Is there a pilot in a pseudopod?

The concept of pilot pseudopodia is reconsidered 30 years after its inauguration (Gerisch, G., Hülser, D., Malchow, D., Wick, U., 1975. Cell communication by periodic cyclic-AMP pulses. Phil. Trans. R. Soc. Lond. B 272, 181-192). The original hypothesis stated that protruding pseudopodia serve as dynamic sensory organelles that aid a cell in perceiving variations of chemoattractant concentration and, consequently, in navigation during chemotaxis. This influential idea is reevaluated in the light of recent findings about the mechanisms governing chemotactic cell motility, morphology and dynamics of pseudopodia, and about molecular constituents and regulators of pseudopod extension and retraction. It is proposed that stimulation by a chemoattractant modulates speed of pseudopod protrusion and thereby increases cell elongation. Elongation further enhances chemotactic sensitivity of the cell to shallow chemoattractant gradients, reinforces cell polarization, and finally leads to suppression of lateral pseudopodia and continuation of cell migration in the gradient direction.     

Recruitment of a specific amoeboid myosin I isoform to the plasma membrane in chemotactic Dictyostelium cells

The Dictyostelium class I myosins, MyoA, -B, -C, and -D, participate in plasma membrane-based cellular processes such as pseudopod extension and macropinocytosis. Given the existence of a high affinity membrane-binding site in the C-terminal tail domain of these motor proteins and their localized site of action at the cortical membrane-cytoskeleton, it was of interest to determine whether each myosin I was directly associated with the plasma membrane. The membrane association of a myosin I heavy chain kinase that regulates the activity of one of the class I myosins, MyoD was also examined. Cellular fractionation experiments revealed that the majority of the Dicyostelium MyoA, -B, -C and -D heavy chains and the kinase are cytosolic. However, a small, but significant, fraction (appr. 7. -15%) of each myosin I and the kinase was associated with the plasma membrane. The level of plasma membrane-associated MyoB, but neither that of MyoC nor MyoD, increases up to 2-fold in highly motile, streaming cells. These results indicate that Dictyostelium specifically recruits myoB to the plasma membrane during directed cell migration, consistent with its known role in pseudopod formation.     

Mechanisms of amoeboid chemotaxis: an evaluation of the cortical expansion model

In this work we evaluate the cortical expansion model for amoeboid chemotaxis with regard to new information about molecular events in the cytoskeleton following chemotactic stimulation of Dictyostelium amoebae. A rapid upshift in the concentration of chemoattractant can be used to synchronize the motile behavior of a large population of cells. This synchrony presents an opportunity to study the biochemical basis of morphological changes such as pseudopod extension that are required for amoeboid chemotaxis. Changes in the composition and activity of the cytoskeleton following stimulation can be measured with precision and correlated with important morphological changes. Such studies demonstrate that activation of actin nucleation is one of the first and most crucial events in the actin cytoskeleton following stimulation. This activation is followed by incorporation of specific actin cross-linking proteins into the cytoskeleton, which are implicated in the extension of pseudopods and filopods. These results, as well as those from studies with mutants deficient in myosin, indicate that cortical expansion, driven by focal actin polymerization, cross-linking and gel osmotic swelling, is an important force for pseudopod extension. It is concluded that whereas three forces, frontal sliding, tail contraction, and cortical expansion may cooperate to produce amoeboid movement, the cortical expansion model offers the simplest explanation of how focal stimulation with a chemoattractant causes polarized pseudopod extension.     

Myosin IB null mutants of Dictyostelium exhibit abnormalities in motility.

Cellular and intracellular motility are compared between normal Dictyostelium amoebae and amoebae lacking myosin IB (DMIB-). DMIB- cells generate elongated cell shapes, form particulate-free pseudopodia filled with F-actin, and exhibit an anterior bias in pseudopod extension in a fashion similar to normal amoebae. DMIB- cells also exhibit a normal response to the addition of the chemoattractant cAMP, including a depression in cellular and intracellular particle velocity, depolymerization of F-actin in pseudopodia, and a concomitant increase in cortical F-actin. DMIB- cells do, however, form lateral pseudopodia roughly three times as frequently as normal cells, turn more often, and exhibit depressed average instantaneous cell velocity. DMIB- cells also exhibit a decrease in the average instantaneous velocity of intracellular particle movement and an increase in the degree of randomness in particle direction. These findings indicate that if there is functional substitution for myosin IB by other myosin I isoforms, it is at best only partial, with myosin IB being necessary for maintenance of the normal rate and persistence of cellular translocation, suppression of lateral pseudopod formation and subsequent turning, rapid intracellular particle motility, and the normal anterograde bias of intracellular particle movement. Furthermore, it is likely that the behavioral abnormalities observed here for DMIB- cells underlie the delay in the onset of chemotactic aggregation, the increase in the time required to complete streaming, and the abnormalities in morphogenesis exhibited by DMIB- cells.     

