Immunochemical analysis of the supramolecular structure of myosin in contractile cytoskeletons of Dictyostelium amoebae

A collection of monoclonal antibodies against Dictyostelium myosin was screened to identify an antibody that could distinguish monomeric from polymeric myosin. An antibody was found that reacted only with monomeric myosin, provided that the antigen-antibody reaction was carried out in solution. This antibody was used in competition radioimmunoassays to probe the supramolecular structure of myosin in Triton-extracted cell models, or cytoskeletons, of Dictyostelium amoebae. The competition assay showed that, as isolated, cytoskeletal myosin was entirely filamentous, but could be converted to monomeric form by increasing the ionic strength of the surrounding buffer. As monomer, it remained associated with the cytoskeleton and could be cycled back to filament form by a second change of buffer. The ability of cytoskeletons to carry out ATP-dependent contraction was examined as a function of the assembly state of myosin. The results suggested that filamentous myosin is responsible for contraction of the cortical filament matrix.     

Multidirectional phototaxis by Dictyostelium discoideum amoebae

Abstract Phototaxis by solitary Dictyostelium discoideum amoebae is known to be complex, the amoebae turning either towards or away from the light, depending on conditions such as light intensity. Having previously shown that amoebal phototaxis can be bidirectional (2 preferred directions either side of the light source), we now report the discovery of multidirectional phototaxis by D. discoideum amoebae, with up to 12 different preferred directions. As in the bidirectional case, multidirectional phototaxis depends on direction-dependent transitions from turning away from to turning towards the light source.     

Prelysosomal acidic vacuoles in Dictyostelium discoideum

We have examined the ameba Dictyostelium discoideum for evidence of a discrete, prelysosomal, acidic receiving compartment in endocytosis. We observed in the cytoplasm abundant round vacuoles with diameters up to 2 microns that concentrated acridine orange by a process inhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). They were therefore taken to be acidic. The vacuoles were observed to fuse nearly quantitatively with primary phagosomes over 30 min and thereby to confer upon them the ability to accumulate acridine orange. The entry into lysosomes of phagocytic cargo occurred later. In the absence of phagocytosis, almost all of the acidic vacuoles rapidly accumulated fluorescent markers that had either been covalently coupled to the cell surface or fed as the soluble dextran conjugate. Therefore, these vacuoles also lie on the pathway of pinocytosis. A prominent subcellular ATPase activity inhibited by 25 microM NBD-Cl co-distributed on sucrose equilibrium density gradients with vacuoles capable of concentrating acridine orange in vitro. The peak was broad and more buoyant than that bearing lysosomal acid hydrolases, which contained only a minor amount of this ATPase. Also migrating in the buoyant peak were internalized plasma membrane markers; e.g., 3H-galactose had been covalently coupled to the surface of intact cells and allowed to enter pinosomes. We conclude that in D. discoideum an extensive prelysosomal vacuolar compartment provides the proton pumps that acidify both phagosomes and pinosomes.     

Giant vacuoles observed in Dictyostelium discoideum

Large intracellular vacuoles, >4 microm in diameter and either round or oval-shaped, were observed infrequently in Dictyostelium discoideum amoebae of axenically-grown strain AX2 (only 1 in 10(6)-10(8)cells). These previously unreported single or multiple 'giant' vacuoles were more common, however, in newly germinated KAX3 cells (0.55% of the population) and AT-K(neg), a strain that lacks an esterase (0.47% of the population). A vacuolar H(+)-ATPase was enriched in their membranes of intracellular giant vacuoles, indicating that the vacuoles were related possibly to both endosomes and the contractile vacuole compartment. When monitored over time, giant vacuoles protruded from, and retracted back into cells under hyperosmotic conditions, suggesting an osmoregulatory role for these vacuoles. Some of the intracellular and protruded giant vacuoles harbored a fluid-phase marker, fluorescein-labeled dextran, implying a pinocytotic origin for the vacuoles.     

