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Circular actin waves separate two distinct areas on the substrate-attached cell surface from each other: an external area from an inner territory that is circumscribed by the wave. These areas differ in composition of actin-associated proteins and of phosphoinositides in the membrane. At the propagating wave, one area is converted into the other. By photo-conversion of Eos-actin and analysis of actin network structures we show that both in the inner territory and the external area the actin network is subject to continuous turnover. To address the question of whether areas in the wave pattern are specified by particular actin polymerizing machines, we locate five members of the formin family to specific regions of the wave landscape using TIRF microscopy and constitutively active formin constructs tagged with fluorescent protein. Formin ForB favors the actin wave and ForG the inner territory, whereas ForA, ForE, and ForH are more strongly recruited to the external area. Fluctuations of membrane binding peculiar to ForB indicate transient states in the specification of membrane domains before differentiation into ForB decorated and depleted ones. Annihilation of the patterns by 1 µM of the formin inhibitor SMIFH2 supports the implication of formins in their generation.

Chemotactic responses of eukaryotic cells require a signal processing system that translates an external gradient of attractant into directed motion. To challenge the response system to its limits, we increased the size of Dictyostelium discoideum cells by using electric-pulse-induced fusion. Large cells formed multiple protrusions at different sites along the gradient of chemoattractant, independently turned towards the gradient and competed with each other. Finally, these cells succeeded to re-establish polarity by coordinating front and tail activities. To analyse the responses, we combined two approaches, one aimed at local responses by visualising the dynamics of Ras activation at the front regions of reorientating cells, the other at global changes of polarity by monitoring front-to-tail-directed actin flow. Asymmetric Ras activation in turning protrusions underscores that gradients can be sensed locally and translated into orientation. Different to cells of normal size, the polarity of large cells is not linked to an increasing front-to-tail gradient of the PIP3-phosphatase PTEN. But even in large cells, the front communicates with the tail through an actin flow that might act as carrier of a protrusion inhibitor., (© 2016. Published by The Company of Biologists Ltd.)

F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave.

Proper regulation of the actin cytoskeleton is essential for cell function and ultimately for survival. Tight control of actin dynamics is required for many cellular processes, including differentiation, proliferation, adhesion, chemotaxis, endocytosis, exocytosis, and multicellular development. Here we describe a putative p21-activated protein kinase, PakD, that regulates the actin cytoskeleton in Dictyostelium discoideum. We found that cells lacking pakD are unable to aggregate and thus unable to develop. Compared to the wild type, cells lacking PakD have decreased membrane extensions, suggesting defective regulation of the actin cytoskeleton. pakD(-) cells show poor chemotaxis toward cyclic AMP (cAMP) but normal chemotaxis toward folate, suggesting that PakD mediates some but not all chemotaxis responses. pakD(-) cells have decreased polarity when placed in a cAMP gradient, indicating that the chemotactic defects of the pakD(-) cells may be due to an impaired cytoskeletal response to cAMP. In addition, while wild-type cells polymerize actin in response to global stimulation by cAMP, pakD(-) cells exhibit F-actin depolymerization under the same conditions. Taken together, the results suggest that PakD is part of a pathway coordinating F-actin organization during development.

We have trapped the catalytic domain of Dictyostelium (Dicty) myosin II in a weak actin-binding conformation by chemically crosslinking two engineered cysteines across the actin-binding cleft, using a bifunctional spin label (BSL). By connecting the lower and upper 50 kDa domains of myosin, the crosslink restricts the conformation of the actin-binding cleft. Crosslinking has no effect on the basal ATPase activity of isolated myosin, but it impairs rigor actin binding and actin-activation of myosin ATPase. EPR spectra of BSL provide insight into actomyosin structural dynamics. BSL is highly immobilized within the actin-binding cleft and is thus exquisitely sensitive to the global orientation and rotational motions of the myosin head. Conventional EPR shows that myosin heads bound to oriented actin filaments are highly disordered with respect to the actin filament axis, in contrast to the nearly crystalline order of myosin heads in rigor. This disorder is similar to that of weakly bound heads induced by ATP, but saturation transfer EPR shows that the disorder of crosslinked myosin is at least 100 times slower. Thus this cleft-crosslinked myosin is remarkably similar, in both actin affinity and rotational dynamics, to SH1-SH2 crosslinked BSL-myosin S1. We conclude that, whether myosin is trapped at the actin-myosin interface or in the force-generating region between the active site and lever arm, the structural state of myosin is intermediate between the weak-binding state preceding phosphate release and the strong-binding state that succeeds it. We propose that it represents the threshold of force generation.