Mutants in the Dictyostelium Arp2/3 complex and chemoattractant-induced actin polymerization

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.     

Identification of actin nucleation activity and polymerization inhibitor in ameboid cells: their regulation by chemotactic stimulation

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.     

Recruitment of a myosin heavy chain kinase to actin-rich protrusions in Dictyostelium

Nonmuscle myosin II plays fundamental roles in cell body translocation during migration and is typically depleted or absent from actin-based cell protrusions such as lamellipodia, but the mechanisms preventing myosin II assembly in such structures have not been identified [1-3]. In Dictyostelium discoideum, myosin II filament assembly is controlled primarily through myosin heavy chain (MHC) phosphorylation. The phosphorylation of sites in the myosin tail domain by myosin heavy chain kinase A (MHCK A) drives the disassembly of myosin II filaments in vitro and in vivo [4]. To better understand the cellular regulation of MHCK A activity, and thus the regulation of myosin II filament assembly, we studied the in vivo localization of native and green fluorescent protein (GFP)-tagged MHCK A. MHCK A redistributes from the cytosol to the cell cortex in response to stimulation of Dictyostelium cells with chemoattractant in an F-actin-dependent manner. During chemotaxis, random migration, and phagocytic/endocytic events, MHCK A is recruited preferentially to actin-rich leading-edge extensions. Given the ability of MHCK A to disassemble myosin II filaments, this localization may represent a fundamental mechanism for disassembling myosin II filaments and preventing localized filament assembly at sites of actin-based protrusion.     

PAKa, a putative PAK family member, is required for cytokinesis and the regulation of the cytoskeleton in Dictyostelium discoideum cells during chemotaxis.

We have identified a Dictyostelium discoideum gene encoding a serine/threonine kinase, PAKa, a putative member of the Ste20/PAK family of p21-activated kinases, with a kinase domain and a long NH(2)-terminal regulatory domain containing an acidic segment, a polyproline domain, and a CRIB domain. PAKa colocalizes with myosin II to the cleavage furrow of dividing cells and the posterior of polarized, chemotaxing cells via its NH(2)-terminal domain. paka null cells are defective in completing cytokinesis in suspension. PAKa is also required for maintaining the direction of cell movement, suppressing lateral pseudopod extension, and proper retraction of the posterior of chemotaxing cells. paka null cells are defective in myosin II assembly, as the myosin II cap in the posterior of chemotaxing cells and myosin II assembly into cytoskeleton upon cAMP stimulation are absent in these cells, while constitutively active PAKa leads to an upregulation of myosin II assembly. PAKa kinase activity against histone 2B is transiently stimulated and PAKa incorporates into the cytoskeleton with kinetics similar to those of myosin II assembly in response to chemoattractant signaling. However, PAKa does not phosphorylate myosin II. We suggest that PAKa is a major regulator of myosin II assembly, but does so by negatively regulating myosin II heavy chain kinase.     

Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain

Dictyostelium amebae have been engineered by homologous recombination of a truncated copy of the myosin heavy chain gene (heavy meromyosin (HMM) cells) and by transformation with a vector encoding an antisense RNA to myosin heavy chain mRNA (mhcA cells) so that they lack native myosin heavy chain protein. In the former case, cells synthesize only the heavy meromyosin portion of the protein and in the latter case they synthesize negligible amounts of the protein. Surprisingly, it was demonstrated that both cell lines are viable and motile. In order to compare the motility of these cells with normal cells, the newly developed computer-assisted Dynamic Morphology System (DMS) was employed. The results demonstrate that the average HMM or mhcA ameba moves at a rate of translocation less than half that of normal cells. It is rounder and less polar than a normal cell, and exhibits a rate of cytoplasmic expansion and contraction roughly half that of normal cells. In a spatial gradient of cAMP, the average ameba of HMM or mhcA exhibits a chemotactic index of +0.10 or less, compared to the chemotactic index of +0.50 exhibited by normal cells. Finally, the initial area, rate of expansion, and final area of pseudopods are roughly half that of normal cells. The five fastest HMM amebae (out of 35 analyzed in detail) moved at an average rate of translocation equal to that of normal amebae, and exhibited an average chemotactic index of +0.34. In addition, the average rate of cytoplasmic flow in fast HMM cells was equal to that of the average normal ameba. However, fast HMM amebae still exhibited the same defects in pseudopod formation that were exhibited by the entire HMM cell population. These results suggest that myosin heavy chain is involved in the "fine tuning" and efficiency of pseudopod formation, but is not essential for the basic behavior of pseudopod expansion.     

Effect of heavy chain phosphorylation on the polymerization and structure of Dictyostelium myosin filaments

In Dictyostelium amebas, myosin appears to be organized into filaments that relocalize during cell division and in response to stimulation by cAMP. To better understand the regulation of myosin assembly, we have studied the polymerization properties of purified Dictyostelium myosin. In 150 mM KCl, the myosin remained in the supernate following centrifugation at 100,000 g. Rotary shadowing showed that this soluble myosin was monomeric and that approximately 80% of the molecules had a single bend 98 nm from the head-tail junction. In very low concentrations of KCl (less than 10 mM) the Dictyostelium myosin was also soluble at 100,000 g. But rather than being monomeric, most of the molecules were associated into dimers or tetramers. At pH 7.5 in 50 mM KCl, dephosphorylated myosin polymerized into filaments whereas myosin phosphorylated to a level of 0.85 mol Pi/mol heavy chain failed to form filaments. The phosphorylated myosin could be induced to form filaments by lowering the pH or by increasing the magnesium concentration to 10 mM. The resulting filaments were bipolar, had blunt ends, and had a uniform length of approximately 0.43 micron. In contrast, filaments formed from fully dephosphorylated myosin were longer, had tapered ends, and aggregated to form very long, threadlike structures. The Dictyostelium myosin had a very low critical concentration for assembly of approximately 5 micrograms/ml, and this value did not appear to be affected by the level of heavy chain phosphorylation. The concentration of polymer at equilibrium, however, was significantly reduced, indicating that heavy chain phosphorylation inhibited the affinity of subunits for each other. Detailed assembly curves revealed that small changes in the concentration of KCl, magnesium, ATP, or H+ strongly influenced the degree of assembly. Thus, changes in both the intracellular milieu and the level of heavy chain phosphorylation may control the location and state of assembly of myosin in response to physiological stimuli.     

Dictyostelium PAKc is required for proper chemotaxis

We have identified a new Dictyostelium p21-activated protein kinase, PAKc, that we demonstrate to be required for proper chemotaxis. PAKc contains a Rac-GTPase binding (CRIB) and autoinhibitory domain, a PAK-related kinase domain, an N-terminal phosphatidylinositol binding domain, and a C-terminal extension related to the Gbetagamma binding domain of Saccharomyces cerevisiae Ste20, the latter two domains being required for PAKc transient localization to the plasma membrane. In response to chemoattractant stimulation, PAKc kinase activity is rapidly and transiently activated, with activity levels peaking at approximately 10 s. pakc null cells exhibit a loss of polarity and produce multiple lateral pseudopodia when placed in a chemoattractant gradient. PAKc preferentially binds the Dictyostelium Rac protein RacB, and point mutations in the conserved CRIB that abrogate this binding result in misregulated kinase activation and chemotaxis defects. We also demonstrate that a null mutation lacking the PAK family member myosin I heavy chain kinase (MIHCK) shows mild chemotaxis defects, including the formation of lateral pseudopodia. A null strain lacking both PAKc and the PAK family member MIHCK exhibits severe loss of cell movement, suggesting that PAKc and MIHCK may cooperate to regulate a common chemotaxis pathway.     

