation ranged from no displacement to about 150 μm/day. The majority of single cells migrated less than 50 μm/day. The phenomenon demonstrates in tissue culture the existence of a form of cellular migration which appears intermediary between single cell locomotion and the deformations of extended sheets of cells [8] in the sense that more than one single cell but less than an entire sheet displace themselves. It also suggests coordination of locomotion between individual cells.     

Guanylate cyclase activity in permeabilized Dictyostelium discoideum cells

Dictyostelium discoideum cells respond to chemoattractants by transient activation of guanylate cyclase. Cyclic GMP is a second messenger that transduces the chemotactic signal. We used an electropermeabilized cell system to investigate the regulation of guanylate cyclase. Enzyme activity in permeabilized cells was dependent on the presence of a nonhydrolysable GTP analogue (e.g., GTPγS), which could not be replaced by GTP, GDP, or GMP. After the initiation of the guanylate cyclase reaction in permeabilized cells only a short burst of activity is observed, because the enzyme is inactivated with a t_1.2 of about 15 s. We show that inactivation is not due to lack of substrate, resealing of the pores in the cell membrane, product inhibition by cGMP, or intrinsic instability of the enzyme. Physiological concentrations of Ca²⁺ ions inhibited the enzyme (half-maximal effect at 0.3 μM), whereas InsP₃ had no effect. Once inactivated, the enzyme could only be reactivated after homogenization of the permeabilized cells and removal of the soluble cell fraction. This suggests that a soluble factor is involved in an autonomous process that inactivates guanylate cyclase and is triggered only after the enzyme is activated. The initial rate of guanylate cyclase activity in permeabilized cells is similar to that in intact, chemotactically activated cells. Moreover, the rate of inactivation of the enzyme in permeabilized cells and that due to adaptation in vivo are about equal. This suggests that the activation and inactivation of guanylate cyclase observed in this permeabilized cell system is related to that of chemotactic activation and adaptation in intact cells. © 1996 Wiley-Liss, Inc.     

Blebbing of Dictyostelium cells in response to chemoattractant

Stimulation of Dictyostelium cells with a high uniform concentration of the chemoattractant cyclic-AMP induces a series of morphological changes, including cell rounding and subsequent extension of pseudopodia in random directions. Here we report that cyclic-AMP also elicits blebs and analyse their mechanism of formation. The surface area and volume of cells remain constant during blebbing indicating that blebs form by the redistribution of cytoplasm and plasma membrane rather than the exocytosis of internal membrane coupled to a swelling of the cell. Blebbing occurs immediately after a rapid rise and fall in submembraneous F-actin, but the blebs themselves contain little F-actin as they expand. A mutant with a partially inactivated Arp2/3 complex has a greatly reduced rise in F-actin content, yet shows a large increase in blebbing. This suggests that bleb formation is not enhanced by the preceding actin dynamics, but is actually inhibited by them. In contrast, cells that lack myosin-II completely fail to bleb. We conclude that bleb expansion is likely to be driven by hydrostatic pressure produced by cortical contraction involving myosin-II. As blebs are induced by chemoattractant, we speculate that hydrostatic pressure is one of the forces driving pseudopod extension during movement up a gradient of cyclic-AMP.     

Fluid-phase uptake and transit in axenic Dictyostelium cells

The main route for fluid-phase uptake in Dictyostelium is macropinocytosis, a process powered by the actin cytoskeleton. Nutrients within the endocytosed fluid are digested and resorbed, disposal of remnants follows by exocytosis. Along the endocytic pathway, membrane fusion and fission events take place at multiple steps. The regulator and effector molecules involved in uptake and transit are largely conserved between higher and lower eukaryotes. This feature, together with its accessibility by molecular genetics, recommend Dictyostelium as a valuable model system for mammalian cells.     

Differentiation of Dictyostelium discoideum cells in suspension culture

Differentiation of Dictyostelium discoideum cells in suspension culture is reported, using a medium containing glucose, albumin, cyclic AMP, EDTA and streptomycin in a phosphate buffer. Production of UDPgalactose:polysaccharide transferase, an enzyme specifically present in prespore cells, and the formation of prespore-specific antigens in more than 60% of the cells, are demonstrated. Differentiation in this medium differs from that previously reported with other suspension systems in that (a) cells form only small, amorphous agglomerates, (b) there is an absolute requirement for cyclic AMP and (c) prior formation of loose cell mounds on a solid substratum is essential for subsequent differentiation in this medium. This last requirement indicates that the differentiation process, giving rise to the prespore-specific enzyme and antigen, can be resolved into two distinct stages, one requiring cell contact on a solid substratum and the other proceeding in small agglomerates incubated in the medium. This medium may be useful for elucidating the role of cyclic AMP and cell contact in slime mould development.     