Actin waves that travel on the planar membrane of a substrate-attached cell underscore the capability of the actin system to assemble into dynamic structures by the recruitment of proteins from the cytoplasm. The waves have no fixed shape, can reverse their direction of propagation and can fuse or divide. Actin waves separate two phases of the plasma membrane that are distinguished by their lipid composition. The area circumscribed by a wave resembles in its phosphoinositide content the interior of a phagocytic cup, leading us to explore the possibility that actin waves are in-plane phagocytic structures generated without the localized stimulus of an attached particle. Consistent with this view, wave-forming cells were found to exhibit a high propensity for taking up particles. Cells fed rod-shaped particles produced elongated phagocytic cups that displayed a zonal pattern that reflected in detail the actin and lipid pattern of free-running actin waves. Neutrophils and macrophages are known to spread on surfaces decorated with immune complexes, a process that has been interpreted as "frustrated" phagocytosis. We suggest that actin waves enable a phagocyte to scan a surface for particles that might be engulfed.

Large-scale gene expression analysis has been applied recently to uncover groups of genes that are co-regulated in particular processes. Here we undertake such an analysis on CAP, a protein that participates in the regulation of the actin cytoskeleton and in cAMP signaling in Dictyostelium. microarray analysis revealed that loss of CAP altered the expression of many cytoskeletal components. One of these, the Rho GDP-dissociation inhibitor RhoGDI1, was analyzed further. RhoGDI1 null cells expressed lower amounts of CAP, which failed to accumulate predominantly at the cell cortex. To further position CAP in the corresponding signal transduction pathways we studied CAP localization and cellular functioning in mutants that have defects in several signaling components. CAP showed correct localization and dynamics in all analyzed strains except in mutants with deficient cAMP dependent protein kinase A activity, where CAP preferentially accumulated in crown shaped structures. Ectopic expression of CAP improved the efficiency of phagocytosis in Gbeta-deficient cells and restored the pinocytosis, morphology and actin distribution defects in a PI3 kinase double mutant (pi3k1/2 null). Our results show that CAP acts at multiple crossroads and links signaling pathways to the actin cytoskeleton either by physical interaction with cytoskeletal components or through regulation of their gene expression.

This chapter will focus on responses that the chemoattractant cyclic AMP elicits in the motility system of Dictyostelium. These cells can be permanently transfected to express cytoskeleton-associated proteins tagged with fluorescent proteins. Multiple proteins that are distinguishable by the excitation and emission spectra of their tags can be simultaneously expressed. This makes it possible to relate the spatial and temporal patterns of their chemoattractant-induced translocation to each other in one cell by a single recording. Since actin polymerization in live cells progresses with velocities of about 3 microm/s, high image frequencies and short acquisition times in the millisecond range are required. Techniques of total internal reflection fluorescence (TIRF) and spinning-disc confocal microscopy provide appropriate temporal and spatial resolution for the analysis of actin dynamics.

We have investigated the role of the Arp2/3 complex in Dictyostelium cell chemotaxis towards cyclic-AMP and in the actin polymerization that is triggered by this chemoattractant. We confirm that the Arp2/3 complex is recruited to the cell perimeter, or into a pseudopod, after cyclic-AMP stimulation and that this is coincident with actin polymerization. This recruitment is inhibited when actin polymerization is blocked using latrunculin suggesting that the complex binds to pre-existing actin filaments, rather than to a membrane associated signaling complex. We show genetically that an intact Arp2/3 complex is essential in Dictyostelium and have produced partially active mutants in two of its subunits. In these mutants both phases of actin polymerization in response to cyclic-AMP are greatly reduced. One mutant projects pseudopodia more slowly than wild type and has impaired chemotaxis, together with slower movement. The second mutant chemotaxes poorly due to an adhesion defect, suggesting that the Arp2/3 complex plays a crucial part in adhering cells to the substratum as they move. We conclude that the Arp2/3 complex largely mediates the actin polymerization response to chemotactic stimulation and contributes to cell motility, pseudopod extension and adhesion in Dictyostelium chemotaxis.

