Intracellular vesicle movement, cAMP and myosin II in Dictyostelium

Dictyostelium amoebae were analyzed before and after rapid addition of 10(-6) M cAMP for cellular motility, dynamic shape changes, and intracellular particle movement. Before cAMP addition, amoebae moved in a persistent anterior fashion and were elongate with F-actin localized predominantly in the anterior pseudopod. Intracellular particles moved rapidly and anteriorly. Within seconds after 10(-6) M cAMP addition, cells stopped translocating, pseudopod formation ceased, intracellular particle movement was depressed, and F-actin was lost from the pseudopod and concomitantly relocalized in the cell cortex. After 10 seconds, expansion zones reappeared but were small and no longer anteriorly localized. Vesicle movement partially rebounded but was no longer anteriorly directed. The myosin II null mutant HS2215 exhibited both depressed cellular translocation and vesicle movement. The addition of cAMP to HS2215 cells did not result in any detectable change in the random, depressed movement of particles. The results with HS2215 suggest that myosin II is essential for (1) rapid cellular translocation, (2) cellular polarity, (3) rapid particle movement, (4) anteriorly directed particle movement, and (5) the cAMP response. Electron micrographs suggest that at least half of the particles examined in this study contain in turn smaller membrane bound vesicles or multilamellar membrane bodies. The possible role of these vesicles is discussed.     

cAMP-mediated inhibition of intracellular particle movement and actin reorganization in Dictyostelium

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.     

Behavior of Dictyostelium amoebae is regulated primarily by the temporal dynamic of the natural cAMP wave.

The instantaneous velocity plots of Dictyostelium discoideum amoebae responding to natural waves and simulated temporal waves of cAMP with periods of 7 min are highly similar. This similarity has been used to deduce the dynamics of a natural wave crossing an amoeba, and the behavior of amoebae has been characterized during the different phases of a natural wave with a computer-assisted dynamic image analyzing system. During the first approximately 150 sec of the front of a natural wave, cells move persistently toward the aggregation center, with high instantaneous velocity and a decreased frequency of lateral pseudopod formation. During the last 30 sec of the front of the wave and the first 30 sec of the back of the wave, there is a "freeze" in cell shape and a dramatic depression in cell motility, pseudopod formation, and intracellular particle movement. During the last 180 sec of the back of the wave, there is a rebound in pseudopod formation, but it is random in direction and leads to no net cellular translocation. The data suggest that all of the behavior of a cell but orientation during the translocation phase is mediated by the temporal dynamics of the wave. The data also suggest that orientation toward the aggregation center occurs early in the front of the wave and that, once oriented, cells move in a blind fashion during the translocation phase.     

The unconventional myosin encoded by the myoA gene plays a role in Dictyostelium motility.

The myoA gene of Dictyostelium is a member of a gene family of unconventional myosins. The myosin Is share homologous head and basic domains, but the myoA gene product lacks the glycine-, proline-, alanine-rich and src homology 3 domains typical of several of the other myosin Is. A mutant strain of Dictyostelium lacking a functional myoA gene was produced by gene targeting, and the motility of this strain in buffer and a spatial gradient of the chemoattractant cyclic AMP was analyzed by computer-assisted methods. The myoA- cells have a normal elongate morphology in buffer but exhibit a decrease in the instantaneous velocity of cellular translocation, an increase in the frequency of lateral pseudopod formation, and an increase in turning. In a spatial gradient, in which the frequency of pseudopod formation is depressed, myoA- cells exhibit positive chemotaxis but still turn several times more frequently than control cells. These results demonstrate that the other members of the unconventional myosin family do not fully compensate for the loss of functional myoA gene product. Surprisingly, the phenotype of the myoA- strain closely resembles that of the myoB- strain, suggesting that both play a role in the frequency of pseudopod formation and turning during cellular translocation.     

