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.

Effects of cAMP on single cell motility in Dictyostelium.

The motility of individual, aggregation-competent amebae of Dictyostelium has been analyzed at different concentrations of cAMP under both nongradient and gradient conditions. The following is demonstrated: (a) concentrations of cAMP greater than 10(-8) M inhibit motility in a concentration-dependent fashion, decrease the frequency but not the degree of turning, and cause rounding in cell shape; (b) no concentration of cAMP stimulates motility, or positive chemokinesis; (c) concentrations of cAMP that stimulate a maximal chemotactic response do not affect motility and concentrations of cAMP that maximally inhibit motility do not stimulate chemotaxis under gradient conditions; and (d) the concentrations of cAMP that inhibit motility are identical under gradient and nongradient conditions.

Filament ring formation in the dimorphic yeast Candida albicans.

Stationary phase cultures of Candida albicans inoculated into fresh medium at 37 degrees C synchronously from buds at pH 4.5 and mycelia at pH 6.5. During bud formation, a filament ring forms just under the plasma membrane at the mother cell-bud junction at roughly the time of evagination. A filament ring also forms in mycelium-forming cells, but it appears later than in a budding cell and it is positioned along the elongating mycelium, on the average 2 microns from the mother cell-mycelium junction. Sections of filament rings in early and late budding cells and in mycelia appear similar. Each contains approximately 11 to 12 filaments equidistant from one another and closely associated with the plasma membrane. In both budding and mycelium-forming cells, the filament ring disappears when the primary septum grows inward. The close temporal and spatial association of the filament ring and the subsequent chitin-containing septum suggests a role for the filament ring in septum formation. In addition, a close temporal correlation is demonstrated between filament ring formation and the time at which cells become committed to bud formation at pH 4.5 and mycelium formation at pH 6.5. The temporal and spatial differences in filament ring formation between the two growth forms also suggest a simple model for the positioning of the filament ring.

Dictyostelium amebae alter motility differently in response to increasing versus decreasing temporal gradients of cAMP.

Using a perfusion chamber, we examined the behavior of individual amebae in increasing and decreasing temporal gradients of cAMP. We demonstrated that amebae respond to increasing temporal gradients of cAMP with stimulated motility and to corresponding decreasing temporal gradients with depressed motility. Depressed motility observed in decreasing temporal gradients corresponded to the inhibited levels observed when cAMP was applied at constant concentrations. These results were consistent with a simple model for the motile behavior of amebae in an early aggregation territory in which nondissipating waves of cAMP originate at the aggregation center and travel outward periodically. We conclude that chemotactically responsive amebae can assess whether a temporal gradient of chemoattractant is increasing or decreasing in the absence of a spatial gradient, and can adjust their motility accordingly.

Tortoise, a novel mitochondrial protein, is required for directional responses of Dictyostelium in chemotactic gradients.

We have identified a novel gene, Tortoise (TorA), that is required for the efficient chemotaxis of Dictyostelium discoideum cells. Cells lacking TorA sense chemoattractant gradients as indicated by the presence of periodic waves of cell shape changes and the localized translocation of cytosolic PH domains to the membrane. However, they are unable to migrate directionally up spatial gradients of cAMP. Cells lacking Mek1 display a similar phenotype. Overexpression of Mek1 in torA- partially restores chemotaxis, whereas overexpression of TorA in mek1- does not rescue the chemotactic phenotype. Regardless of the genetic background, TorA overexpressing cells stop growing when separated from a substrate. Surprisingly, TorA-green fluorescent protein (GFP) is clustered near one end of mitochondria. Deletion analysis of the TorA protein reveals distinct regions for chemotactic function, mitochondrial localization, and the formation of clusters. TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker. Cells overexpressing TorA contain many more of these structures than do wild-type cells. These TorA-containing structures resist extraction with Triton X-100, which dissolves the mitochondria. The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.

Inhibition of a nutrient-dependent pinocytosis in Dictyostelium discoideum by the amino acid analogue hadacidin.

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.

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.

Ponticulin plays a role in the positional stabilization of pseudopods.