At the leading edge of a motile cell, actin polymerizes in close apposition to the plasma membrane. Here we ask how the machinery for force generation at a leading edge is established de novo after the global depolymerization of actin. The depolymerization is accomplished by latrunculin A, and the reorganization of actin upon removal of the drug is visualized in Dictyostelium cells by total internal reflection fluorescence microscopy. The actin filament system is reorganized in three steps. First, F-actin assembles into globular complexes that move along the bottom surface of the cells at velocities up to 10 microm/min. These clusters are transient structures that eventually disassemble, fuse, or divide. In a second step, clusters merge into a contiguous zone at the cell border that spreads and gives rise to actin waves traveling on a planar membrane. Finally, normal cell shape and motility are resumed. These data show that the initiation of actin polymerization is separated in Dictyostelium from front protrusion, and that the coupling of polymerization to protrusion is a later step in the reconstitution of a leading edge.

Hypertonic stimulation induced association of S-adenosyl-L-homocysteine hydrolase (SAHH) with the F-actin-rich cell cortex in Dictyostelium. At intermediate, but not higher, levels of hypertonicity, SAHH further translocated from the cortex to the cytosol in company with a fraction of actin and cofilin. At the same time the cells rounded up. Acidification of the cells stimulated both the cell rounding and the translocation of actin and SAHH, whereas alkalinization retarded these responses, suggesting that cellular pH is involved in their control. On the other hand, mutant analysis suggested that neither cGMP signaling nor conventional myosin is required.

Experiments in amoebae and neutrophils have shown that local accumulations of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] mediate the ability of cells to migrate during gradient sensing. To define the nature of this response, we subjected Dictyostelium discoideum cells to measurable temporal and spatial chemotactic inputs and analyzed the accumulation of PI(3,4,5)P(3) on the membrane, as well as the recruitment of the enzymes phosphoinositide 3-kinase and PTEN. In latrunculin-treated cells, spatial gradients elicited a PI(3,4,5)P(3) response only on the front portion of the cell where the response increased more steeply than the gradient and did not depend on its absolute concentration. Phosphoinositide 3-kinase bound to the membrane only at the front, although it was less sharply localized than PI(3,4,5)P(3). Membrane-bound PTEN was highest at the rear and varied inversely with receptor occupancy. The localization of PI(3,4,5)P(3) was enhanced further in untreated polarized cells containing an intact cytoskeleton. Interestingly, the treated cells could respond to two independent gradients simultaneously, demonstrating that a response at the front does not necessarily inhibit the back. Combinations of temporal and spatial stimuli provided evidence of an inhibitory process and showed that a gradient generates a persistent steady-state response independent of a previous history of exposure to chemoattractant. These results support a local excitation/global inhibition model and argue against other schemes proposed to explain directional sensing.

We found that amoeboid cells of Dictyostelium are induced by a millimolar concentration of quinine to form a rapidly elongating, cylindrical protrusion, which often led to sustained locomotion of the cells. Formation of the protrusion was initiated by fusion of a contractile vacuole with the cell membrane. During protrusion extension, a patch of the contractile vacuole membrane stayed undiffused on the leading edge of the protrusion for over 30 seconds. Protrusion formation was not inhibited by high osmolarity of the external medium (at least up to 400 mosM). By contrast, mutant cells lacking myosin II (mhc(-) cells) failed to extend protrusions upon exposure to quinine. When GFP-myosin-expressing cells were exposed to quinine, GFP-myosin was accumulated in the cell periphery forming a layer under the cell membrane, but a newly formed protrusion was initially devoid of a GFP-myosin layer, which gradually formed and extended from the base of the protrusion. F-actin was absent in the leading front of the protrusion during the period of its rapid elongation, and the formation of a layer of F-actin in the front was closely correlated with its slowing-down or retraction. Periodical or continuous detachment of the F-actin layer from the apical membrane of the protrusion, accompanied by a transient increase in the elongation speed at the site of detachment, was observed in some of the protrusions. The detached F-actin layers, which formed a spiral layer of F-actin in the case of continuous detachment, moved in the opposite direction of protrusion elongation. In the presence of both cytochalasin A and quinine, the protrusions formed were not cylindrical but spherical, which swallowed up the entire cellular contents. The estimated bulk flux into the expanding spherical protrusions of such cells was four-times higher than the flux into the elongating cylindrical protrusions of the cells treated with quinine alone. These results indicate that the force responsible for the quinine-induced protrusion is mainly due to contraction of the cell body, which requires normal myosin II functions, while actin polymerization is important in restricting the direction of its expansion. We will discuss the possible significance of tail contraction in cell movement in the multicellular phase of Dictyostelium development, where cell locomotion similar to that induced by quinine is often observed without quinine treatment, and in protrusion elongation in general. Movies available on-line