Chemoattractant-mediated increases in cGMP induce changes in Dictyostelium myosin II heavy chain-specific protein kinase C activities

Myosin II heavy chain (MHC)-specific protein kinase C (MHC-PKC) isolated from the ameba, Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cAMP (Abu- Elneel et al. 1996. J. Biol. Chem. 271:977- 984). Recent studies have indicated that cAMP-induced cGMP accumulation plays a role in the regulation of myosin II phosphorylation and localization (Liu, G., and P. Newell. 1991. J. Cell. Sci. 98: 483-490). This report describes the roles of cAMP and cGMP in the regulation of MHC-PKC membrane association, phosphorylation, and activity (hereafter termed MHC-PKC activities). cAMP stimulation of Dictyostelium cells resulted in translocation of MHC-PKC from the cytosol to the membrane fraction, as well as increasing in MHC-PKC phosphorylation and in its kinase activity. We present evidence that MHC is phosphorylated by MHC-PKC in the cell cortex which leads to myosin II dissociation from the cytoskeleton. Use of Dictyostelium mutants that exhibit aberrant cAMP- induced increases in cGMP accumulation revealed that MHC-PKC activities are regulated by cGMP. Dictyostelium streamer F mutant (stmF), which produces a prolonged peak of cGMP accumulation upon cAMP stimulation, exhibits prolonged increases in MHC-PKC activities. In contrast, Dictyostelium KI-10 mutant that lacks the normal cAMP-induced cGMP response, or KI-4 mutant that shows nearly normal cAMP-induced cGMP response but has aberrant cGMP binding activity, show no changes in MHC- PKC activities. We provide evidence that cGMP may affect MHC-PKC activities via the activation of cGMP-dependent protein kinase which, in turn, phosphorylates MHC-PKC. The results presented here indicate that cAMP-induced cGMP accumulation regulates myosin II phosphorylation and localization via the regulation of MHC-PKC.     

WASP-interacting protein is important for actin filament elongation and prompt pseudopod formation in response to a dynamic chemoattractant gradient

The role of WASP-interacting protein (WIP) in the process of F-actin assembly during chemotaxis of Dictyostelium was examined. Mutations of the WH1 domain of WASP led to a reduction in binding to WIPa, a newly identified homolog of mammalian WIP, a reduction of F-actin polymerization at the leading edge, and a reduction in chemotactic efficiency. WIPa localizes to sites of new pseudopod protrusion and colocalizes with WASP at the leading edge. WIPa increases F-actin elongation in vivo and in vitro in a WASP-dependent manner. WIPa translocates to the cortical membrane upon uniform cAMP stimulation in a time course that parallels F-actin polymerization. WIPa-overexpressing cells exhibit multiple microspike formation and defects in chemotactic efficiency due to frequent changes of direction. Reduced expression of WIPa by expressing a hairpin WIPa (hp WIPa) construct resulted in more polarized cells that exhibit a delayed response to a new chemoattractant source due to delayed extension of pseudopod toward the new gradient. These results suggest that WIPa is required for new pseudopod protrusion and prompt reorientation of cells toward a new gradient by initiating localized bursts of actin polymerization and/or elongation.     

Guanylyl cyclase protein and cGMP product independently control front and back of chemotaxing Dictyostelium cells

Chemotaxis of amoeboid cells is driven by actin filaments in leading pseudopodia and actin-myosin filaments in the back and at the side of the cell to suppress pseudopodia. In Dictyostelium, cGMP plays an important role during chemotaxis and is produced predominantly by a soluble guanylyl cyclase (sGC). The sGC protein is enriched in extending pseudopodia at the leading edge of the cell during chemotaxis. We show here that the sGC protein and the cGMP product have different functions during chemotaxis, using two mutants that lose either catalytic activity (sGCDelta cat) or localization to the leading edge (sGCDeltaN). Cells expressing sGCDeltaN exhibit excellent cGMP formation and myosin localization in the back of the cell, but they exhibit poor orientation at the leading edge. Cells expressing the catalytically dead sGCDelta cat mutant show poor myosin localization at the back, but excellent localization of the sGC protein at the leading edge, where it enhances the probability that a new pseudopod is made in proximity to previous pseudopodia, resulting in a decrease of the degree of turning. Thus cGMP suppresses pseudopod formation in the back of the cell, whereas the sGC protein refines pseudopod formation at the leading edge.     