Ponticulin is a 17-kD glycoprotein that represents a major high affinity link between the plasma membrane and the cortical actin network of Dictyostelium. To assess the role of ponticulin in pseudopod extension and retraction, the motile behavior of two independently generated mutants lacking ponticulin was analyzed using computer-assisted two- and three-dimensional motion analysis systems. More than half of the lateral pseudopods formed off the substratum by ponticulin-minus cells slipped relative to the substratum during extension and retraction. In contrast, all pseudopods formed off the substratum by wild-type cells were positionally fixed in relation to the substratum. Ponticulin-minus cells also formed a greater proportion of both anterior and lateral pseudopods off the substratum and absorbed a greater proportion of lateral pseudopods into the uropod than wild-type cells. In a spatial gradient of cAMP, ponticulin-minus cells were less efficient in tracking the source of chemoattractant. Since ponticulin-minus cells extend and retract pseudopods with the same time course as wild-type cells, these behavioral defects in ponticulin-minus cells appear to be the consequence of pseudopod slippage. These results demonstrate that pseudopods formed off the substratum by wild-type cells are positionally fixed in relation to the substratum, that ponticulin is required for positional stabilization, and that the loss of ponticulin and the concomitant loss of positional stability of pseudopods correlate with a decrease in the efficiency of chemotaxis.

Timers in developing systems.

Conditional methods are proposed for investigating the number and relationships of processes that are rate-limiting for the genesis of consecutive stages in a developmental sequence. These methods depend on the differential sensitivity of "timer" pathways to small changes in temperature and can be applied to any developmental sequence in which discrete stages can be reproducibly monitored with time. We have applied the methods to multicellular morphogenesis in the slime mold Dictyostelium discoideum and have obtained an unexpected tentative scheme for timer relationships. A minimum of six timers has been delineated, each specific for at least one morphological stage. The majority of these timers appear to be in parallel.

High-frequency switching of colony morphology in Candida albicans.

The pathogenic yeast Candida albicans switches heritably and at high frequency between at least seven general phenotypes identified by colony morphology on agar. Spontaneous conversion from the original smooth to variant phenotypes (star, ring, irregular wrinkle, hat, stipple, and fuzzy) occurs at a combined frequency of 1.4 X 10(-4), but is increased 200 times by a low dose of ultraviolet light that kills less than 10 percent of the cells. After the initial conversion, cells switch spontaneously to other phenotypes at a combined frequency of 2 X 10(-2). Switching is therefore heritable, but also reversible at high frequency. The genetic basis of this newly discovered process and its possible role in Candida pathogenesis are considered.

A role for myosin VII in dynamic cell adhesion.

BACKGROUND: The initial stages of phagocytosis and cell motility resemble each other. The extension of a pseudopod at the leading edge of a migratory cell and the formation of a phagocytic cup are actin dependent, and each rely on the plasma membrane adhering to a surface during dynamic extension. RESULTS: A myosin VII null mutant exhibited a drastic loss of adhesion to particles, consistent with the extent of an observed decrease in particle uptake. Additionally, cell-cell adhesion and the adhesion of the leading edge to the substratum during cell migration were defective in the myosin VII null cells. GFP-myosin VII rescued the phagocytosis defect of the null mutant and was distributed in the cytosol and recruited to the cortical cytoskeleton, where it appeared to be enriched at the tips of filopods. It was also localized to phagocytic cups, but only during the initial stages of particle engulfment. During migration, GFP-myosin VII is found at the leading edge of the cell. CONCLUSIONS: Myosin VII plays an important role in mediating the initial binding of cells to substrata, a novel role for an unconventional myosin.

High-frequency phenotypic switching in Candida albicans.

Most strains of Candida albicans are capable of switching spontaneously and at high frequencies between a number of phenotypes distinguished by colony morphology. Unlike switching in many other microbial pathogens, switching in C. albicans is pleiotropic, affecting several morphological and physiological parameters. Recently, the first phase-specific genes were identified and shown to be regulated at the level of gene transcription.

Functional analysis of the promoter of the phase-specific WH11 gene of Candida albicans.

Candida albicans WO-1 switches spontaneously, frequently, and reversibly between a hemispherical white and a flat gray (opaque) colony-forming phenotype. This transition affects a number of morphological and physiological parameters and involves the activation and deactivation of phase-specific genes. The WH11 gene is transcribed in the white but not the opaque phase. A chimeric WH11-firefly luciferase gene containing the 5' upstream region of WH11 was demonstrated to be under phase regulation regardless of the site of integration, and a series of promoter deletion constructs was used to delineate two white-phase-specific transcription activation domains. Gel retardation experiments with the individual distal or proximal domain and white-phase or opaque-phase protein extract demonstrated the formation of one distal white-phase-specific complex and two proximal white-phase-specific complexes. Specific subfragments were tested for their ability to compete with the entire domain in the formation of complexes with white-phase protein extract in order to map the proximal domain sequence involved in white-phase-specific complex formation. Our results indicate that white-phase-specific transcription of WH11 is positively regulated by trans-acting factors interacting with two cis-acting activation sequences in the WH11 promoter.