In the presence of germination signals, dormant spores of Dictyostelium discoideum rapidly germinate to start a new life cycle. Previously we have shown that half of the actin molecules in spores are maintained in a tyrosine-phosphorylated state, and a decline of the actin phosphorylation levels is a prerequisite for spore swelling. In this study, we have established d-glucose as a trigger molecule for the actin dephosphorylation. Present in a nutrient germination medium, d-glucose both may act as a trigger molecule and/or may serve as a substrate within a pathway for actin dephosphorylation depending upon spore age. However, the glucose-induced actin dephosphorylation was insufficient for spores to swell. Other factors in the nutrient medium were required for complete germination of young spores aged 1 to 5 days. In contrast, dispersion in nonnutrient buffer was necessary and sufficient for a decline of actin phosphorylation levels and even the emergence of amoebae in older spores (6 days and beyond). Moreover, the dephosphorylation pathway in the older spores was independent of energy production. We propose that the diversification of the actin dephosphorylation pathway may enable spores to increase their probability of germination upon spore aging., (Copyright 2000 Academic Press.)

We have electroporated Dictyostelium amoebae with fluorescent phalloidin in order to visualize the localization and behavior of F-actin filaments in living cells. Immediately after electroporation with phalloidin, cells became round and showed bright staining in the cortical region. Over time, the cortical staining disappeared and was replaced by a large aggregate of actin filaments. The aggregates were predominantly localized to the apical posterior of actively moving cells and in the middle of dividing cells or stationary AX4 cells. Mutants lacking myosin II or ABP-120 also formed actin aggregates; however, the rate of formation of aggregates was slower in myosin II mutant cells. In order to investigate this phenomenon further, we have used jasplakinolide, a membrane-permeable drug that also stabilizes F-actin filaments. Cells treated with jasplakinolide formed actin aggregates in a concentration-dependent manner. Drug treatment led to an increase in the proportion of actin associated with the cytoskeleton. Jasplakinolide-treated cells were still motile; however, their rate of movement was less than that of untreated cells. Cytochalasin B and nocodazole had inhibitory effects on aggregate formation, while azide blocked the process completely. We hypothesize that aggregates are formed from the cortical flow of F-actin filaments. These filaments would normally be depolymerized but are artificially stabilized by phalloidin or jasplakinolide binding. The localization of the aggregate is likely to be an indication of the direction of cortical flow.

The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.

Background: The microfilament system in the cortex of highly motile cells, such as neutrophils and cells of the eukaryotic microorganism Dictyostelium discoideum, is subject to rapid re-organization, both spontaneously and in response to external signals. In particular, actin polymerization induced by a gradient of chemoattractant leads to local accumulation of filamentous actin and protrusion of a 'leading edge' of the cell in the direction of the gradient. In order to study the dynamics of actin in these processes, actin was tagged at its amino terminus with green fluorescent protein (GFP) and observed with fluorescence microscopy in living cells of D. discoideum., Results: Purified GFP-actin was capable of copolymerizing with actin. In the transfected cells of D. discoideum studied, GFP-actin made up 10-20% of the total actin. Microfilaments containing GFP-actin were capable of generating force with myosin in an in vitro assay. Observations of single living cells using fluorescence microscopy showed that the fusion protein was enriched in cell projections, including filopodia and leading edges, and that the fusion protein reflected the dynamics of the microfilament system in cells that were freely moving, being chemotactically stimulated, or aggregated. When confocal sections of fixed cells containing GFP-actin were labeled with fluorescent phalloidin, which binds only to filamentous actin, there was a correlation between the areas of GFP-actin and phalloidin fluorescence, but there were distinct sites in which GFP-actin was more prominent., Conclusions: Double labeling with GFP-actin and other probes provides an indication of the various states of actin in motile cells. A major portion of the actin assemblies visualized using GFP-actin are networks or bundles of filamentous actin. Other clusters of GFP-actin might represent stores of monomeric actin in the form of complexes with actin-sequestering proteins.