Myosin II heavy chain null mutant of Dictyostelium exhibits defective intracellular particle movement

Both cellular motility and intracellular particle movement are compared between normal Dictyostelium amebae of strain AX4 and amebae of a myosin II heavy chain null mutant, HS2215, using the computer assisted "Dynamic Morphology System." In AX4 cells rapidly translocating in buffer, cytoplasmic expansion is apical and the majority of intracellular particles move anteriorly, towards the site of expansion. When these cells are pulsed with 10(-6) M cAMP, the peak concentration of the natural cAMP wave, cells stop translocating and average particle velocity decreases threefold within 2-4 s after cAMP addition. After 8 s, there is a partial rebound both in cytoplasmic expansion and particle velocity, but in both cases, original apical polarity is lost. In HS2215 cells in buffer, both cellular translocation and average particle velocity are already at the depressed levels observed in normal cells immediately after cAMP addition, and no anterior bias is observed in either the direction of cytoplasmic expansion or the direction of particle movement. The addition of cAMP to myosin-minus cells results in no additional effect. The results demonstrate that myosin II is necessary for (a) the rapid rate of intracellular particle movement, (b) the biased anterior directionality of particle movement, and (c) the rapid inhibition of particle movement by cAMP.     

Myosin I phosphorylation is increased by chemotactic stimulation

Directed cell migration occurs in response to extracellular cues. Following stimulation of a cell with chemoattractant, a significant rearrangement of the actin cytoskeleton is mediated by intracellular signaling pathways and results in polarization of the cell and movement via pseudopod extension. Amoeboid myosin Is play a critical role in regulating pseudopod formation in Dictyostelium, and their activity is activated by heavy chain phosphorylation. The effect of chemotactic stimulation on the in vivo phosphorylation level of a Dictyostelium myosin I, myoB, was tested. The myoB heavy chain is phosphorylated in vivo on serine 322 (the myosin TEDS rule phosphorylation site) in chemotactically competent cells. The level of myoB phosphorylation increases following stimulation of starving cells with the chemoattractant cAMP. A 3-fold peak increase in the level of phosphorylation is observed at 60 s following stimulation, a time at which the Dictyostelium cell actively extends pseudopodia. These findings suggest that chemotactic stimulation results in increased myoB activity via heavy chain phosphorylation and contributes to the global extension of pseudopodia that occurs prior to polarization and directed motility.     

Chemotaxis: signalling modules join hands at front and tail

Chemotaxis is the result of a refined interplay among various intracellular molecules that process spatial and temporal information. Here we present a modular scheme of the complex interactions between the front and the back of cells that allows them to navigate. First, at the front of the cell, activated Rho-type GTPases induce actin polymerization and pseudopod formation. Second, phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) is produced in a patch at the leading edge, where it binds pleckstrin-homology-domain-containing proteins, which enhance actin polymerization and translocation of the pseudopod. Third, in Dictyostelium amoebae, a cyclic-GMP-signalling cascade has been identified that regulates myosin filament formation in the posterior of the cell, thereby inhibiting the formation of lateral pseudopodia that could misdirect the cell.     

Sphingosine-1-phosphate plays a role in the suppression of lateral pseudopod formation during Dictyostelium discoideum cell migration and chemotaxis

Sphingosine-1-phosphate (S-1-P) is a bioactive lipid that plays a role in diverse biological processes. It functions both as an extracellular ligand through a family of high-affinity G-protein-coupled receptors, and intracellularly as a second messenger. A growing body of evidence has implicated S-1-P in controlling cell movement and chemotaxis in cultured mammalian cells. Mutant D. discoideum cells, in which the gene encoding the S-1-P lyase had been specifically disrupted by homologous recombination, previously were shown to be defective in pseudopod formation, suggesting that a resulting defect might exist in motility and/or chemotaxis. To test this prediction, we analyzed the behavior of mutant cells in buffer, and in both spatial and temporal gradients of the chemoattractant cAMP, using computer-assisted 2-D and 3-D motion analysis systems. Under all conditions, S-1-P lyase null mutants were unable to suppress lateral pseudopod formation like wild-type control cells. This resulted in a reduction in velocity in buffer and spatial gradients of cAMP. Mutant cells exhibited positive chemotaxis in spatial gradients of cAMP, but did so with lowered efficiency, again because of their inability to suppress lateral pseudopod formation. Mutant cells responded normally to simulated temporal waves of cAMP but mimicked the temporal dynamics of natural chemotactic waves. The effect must be intracellular since no homologs of the S-1-P receptors have been identified in the Dictyostelium genome. The defects in the S-1-P lyase null mutants were similar to those seen in mutants lacking the genes for myosin IA, myosin IB, and clathrin, indicating that S-1-P signaling may play a role in modulating the activity or organization of these cytoskeletal elements in the regulation of lateral pseudopod formation.     