Transcription of the gene for a pepsinogen, PEP1, is regulated by white-opaque switching in Candida albicans.

Cells of Candida albicans WO-1 spontaneously switch between a white and opaque CFU, and this phase transition involves a dramatic change in cellular phenotype. By using a differential hybridization screen, an opaque-specific cDNA, Op1a, which represents the transcript of a gene regulated by switching, has been isolated. The gene for Op1a is transcribed by opaque but not by white cells. The nucleotide sequence of the Op1a cDNA reveals over 99% base homology with an acid protease gene of C. albicans, and the predicted amino acid sequence demonstrates that the product of this gene is a member of the family of pepsinogens, which possess a hydrophobic leader sequence for secretion and two catalytic aspartate domains. Southern blots of both genomic DNA digested with 14 different endonucleases and electrophoretically separated chromosomes were probed with the Op1a cDNA. No polymorphisms were detected in either case between white and opaque cells, suggesting that no genomic reorganization occurs in the proximity of the gene during the white-opaque transition. Although transcription of Op1a correlates with the high levels of extracellular protease activity in opaque cell cultures and the absence of activity in white cell cultures, stimulation of extracellular protease activity by addition of serum albumin is not accompanied by Op1a transcription in cultures of WO-1 white cells or cultures of two additional clinical isolates of C. albicans, suggesting that expression of one or more other protease genes is stimulated in these cases. The results demonstrate that transcription of the Op1a gene is under the rigid control of switching in strain WO-1.

Chemotaxis to cAMP and slug migration in Dictyostelium both depend on migA, a BTB protein.

Chemotaxis in natural aggregation territories and in a chamber with an imposed gradient of cyclic AMP (cAMP) was found to be defective in a mutant strain of Dictyostelium discoideum that forms slugs unable to migrate. This strain was selected from a population of cells mutagenized by random insertion of plasmids facilitated by introduction of restriction enzyme (a method termed restriction enzyme-mediated integration). We picked this strain because it formed small misshapen fruiting bodies. After isolation of portions of the gene as regions flanking the inserted plasmid, we were able to regenerate the original genetic defect in a fresh host and show that it is responsible for the developmental defects. Transformation of this recapitulated mutant strain with a construct carrying the full-length migA gene and its upstream regulatory region rescued the defects. The sequence of the full-length gene revealed that it encodes a novel protein with a BTB domain near the N terminus that may be involved in protein-protein interactions. The migA gene is expressed at low levels in all cells during aggregation and then appears to be restricted to prestalk cells as a consequence of rapid turnover in prespore cells. Although migA- cells have a dramatically reduced chemotactic index to cAMP and an abnormal pattern of aggregation in natural waves of cAMP, they are completely normal in size, shape, and ability to translocate in the absence of any chemotactic signal. They respond behaviorally to the rapid addition of high levels of cAMP in a manner indicative of intact circuitry connecting receptor occupancy to restructuring of the cytoskeleton. Actin polymerization in response to cAMP is also normal in the mutant cells. The defects at both the aggregation and slug stage are cell autonomous. The MigA protein therefore is necessary for efficiently assessing chemical gradients, and its absence results in defective chemotaxis and slug migration.

Re-expression of ABP-120 rescues cytoskeletal, motility, and phagocytosis defects of ABP-120- Dictyostelium mutants.

The actin binding protein ABP-120 has been proposed to cross-link actin filaments in nascent pseudopods, in a step required for normal pseudopod extension in motile Dictyostelium amoebae. To test this hypothesis, cell lines that lack ABP-120 were created independently either by chemical mutagenesis or homologous recombination. Different phenotypes were reported in these two studies. The chemical mutant shows only a subtle defect in actin cross-linking, while the homologous recombinant mutants show profound defects in actin cross-linking, cytoskeletal structure, pseudopod number and size, cell motility and chemotaxis and, as shown here, phagocytosis. To resolve the controversy as to what the ABP-120- phenotype is, ABP-120 was re-expressed in an ABP-120- cell line created by homologous recombination. Two independently "rescued" cell lines that express wild-type levels of ABP-120 were analyzed. In both rescued cell lines, actin incorporation into the cytoskeleton, pseudopod formation, cell morphology, instantaneous velocity, phagocytosis, and chemotaxis were restored to wild-type levels. There is no alteration in the expression levels of several related actin binding proteins in either the original ABP-120- cell line or in the rescued cell lines, leading to the conclusion that neither the aberrant phenotype observed in ABP-120- cells nor the normal phenotype reasserted in rescued cells can be attributed to alterations in the levels of other abundant and related actin binding proteins. Re-expression of ABP-120 in ABP-120- cells reestablishes normal structural and behavioral parameters, demonstrating that the severity and properties of the structural and behavioral defects of ABP-120- cell lines produced by homologous recombination are the direct result of the absence of ABP-120.