Myosin II dynamics in Dictyostelium: determinants for filament assembly and translocation to the cell cortex during chemoattractant responses

In the simple amoeba Dictyostelium discoideum, myosin II filament assembly is regulated primarily by the action of a set of myosin heavy chain (MHC) kinases and by MHC phosphatase activity. Chemoattractant signals acting via G-protein coupled receptors lead to rapid recruitment of myosin II to the cell cortex, but the structural determinants on myosin necessary for translocation and the second messengers upstream of MHC kinases and phosphatases are not well understood. We report here the use of GFP-myosin II fusions to characterize the domains necessary for myosin II filament assembly and cytoskeletal recruitment during responses to global stimulation with the developmental chemoattractant cAMP. Analysis performed with GFP-myosin fusions, and with latrunculin A-treated cells, demonstrated that F-actin binding via the myosin motor domain together with concomitant filament assembly mediates the rapid cortical translocation observed in response to chemoattractant stimulation. A "headless" GFP-myosin construct lacking the motor domain was unable to translocate to the cell cortex in response to chemoattractant stimulation, suggesting that myosin motor-based motility may drive translocation. This lack of localization contrasts with previous work demonstrating accumulation of the same construct in the cleavage furrow of dividing cells, suggesting that recruitment signals and interactions during cytokinesis differ from those during chemoattractant responses. Evaluating upstream signaling, we find that iplA null mutants, devoid of regulated calcium fluxes during chemoattractant stimulation, display full normal chemoattractant-stimulated myosin assembly and translocation. These results indicate that calcium transients are not necessary for chemoattractant-regulated myosin II filament assembly and translocation.     

Expression in Escherichia coli of a functional Dictyostelium myosin tail fragment

The amino acid sequence of the myosin tail determines the specific manner in which myosin molecules are packed into the myosin filament, but the details of the molecular interactions are not known. Expression of genetically engineered myosin tail fragments would enable a study of the sequences important for myosin filament formation and its regulation. We report here the expression in Escherichia coli of a 1.5- kb fragment of the Dictyostelium myosin heavy chain gene coding for a 58-kD fragment of the myosin tail. The expressed protein (DdLMM-58) was purified to homogeneity from the soluble fraction of E. coli extracts. The expressed protein was found to be functional by the following criteria: (a) it appears in the electron microscope as a 74-nm-long rod, the predicted length for an alpha-helical coiled coil of 500 amino acids; (b) it assembles into filamentous structures that show the typical axial periodicity of 14 nm found in muscle myosin native filaments; (c) its assembly into filaments shows the same ionic strength dependence as Dictyostelium myosin; (d) it serves as a substrate for the Dictyostelium myosin heavy chain kinase which phosphorylates myosin in response to chemotactic signaling; (e) in its phosphorylated form it has the same phosphoamino acids and similar phosphopeptide maps to those of phosphorylated Dictyostelium myosin heavy chain; (f) it competes with myosin for the heavy chain kinase. Thus, all the information required for filament formation and phosphorylation is contained within this expressed protein.     

Chemoattractant-elicited increases in Dictyostelium myosin phosphorylation are due to changes in myosin localization and increases in kinase activity

We previously reported (Berlot, C. H., Spudich, J. A., and Devreotes, P. N. (1985) Cell 43, 307-314) that cAMP stimulation of chemotactically competent Dictyostelium amoebae causes transient increases in phosphorylation of the myosin heavy chain and 18,000-dalton light chain in vivo and in vitro. In this report we investigate the mechanisms involved in these changes in phosphorylation. In the case of heavy chain phosphorylation, the amount of substrate available for phosphorylation appears to be the major factor regulating the in vitro phosphorylation rate. Almost all heavy chain kinase activity is insoluble in Triton X-100, and the increase in the heavy chain phosphorylation rate in vitro parallels an increase in Triton insolubility of myosin. Changes in heavy chain phosphatase activity are not involved in the changes in the in vitro phosphorylation rate. In the case of light chain phosphorylation, increases in the vitro phosphorylation rate occur under conditions where the amount of substrate available for phosphorylation is constant and phosphatase activity is undetectable, implicating light chain kinase activation as the means of regulation. The specificity of the myosin kinases operating in vivo and in vitro was explored using phosphoamino acid and chymotryptic phosphopeptide analysis. The light chain is phosphorylated on serine both in vivo and in vitro, and phosphopeptide maps of the light chain phosphorylated in vivo and in vitro are indistinguishable. In the case of the heavy chain, both serine and threonine are phosphorylated in vivo and in vitro, although the cAMP-stimulated increases in phosphorylation occur primarily on threonine. Phosphopeptide maps of the heavy chain show that the peptides phosphorylated in vitro represent a major subset of those phosphorylated in vivo. The kinetics of the transient increases in myosin phosphorylation rates observed in vitro can be predicted quantitatively from the in vivo myosin phosphorylation data assuming that there is a constant phosphatase activity.     