The actomyosin cytoskeleton of amoebae of the cellular slime molds acrasis rosea and protostelium mycophaga: structure, biochemical properties, and function

In amoebae of the cellular slime molds (mycetozoans) Acrasis rosea and Protostelium mycophaga, bundles of F-actin radiate from the endoplasm-ectoplasm interface into the pseudopodia, where G-actin is also located. We conclude that these actin bundles form a core scaffold driving pseudopod extension which is subsequently completed by filling with a more loosely organized meshwork of F-actin. Some bipolar, elongate amoebae of A. rosea also contained long bundles of F-actin that traverse the cells lengthwise and remotely resemble stress fibers. Rodlets of F-actin were scattered in the body of amoebae of A. rosea or formed star-shaped or polygonal complexes near or around contractile vacuoles, where they may play a role in contraction. In total protein extracts analyzed by SDS-PAGE and immunoblots the actins migrated like the rabbit skeletal muscle control. The relative proportion of actin in total protein extracts was 7.9% for A. rosea and 34.5% for P. mycophaga. We detected four or five isoactins in extracts of both species and we determined that the genome of each species contains approximately six actin genes. Whether they are all expressed or if posttranslational modifications occur remains to be determined. Myosin II was enriched in actomyosin extracts; its Mr was 187.8 kDa for A. rosea and 220.7 kDa for P. mycophaga. Cell models ("ghosts") contracted upon the addition of ATP. We conclude that amoebae of A. rosea and P. mycophaga, although behaving differently from those of Dictyostelium discoideum, contain the basic repertoire of molecules that enable pseudopod extension by actin polymerization and ATP-induced contraction of the cell cortex. Copyright 1998 Academic Press.     

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.     

A developmentally regulated Na-H exchanger in Dictyostelium discoideum is necessary for cell polarity during chemotaxis

Increased intracellular H(+) efflux is speculated to be an evolutionarily conserved mechanism necessary for rapid assembly of cytoskeletal filaments and for morphological polarity during cell motility. In Dictyostelium discoideum, increased intracellular pH through undefined transport mechanisms plays a key role in directed cell movement. We report that a developmentally regulated Na-H exchanger in Dictyostelium discoideum (DdNHE1) localizes to the leading edge of polarized cells and is necessary for intracellular pH homeostasis and for efficient chemotaxis. Starved DdNHE1-null cells (Ddnhe1(-)) differentiate, and in response to the chemoattractant cAMP they retain directional sensing; however, they cannot attain a polarized morphology, but instead extend mislocalized pseudopodia around the cell and exhibit decreased velocity. Consistent with impaired polarity, in response to chemoattractant, Ddnhe1(-) cells lack a leading edge localization of F-actin and have significantly attenuated de novo F-actin polymerization but increased abundance of membrane-associated phosphatidylinositol 3,4,5-trisphosphate (PI((3,4,5))P(3)). These findings indicate that during chemotaxis DdNHE1 is necessary for establishing the kinetics of actin polymerization and PI((3,4,5))P(3) production and for attaining a polarized phenotype.     

Role of spectrin in Amoeba proteus, as studied by microinjection of anti-spectrin monoclonal antibodies.

Spectrin is a major protein accounting for about 5% of whole-cell proteins in Amoeba proteus, and the precipitation of spectrin by intracellular injection of purified anti-spectrin monoclonal antibodies has a profound effect on cell morphology, motility, and movement-related cell activities in amoebae. Thus, amoebae injected with anti-spectrin antibodies show drastic changes in their shape and movement, suggesting that amoeba spectrin plays an important structural role, unlike nonerythroid spectrins in other cells. However, precipitation of spectrin does not affect the distribution of F-actin in amoebae.     