Actin is known to be phosphorylated at tyrosine, serine, or threonine residues in various cells. In cells of Dictyostelium discoideum, a rise in the tyrosine phosphorylation of actin is observed in response to ATP depletion. An actin fraction rich in phosphotyrosine was obtained by chromatography on the weak anion exchanger Mono-P. Mass spectrometry and amino acid sequencing of protease cleavage products indicated that a single tyrosine residue was phosphorylated. Localization of this residue to position 53 of the actin sequence attributed the modification to a site that is critical for the capability of actin to polymerize. Induction of the tyrosine phosphorylation by heat shock and Cd2+ ions indicates that this modification of actin is implicated in the response of Dictyostelium cells to stress.

'Contact regions' are plasma membrane domains derived from areas of intercellular contact between aggregating Dictyostelium amebae (H.M. Ingalls et al. (1986). Proc. Nat. Acad. Sci. USA 83, 4779). Purified contact regions contain a prominent actin-binding protein with an M(r) of 34,000. Immunoblotting with monoclonal antibodies identifies this polypeptide as a 34,000 M(r) actin-bundling protein (known as 30 kDa protein), previously shown to be enriched in filopodia (M. Fechheimer (1987). J. Cell Biol. 104, 1539). About four times more 30 kDa protein by mass is associated with contact regions than is found in total plasma membranes isolated from aggregating cells. In agreement with these observations, immunostaining of the 30 kDa protein in aggregating cells reveals a prominent localization along the plasma membrane at sites of intercellular contact. By contrast, alpha-actinin does not appear to be significantly enriched at sites of cell to cell contact. Binding experiments using purified plasma membranes, actin and 30 kDa protein indicate that the 30 kDa protein is associated with the plasma membrane primarily through interactions with actin filaments. Calcium ions are known to decrease the interaction of actin with 30 kDa protein in solution. Surprisingly, membrane-associated complexes of actin and the 30 kDa protein are much less sensitive to dissociation by micromolar levels of free calcium ions than are complexes in solutions lacking membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

When oxidative phosphorylation is inhibited in cells of Dictyostelium discoideum, the phosphorylation of tyrosine residues on actin is strongly increased. This increase is fully reversible. Under the same conditions the amoeboid cells undergo a series of shape changes. Within three minutes the pseudopods are withdrawn and replaced by cell surface blebs. Subsequently, the cells are rounding up to become immobile. In parallel with the changes in cell shape, the distribution of actin filaments is grossly altered within the cells. The cortical network of actin filaments of normal cells is broken down, and the F-actin forms large, irregular clusters deep within the cytoplasm. In these clusters the actin is associated with myosin II and with the heterodimeric F-actin capping protein cap32/34. After restoration of oxidative phosphorylation the actin returns within less than four minutes to its normal cortical position. A causal relationship between tyrosine phosphorylation and changes in the distribution of actin remains to be established. The rearrangements in the actin system that result from the inhibition of oxidative phosphorylation indicate that the organisation of this system and its maintenance in a functional state depend on the continuous supply of energy by ATP.

The distribution of actin and myosin II was studied by immunofluorescence microscopy in Dictyostelium discoideum cells that had been stimulated with a chemoattractant, cAMP. The amounts of actin but not of myosin II increased in the cortical region of cells after 5-10 seconds of stimulation by cAMP at 21 degrees C (the first peak). After a transient decrease in the amount of actin in the cortical region, the amounts of both actin and myosin II increased in the cortical region after 25-35 seconds of stimulation (the second peak). The amounts of myosin II decreased in the cortical region thereafter but actin became localized in the pseudopods. The stimulated cells extended many short projections that contained actin at the time of the first peak and the cell bodies contracted at the time of the second peak. Foci in the cortical actin network decreased in number at 20-30 sec and recovered thereafter. A quantitative examination using a fluorocytometer revealed that the amounts of actin filaments in the cells increased at both peaks. The calcium ionophore A23187 and Ca2+ ion induced the reorganization of actin and myosin II to a certain extent. Furthermore, Ca2+ ions inhibited the return of myosin II to the endoplasm in cAMP-stimulated cells after preincubation with A23187. Thus, Ca2+ ions may play a dual role, being involved in the regulation of both the initiation and the termination of the reorganization of cytoskeletons. The cAMP-stimulated responses of cytoskeletons were not inhibited by EGTA, La3+ ions, or ruthenium red but were inhibited by preincubation with BAPTA-AM.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemotactic stimulation of Dictyostelium discoideum amoebae with pulses of cAMP or folate causes a series of rapid changes in the amount of actin protein associated with the Triton-insoluble cytoskeleton. The first of these changes occurs within 3 sec. of stimulation. The changes are dose-dependent and are within the physiological range of concentrations of cAMP or folate eliciting chemotaxis. These effects on the cytoskeleton show a pattern of regulation during development matching the respective chemotactic sensitivities of D.discoideum to cAMP (most sensitive at 4-8 hr of development) and to folate (rapidly decreasing sensitivity over 0-4 hr). At twelve hr, however, the responsiveness to folate unexpectedly reappears, suggesting a function of folate later in development than previously reported.