Signal transduction and motility ofDictyostelium

This review is concerned with the roles of cyclic GMP and Ca²⁺ ions in signal transduction for chemotaxis ofDictyostelium. These molecules are involved in signalling between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Evidence is presented for uptake and/or eflux of Ca²⁺ being regulated by cyclic GMP. The link between Ca²⁺, cyclic GMP and chemotactic cell movement has been explored using streamer F mutants whose primary defect is in the structural gene for the cyclic GMP-specific phosphodiesterase. This mutation causes the mutants to produce an abnormally prolonged peak of cyclic GMP accumulation in response to stimulation with the chemoattractant cyclic AMP. The production and relay of cyclic AMP signals is normal in these mutants, but certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca²⁺ uptake, myosin II association with the cytoskeleton and regulation of both myosin heavy and light chain phosphorylation. These changes can be correlated with changes in the shape of the amoebae after chemotactic stimulation. Other mutants in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses.A model is described in which cyclic GMP (directly or indirectly via Ca²⁺) regulates accumulation of myosin II on the cytoskeleton by regulating phosphorylation of the myosin heavy and light chain kinases.     

Local and spatially coordinated movements in Dictyostelium discoideum amoebae during chemotaxis

We have studied chemotaxis by individual Dictyostelium discoideum amoebae using strong, local gradients of the chemoattractant cyclic AMP. Gradients were provided by diffusion of cyclic AMP from a microneedle, which could be positioned at various points around the cell. Responses to changes in the gradient indicate how the cell is structurally organized for chemotactic movement. There is a polarity in the responsiveness of the surface to stimulation by cyclic AMP along the length of the amoeba. Furthermore, two aspects of chemotactic movement can be distinguished. The first response to cyclic AMP is a locally generated extension of a hyaline pseudopod from the region of the surface nearest the stimulus. The second response, the flow of cytoplasm in the direction of the stimulus, is coordinated and separate from the first response. The coordination appears to depend on the nucleus or on the microtubule-organizing center.     

Actin activation of myosin heavy chain kinase A in Dictyostelium: a biochemical mechanism for the spatial regulation of myosin II filament disassembly

Studies in Dictyostelium discoideum have established that the cycle of myosin II bipolar filament assembly and disassembly controls the temporal and spatial localization of myosin II during critical cellular processes, such as cytokinesis and cell locomotion. Myosin heavy chain kinase A (MHCK A) is a key enzyme regulating myosin II filament disassembly through myosin heavy chain phosphorylation in Dictyostelium. Under various cellular conditions, MHCK A is recruited to actin-rich cortical sites and is preferentially enriched at sites of pseudopod formation, and thus MHCK A is proposed to play a role in regulating localized disassembly of myosin II filaments in the cell. MHCK A possesses an aminoterminal coiled-coil domain that participates in the oligomerization, cellular localization, and actin binding activities of the kinase. In the current study, we show that the interaction between the coiled-coil domain of MHCK A and filamentous actin leads to an approximately 40-fold increase in the initial rate of kinase catalytic activity. Actin-mediated activation of MHCK A involves increased rates of kinase autophosphorylation and requires the presence of the coiled-coil domain. Structure-function analyses revealed that the coiled-coil domain alone binds to actin filaments (apparent K(D) = 0.9 microm) and thus mediates the direct interaction with F-actin required for MHCK A activation. Collectively, these results indicate that MHCK A recruitment to actin-rich sites could lead to localized activation of the kinase via direct interaction with actin filaments, and thus this mode of kinase regulation may represent an important mechanism by which the cell achieves localized disassembly of myosin II filaments required for specific changes in cell shape.