Caenorhabditis elegans spermatozoan locomotion: amoeboid movement with almost no actin

The pseudopods of Caenorhabditis elegans spermatozoa move actively causing some cells to translocate when the sperm are dissected into a low osmotic strength buffered salts solution. On time-lapse video tapes, pseudopodial projections can be seen moving at 20-45 micrometers/min from the tip to the base of the pseudopod. This movement occurs whether or not the cell is attached to a substrate. Translocation of the cell is dependent on the substrate. Some spermatozoa translocate on acid-washed glass, but a better substrate is prepared by drying an extract of Ascaris uteri (the normal site of nematode sperm motility) onto glass slides. On this substrate more than half the spermatozoa translocate at a velocity (21 micrometers/min) similar to that observed in vivo. Translocating cells attach to the substrate by their pseudopodial projections. They always move toward the pseudopod; changes in direction are caused by changes in pseudopod shape that determine points of detachment and reattachment of the cell to the substrate. Actin comprises less than 0.02% of the proteins in sperm, and myosin is undetectable. No microfilaments are found in the sperm. Immunohistochemistry shows that some actin is localized in patches in the pseudopod. The movement of spermatozoa is unaffected by cytochalasins, however, so there is no evidence that actin participates in locomotion. Fertilization-defective mutants in genes fer-2, fer-4, and fer-6 produce spermatozoa with defective pseudopodial projections, and these spermatozoa are largely immotile. Mutants in the spermatozoa do not translocate. Thus pseudopod movement is correlated with the presence of normal projections. Twelve mutants with defective muscles have spermatozoa with normal movement, so these genes do not specify products needed for both muscle and nonmuscle cell motility.     

Characterization of Amoeba proteus myosin VI immunoanalog

Amoeba proteus, the highly motile free-living unicellular organism, has been widely used as a model to study cell motility. However, molecular mechanisms underlying its unique locomotion and intracellular actin-based-only trafficking remain poorly understood. A search for myosin motors responsible for vesicular transport in these giant cells resulted in detection of 130-kDa protein interacting with several polyclonal antibodies against different tail regions of human and chicken myosin VI. This protein was binding to actin in the ATP-dependent manner, and immunoprecipitated with anti-myosin VI antibodies. In order to characterize its possible functions in vivo, its cellular distribution and colocalization with actin filaments and dynamin II during migration and pinocytosis were examined. In migrating amoebae, myosin VI immunoanalog localized to vesicular structures, particularly within the perinuclear and sub-plasma membrane areas, and colocalized with dynamin II immunoanalog and actin filaments. The colocalization was even more evident in pinocytotic cells as proteins concentrated within pinocytotic pseudopodia. Moreover, dynamin II and myosin VI immunoanalogs cosedimented with actin filaments, and were found on the same isolated vesicles. Blocking endogenous myosin VI immunoanalog with anti-myosin VI antibodies inhibited the rate of pseudopodia protrusion (about 19% decrease) and uroidal retraction (about 28% decrease) but did not affect cell morphology and the manner of cell migration. Treatment with anti-human dynamin II antibodies led to changes in directionality of amebae migration and affected the rate of only uroidal translocation (about 30% inhibition). These results indicate that myosin VI immunoanalog is expressed in protist Amoeba proteus and may be involved in vesicle translocation and cell locomotion.     

Germinal center-specific protein human germinal center associated lymphoma directly interacts with both myosin and actin and increases the binding of myosin to actin

Human germinal center associated lymphoma (HGAL) is a germinal center-specific gene whose expression correlates with a favorable prognosis in patients with diffuse large B-cell and classic Hodgkin lymphomas. HGAL is involved in negative regulation of lymphocyte motility. The movement of lymphocytes is directly driven by actin polymerization and actin-myosin interactions. We demonstrate that HGAL interacts directly and independently with both actin and myosin and delineate the HGAL and myosin domains responsible for the interaction. Furthermore, we show that HGAL increases the binding of myosin to F-actin and inhibits the ability of myosin to translocate actin by reducing the maximal velocity of myosin head/actin movement. No effects of HGAL on actomyosin ATPase activity and the rate of actin polymerization from G-actin to F-actin were observed. These findings reveal a new mechanism underlying the inhibitory effects of germinal center-specific HGAL protein on lymphocyte and lymphoma cell motility.     