Before addition of cAMP, Dictyostelum amoebae rapidly translocating in buffer are elongate, exhibit expansion zones primarily at the anterior end and filamentous actin (F-actin) localization primarily in the anterior pseudopodia. Intracellular particle movement is primarily in the anterior direction, and the average rate of particle movement is roughly five times the rate of cellular translocation. Within seconds after the addition of 10(-6)M cAMP, there is a dramatic suppression of cellular translocation, an inhibition of pseudopod formation, a freeze in cellular morphology, a dramatic depression in intracellular particle movement, loss of F-actin localization in pseudopodia concomitant with relocalization of F-actin in the general cytoplasmic cortex under the plasma membrane, and a doubling of F-actin content. After 10 s, expansion zones are again visible at the cell perimeter, but they no longer are localized in the original anterior portion of the cell. There is a slight rebound in particle movement after 10 s, but particles with persistent tracks now show no directionality towards the original anterior portion of the cell, as they did before cAMP addition. Finally, in parallel with the resumption of peripheral expansion and the small rebound in particle movement, there is a decrease in total cellular F-actin to the untreated level. The pattern of microtubule organization is unaffected by the addition of cAMP.

Actin polymerization occurs in amebae of Dictyostelium discoideum after chemotactic stimulation (Hall, A. L., A. Schlein, and J. Condeelis. 1988. J. Cell. Biochem. 37:285-299). When cells are lysed with Triton X-100 during stimulation, an actin nucleation activity is detected in lysates by measuring the rate of pyrene-labeled actin polymerization. This stimulated nucleation activity is closely correlated with actin polymerization observed in vivo in its kinetics, developmental regulation, and cytochalasin D sensitivity. Actin polymerization is coordinate with pseudopod extension in synchronized populations of cells and is correlated with the accumulation of F actin in pseudopods. The stimulated actin nucleation activity is present in low-speed pellets from Triton lysates (cytoskeletons) within 3 s of stimulation and is stable compared with the nucleation activity of whole cell lysates. Low-speed supernatants contain a reversible inhibitor of the actin nucleation activity that is itself regulated by chemotactic stimulation. Neither activity requires Ca2+ and both are fully expressed in 10 mM EGTA. Fractions containing the inhibitor do not sever actin filaments but do inhibit actin polymerization that is seeded by fragments of purified F actin. These results indicate that chemotactic stimulation of Dictyostelium discoideum generates both an actin-nucleating activity and an actin-polymerization inhibitor, and suggest that the parallel regulation of these two activities leads to the transient phases of actin polymerization observed in vivo. The different compartmentation of these two activities may account for polarized pseudopod extension in gradients of chemoattractant.

We report changes in actin associated with the Triton-insoluble cytoskeleton following chemotactic stimulation in five species of cellular slime mould, Dictyostelium discoideum, D. mucoroides, D. purpureum, D. lacteum and Polysphondylium violaceum. Stimulation of amoebae with the chemoattractant specific for each species elicits a rapid increase in cytoskeletal actin content with a peak at 5 s, probably corresponding to pseudopodium formation. Subsequent changes consist of a second discrete peak of actin accumulation and, in at least two species, D. discoideum and P. violaceum, a third peak. We correlate these later changes in actin content with the changes in amoebal shape that have been reported to be a consequence of chemotactic stimulation. We have also investigated species variation in actin content and find D. purpureum to have a remarkably low cytoskeletal actin content in comparison to other species. Whole cell and cytoskeletal actin levels during the first 12 h of D. discoideum development have also been analysed. While little change is found in total protein content, whole cell actin shows an abrupt decline during aggregation and this is accompanied by an increase in cytoskeletal actin, amounting to 100% over 0-12 h of development. These results are discussed in the light of a possible changing role for actin during the course of development, from being involved in the dynamic events of chemotaxis to assuming a more structural role in the multicellular aggregate.