The internal phosphodiesterase RegA is essential for the suppression of lateral pseudopods during Dictyostelium chemotaxis.

Dictyostelium strains in which the gene encoding the cytoplasmic cAMP phosphodiesterase RegA is inactivated form small aggregates. This defect was corrected by introducing copies of the wild-type regA gene, indicating that the defect was solely the consequence of the loss of the phosphodiesterase. Using a computer-assisted motion analysis system, regA(-) mutant cells were found to show little sense of direction during aggregation. When labeled wild-type cells were followed in a field of aggregating regA(-) cells, they also failed to move in an orderly direction, indicating that signaling was impaired in mutant cell cultures. However, when labeled regA(-) cells were followed in a field of aggregating wild-type cells, they again failed to move in an orderly manner, primarily in the deduced fronts of waves, indicating that the chemotactic response was also impaired. Since wild-type cells must assess both the increasing spatial gradient and the increasing temporal gradient of cAMP in the front of a natural wave, the behavior of regA(-) cells was motion analyzed first in simulated temporal waves in the absence of spatial gradients and then was analyzed in spatial gradients in the absence of temporal waves. Our results demonstrate that RegA is involved neither in assessing the direction of a spatial gradient of cAMP nor in distinguishing between increasing and decreasing temporal gradients of cAMP. However, RegA is essential for specifically suppressing lateral pseudopod formation during the response to an increasing temporal gradient of cAMP, a necessary component of natural chemotaxis. We discuss the possibility that RegA functions in a network that regulates myosin phosphorylation by controlling internal cAMP levels, and, in support of that hypothesis, we demonstrate that myosin II does not localize in a normal manner to the cortex of regA(-) cells in an increasing temporal gradient of cAMP.

Reporters for the analysis of gene regulation in fungi pathogenic to man.

In the past few years, highly sensitive gene reporters have been developed for the infectious fungi including gene reporters with altered codon usage. The tools are, therefore, now at hand for functionally characterizing the promoters of genes regulated by the bud-hypha transition, high frequency switching and cues from the cellular environment.

Transcription and division inhibitors in the medium of stationary phase cultures of the slime mold Dictyostelium discoideum.

When cell-free medium of a stationary phase culture is added to an exponentially multiplying culture, it blocks further cell division and depresses the level of nuclear transcription. Furthermore, when cell-free medium of a stationary phase culture is added directly to an in vitro transcription system containing nuclei isolated from exponentially multiplying amebae, it completely inhibits "late" incorporation (20-60 min reaction time), which is predominantly alpha-amanitin resistant (ribosomal-like). The stationary phase medium however, affects neither "early" alpha-amanitin resistant incorporation (0-20 min reaction time) nor alpha-amanitin sensitive incorporation. Inhibition of late in vitro transcription is not due to a nucleotidase, nor is it due to changes in the pH, ionic strength, or osmolarity of the reaction mixture.

The use of computers in understanding how animal cells crawl.

Amoeboid cell motility is a complex three-dimensional process which involves pseudopod expansion, cellular translocation, and, in some cases, pseudopod retraction and complex interactions between the ventral surface of the pseudopod and substratum. In order to quantify the basic behavior of amoeboid cells and the dynamics of pseudopod extension and retraction, sophisticated two-dimensional and three-dimensional computer-assisted motion analysis systems have been developed which reconstruct digitized images and compute motility and dynamics morphology parameters. These systems provide a wealth of information of how amoeboid cells crawl and they have begun to be utilized (1) to elucidate the basic rules of amoeboid movement, (2) to identify the behavioral defects of cytoskeletal mutants, and (3) to elucidate the mechanism of chemotaxis. In addition, these systems represent powerful tools for analyzing the effects of drugs on cell behavior, most notably that of white blood cells and neoplastic cells. Since computer-assisted motion analysis is a relatively young field, the technologies are still evolving and have been underutilized in most areas involving cell motility. This review, which includes a description of these technologies and examples of their application, will hopefully serve as an impetus for expanded use.