A Model for a Correlated Random Walk Based on the Ordered Extension of Pseudopodia

Cell migration in the absence of external cues is well described by a correlated random walk. Most single cells move by extending protrusions called pseudopodia. To deduce how cells walk, we have analyzed the formation of pseudopodia by Dictyostelium cells. We have observed that the formation of pseudopodia is highly ordered with two types of pseudopodia: First, de novo formation of pseudopodia at random positions on the cell body, and therefore in random directions. Second, pseudopod splitting near the tip of the current pseudopod in alternating right/left directions, leading to a persistent zig-zag trajectory. Here we analyzed the probability frequency distributions of the angles between pseudopodia and used this information to design a stochastic model for cell movement. Monte Carlo simulations show that the critical elements are the ratio of persistent splitting pseudopodia relative to random de novo pseudopodia, the Left/Right alternation, the angle between pseudopodia and the variance of this angle. Experiments confirm predictions of the model, showing reduced persistence in mutants that are defective in pseudopod splitting and in mutants with an irregular cell surface.     

Food Searching Strategy of Amoeboid Cells by Starvation Induced Run Length Extension

Food searching strategies of animals are key to their success in heterogeneous environments. The optimal search strategy may include specialized random walks such as Levy walks with heavy power-law tail distributions, or persistent walks with preferred movement in a similar direction. We have investigated the movement of the soil amoebae Dictyostelium searching for food. Dictyostelium cells move by extending pseudopodia, either in the direction of the previous pseudopod (persistent step) or in a different direction (turn). The analysis of ∼4000 pseudopodia reveals that step and turn pseudopodia are drawn from a probability distribution that is determined by cGMP/PLA2 signaling pathways. Starvation activates these pathways thereby suppressing turns and inducing steps. As a consequence, starved cells make very long nearly straight runs and disperse over ∼30-fold larger areas, without extending more or larger pseudopodia than vegetative cells. This ‘win-stay/lose-shift’ strategy for food searching is called Starvation Induced Run-length Extension. The SIRE walk explains very well the observed differences in search behavior between fed and starving organisms such as bumble-bees, flower bug, hoverfly and zooplankton.     

Navigation of Chemotactic Cells by Parallel Signaling to Pseudopod Persistence and Orientation

The mechanism of chemotaxis is one of the most interesting issues in modern cell biology. Recent work shows that shallow chemoattractant gradients do not induce the generation of pseudopods, as has been predicted in many models. This poses the question of how else cells can steer towards chemoattractants. Here we use a new computational algorithm to analyze the extension of pseudopods by Dictyostelium cells. We show that a shallow gradient of cAMP induces a small bias in the direction of pseudopod extension, without significantly affecting parameters such as pseudopod frequency or size. Persistent movement, caused by alternating left/right splitting of existing pseudopodia, amplifies the effects of this bias by up to 5-fold. Known players in chemotactic pathways play contrasting parts in this mechanism; PLA2 and cGMP signal to the cytoskeleton to regulate the splitting process, while PI 3-kinase and soluble guanylyl cyclase mediate the directional bias. The coordinated regulation of pseudopod generation, orientation and persistence by multiple signaling pathways allows eukaryotic cells to detect extremely shallow gradients.     

In vitro motility of skeletal muscle myosin and its proteolytic fragments

We have compared actin-activated myosin ATPase activity, myosin binding to actin, and the velocity of myosin-induced actin sliding in order to understand the mechanism of myosin motility. In our in vitro assay, F-actin slides at a constant velocity, regardless of length. The F-actin could slide over myosin heads at KCl concentrations below a critical value (60 mM with myosin and HMM, 100 mM with S-1), and the sliding velocities were quite similar below the critical KCl concentration. However, at KCl concentrations close to the critical value, the sliding F-actin is attached to only one or a few particular points on the surface, each of which perhaps consists of a single head of myosin. The KATPase values for actin-activated ATPase were approximately 300 microM for S-1 and approximately 200 microM with HMM below the critical KCl concentration, and approximately 5,000 microM above the critical KCl concentration. This increase in KATPase is due to a drastic reduction in the binding affinity of myosin heads to F-actin, as determined by a proteolytic digestion method and direct observation by fluorescence microscopy. We also show that the Vmax of actin-activated myosin ATPase activity decreases steadily with increasing KCl concentration, even though the velocity of F-actin sliding remains unchanged. This result provides evidence that the ATPase activity is not necessarily linked to motility. We discuss possible models that do not require a tight coupling between myosin ATPase and motility.