Reduction of nucleolar size in reaggregated Dictyostelium cells

The ultra-structure of the nucleolus in Dictyostelium discoideum cells was studied by electron microscopy. Large nucleoli on the periphery of the nucleus in cells of the multi-cellular pseudoplasmodium (slug) were maintained during long migration. Disaggregation of the slug cells induced a reduction in the size of the large nucleoli. The size of the reduced nucleoli in the reaggregated cells were maintained during the long migration and culmination of reconstructed slug. The electron density of the cytoplasm clearly distinguishes the prespore from the prestalk region, and it takes about 6 h for the complete recovery of cell-to-cell contact after reaggregation.     

Input-output relationship in galvanotactic response of Dictyostelium cells

Under a direct current electric field, Dictyostelium cells exhibit migration towards the cathode. To determine the input-output relationship of the cell's galvanotactic response, we developed an experimental instrument in which electric signals applied to the cells are highly reproducible and the motile response are analyzed quantitatively. With no electric field, the cells moved randomly in all directions. Upon applying an electric field, cell migration speeds became about 1.3 times faster than those in the absence of an electric field. Such kinetic effects of electric fields on the migration were observed for cells stimulated between 0.25 and 10 V/cm of the field strength. The directions of cell migrations were biased toward the cathode in a positive manner with field strength, showing galvanotactic response in a dose-dependent manner. Quantitative analysis of the relationship between field strengths and directional movements revealed that the biased movements of the cells depend on the square of electric field strength, which can be described by one simple phenomenological equation. The threshold strength for the galvanotaxis was between 0.25 and 1 V/cm. Galvanotactic efficiency reached to half-maximum at 2.6 V/cm, which corresponds to an approximate 8 mV voltage difference between the cathode and anode direction of 10 microm wide, round cells. Based on these results, possible mechanisms of galvanotaxis in Dictyostelium cells were discussed. This development of experimental system, together with its good microscopic accessibility for intracellular signaling molecules, makes Dictyostelium cells attractive as a model organism for elucidating stochastic processes in the signaling systems responsible for cell motility and its regulations.     

Ruffling and locomotion in Rana pipiens gastrula cells.

Embryonic amphibian cells move during gastrulation, even though they are in contact with many neighboring cells. The behavior of these cells in vitro with respect to cell movement and contact inhibition is thus of interest. Cultures of isolated presumptive mesodermal cells of early Rana pipiens gastrulae were sealed with a coverslip and filmed under phase contrast at 16 frames/min. At the end of 30 min in vitro, cells settle to the substratum and form fan-like lamellipodia which are sites of cell attachment. Ruffling is qualitatively similar to that seen in many chick and mammalian cell types in vitro. Ruffles lift up and move back from marginal extensions of cells. When lamellipodia are symmetrically arranged around the cell periphery, no net translocation of the cell occurs. In contrast, when cells have a dominant lamellipodium (larger and/or more active), movement occurs in that direction. Cells may exhibit complex margins composed of microspikes, ruffles, and hyaline extensions of the cell draped between microspikes. When cells come in contact there is a local paralysis of ruffling. When cells lose contact, a broad ruffling lamellipodium often appears immediately at the former sites of contact.     

Novel cellular tracks of migrating Dictyostelium cells

After Dictyostelium cells were settled on a coverslip and allowed to migrate freely on the surface, they were stained with fluorescently labeled Concanavalin A. Tracks with distinct patterns that consist of dots and short fibers were observed behind the cells. In this study, we refer to these tracks as "cellular tracks", CTs for short. We characterized the biological effect of CTs on cell behavior and development. CTs decreased the strength of cell-substratum adhesion, increased the velocity of cell migration, but did not affect growth of cells. CTs also promoted cell aggregation. When pre-aggregation cells touched the CTs of other cells, they avoided or orthogonally crossed them, but did not migrate along them. These observations suggest that the CTs of pre-aggregation cells prompts cells to disperse uniformly on substratum and may enable cells to sense cell density. On the other hand, when aggregation-competent cells touched the CTs of other aggregation-competent cells, a half of them migrated along the CTs. Pre-aggregation cells did not migrate along the CTs of aggregation-competent cells. The CTs of aggregation-competent cells may help the cells to aggregate toward the aggregation center.     

Adenylate cyclase activity in permeabilized cells from Dictyostelium discoideum

Aggregation competent cells of Dictyostelium discoideum have been permeabilized using the method previously developed for mammalian cells by Aragón et al. (Proc. Natl. Acad. Sci. USA (1980) 77, 6324). The permeabilization allows the “in situ” assay of adenylate cyclase. The enzyme activity is proportional with the amount of cells and the time of incubation. The enzyme exhibits non Michaelian kinetics towards its substrate ATP as recently reported for the enzyme assayed in crude extracts. Permeabilized cells contain lower levels of phosphodies-terase activity as compared with cellular homogenates. The similar properties of the adenylate cyclase from cell homogenates and permeabilized cells, as well as the low phosphodiesterase activity, makes the permeabilization method of great interest to study the regulation of adenylate cyclase during the differentiation of Didiscoideum.