In this study we investigated concanavalin A (Con A) induced changes in the locations of actin, myosin, 120K, and 95K (alpha-actinin) to determine the extent to which actin and myosin are reorganized during capping and the roles that 120K and 95K might play in this reorganization. We observed the location of each protein by indirect immunofluorescence using affinity purified antibodies. Four morphological states were distinguished in vegetative Dictyostelium amebae: ameboid cells before Con A binding, patched cells, capped cells, and ameboid cells with caps. The location of each protein was distinct in ameboid cells both before and after capping Actin and 120K were found in the cell cortex usually associated with surface projections, and myosin and 95K were diffusely distributed. Myosin was excluded from surface projections in ameboid cells. During patching, all four proteins were localized below Con A patches. During capping, actin, myosin, and 95K protein moved with the Con A patches into the cap whereas 120K protein was excluded from the cap. During the late stages of cap formation actin and myosin were progressively lost from the cap, and 120K became concentrated in new actin-filled projections that formed away from the cap. However, 95K remained tightly associated with the cap. Poisoning cells with sodium azide inhibited capping but not patching of ligand. In azide-poisoned cells, myosin and 95K did not co-patch with Con A, whereas copatching of 120K and actin with Con A occurred as usual. Our results support the hypothesis that capping is an actomyosin-mediated motile event that involves a sliding interaction between actin filaments, which are anchored through the membrane to ligand patches, and myosin in the cortex. They are also consistent with a role for 120K in the formation of surface projections by promoting growth and/or cross-linking of actin filaments within projections, and with a role for 95K in regulating actomyosin-mediated contractility, earlier proposals based on the in vitro properties of these two proteins (Condeelis, J., M. Vahey, J. M. Carboni, J. DeMey, S. Ogihara, 1984, J. Cell Biol., 99:119s-126s).

Triton-insoluble cytoskeletons were isolated from Dictyostelium discoideum AX3 cells prior to and following stimulation with 2'deoxy cyclic adenosine monophosphate (cAMP). Temporal changes in the content of actin and a 120,000 dalton actin-binding protein (ABP-120) in cytoskeletons following stimulation were monitored. Both actin and ABP-120 were incorporated into the cytoskeleton at 30-40 seconds following stimulation, which is cotemporal with the onset of pseudopod extension during stimulation of amoebae with chemoattractants. Changes in the content of total cytoskeletal protein and cytoskeletal myosin were determined under the same experimental conditions as controls. These proteins exhibited different kinetics from those of cytoskeletal ABP-120 and actin following the addition of 2'deoxy cAMP. The authors concluded that the association of ABP-120 with the cytoskeleton is regulated during cAMP signalling. Furthermore, these results indicate that ABP-120 is involved in cross-linking newly assembled actin filaments into the cytoskeleton during chemoattractant-stimulated pseudopod extension.

Electron microscopic evidence demonstrated that dimethyl sulfoxide (DMSO) induces formation of giant intranuclear microfilament bundles in the interphase nucleus of a cellular slime mold, Dictyostelium. These giant bundles are approximately giant bundles are approximately 3 micrometer long, 0.85 micrometer wide, and composed of microfilaments 6 nm in diameter. Studies in which glycerinated cells were used showed that these microfilaments bind rabbit skeletal muscle heavy meromyosin, forming typical decorated "arrowhead" structures, and that this binding can be reverted by Mg-adenosine triphosphate. These data verify that the intranuclear microfilaments are the contractile protein actin, and that DMSO affects intranuclear actin, inducing the formation of such giant bundles. The intranuclear actin bundles appear at any developmental stage in two different species of cellular slime molds after treatment with DMSO. The native form of the intranuclear actin molecules and their possible functions are discussed, and it is proposed that the contractile protein has essential functions in the cell nucleus.