Cytoimmunofluorescent localization of severin in Dictyostelium amoebae

Severin is a 40-kDa Ca2+-activated protein from Dictyostelium that rapidly fragments and disassembles actin filaments in vitro (S.S. Brown, K. Yamamoto, and J.A. Spudich, J. Cell Biol. 93, 205-210, 1982; and K. Yamamoto, J.D. Pardee, J. Reidler, L. Stryer, and J.A. Spudich. J. Cell Biol. 95, 711-719, 1982). To determine if severin is colocalized with actin filaments in vivo, we have used the agar-overlay technique of S. Yumura, H. Mori, and Y. Fukui (J. Cell Biol. 99, 894-899, 1984) to examine the intracellular locations of severin and F-actin in vegetative Dictyostelium amoebae. In rounded cells taken from suspension culture severin colocalized with F-actin at cortical edges while maintaining an endoplasmic presence. Both severin and F-actin were present throughout nascent pseudopods of motile cells, while severin appeared concentrated at the leading edge of fully developed pseudopods. Amoebae feeding on a bacterial lawn formed large phagocytic vesicles that were surrounded by an extensive cell cortex rich in severin. Streaming cells entering aggregates during the Dictyostelium developmental cycle showed severin staining throughout the cytoplasm with F-actin at the cortex. The preferential localization of severin in cytoplasmic regions of vegetative cells undergoing extensive actin cytoskeletal rearrangement prompts consideration of a role for severin-mediated disruption of actin filament networks during pseudopod extension and phagocytosis.     

Functional genomics of the social amoebae, Dictyostelium discoideum

Dictyostelium discoideum is one of the simplest organisms to form a multicellular structure, and it offers several advantages as a model. In order to understand the genetic basis of the multicellular development, a comprehensive analysis of cDNAs is being performed. To date, about 75,000 ESTs have been collected at different stages of development. They have been assembled into about 6,400 independent sequences that represent 70-80% of all of the expected genes in D. discoideum. The results are available on the Internet. In addition to structural analyses, functional analyses of the temporal and spatial expression patterns and gene targeting are being carried out. Furthermore, there are plans to combine the information that is obtained from the cDNA, Genome, and Proteome Projects, as well as the published results, into an integrated database, DictyBase.     

Efficient transformation of Dictyostelium discoideum amoebae.

We have transformed Dictyostelium discoideum amoebae by using derivatives of a plasmid, pAG60, which was designed for transformation of mammalian cells. The plasmid carries the promoter region of the herpes simplex virus type 1 thymidine kinase gene linked to the bacterial gene kan, which codes for the enzyme aminoglycoside 3'-phosphotransferase. kan is derived from the Tn5 transposon. Expression of the phosphotransferase permits direct selection of transformed cells by their resistance to the antibiotic G-418. pAG60 is incapable of transforming D. discoideum but is made transformation proficient by cloning D. discoideum sequences into the tetracycline resistance gene. The majority of transformed cells grow and develop normally and differentiate to give G-418-resistant spores. These transformants are unstable and rapidly lose their G-418-resistance during growth in the absence of antibiotic selection. Southern blots show that these unstable G-418-resistant transformants carry the pBR322 and kan sequences of pAG60. The pAG60-D. discoideum recombinant plasmids used for transformation were constructed in a way that might make them mutagenic. We have isolated several developmental mutants after transformation of D. discoideum with libraries of pAG60-D. discoideum recombinant plasmids. These mutants are G-418 resistant and carry pAG60 in their nuclear DNA. We recovered a pAG60-D. discoideum recombinant plasmid from several developmental mutants. This plasmid transforms D. discoideum at an elevated frequency and integrates into the nuclear genome. We speculate that integration can result in insertional inactivation of genes that are essential for differentiation but not for growth. Mutagenic transformation occurred only if the transforming plasmid had homology with D. discoideum nuclear DNA. A mammalian cell transformation vector, pSV2-neo, carried no D. discoideum sequences and was able to transform. However, pSV2-neo transformation was not mutagenic. These results suggest that direct inactivation and recovery of genes that are essential for differentiation of D. discoideum will be possible.     

Myosin filaments in cytoskeletons of Dictyostelium amoebae

Cytoskeletons were prepared from vegetative amoebae of Dictyostelium discoideum by extraction with Triton X-100. The cytoskeletons were suspended in buffers known to induce the assembly or disassembly of myosin filaments. The samples were fixed, and thin sections were examined by transmission electron microscopy. In both types of buffers, myosin-containing cytoskeletons exhibited a ring of densely staining proteinaceous material within the cortical filament matrix; this ring was not observed in myosin-free cytoskeletons. When myosin-containing cytoskeletons were placed in buffers that induced myosin polymerization, the ring appeared as an array of rodlike filaments approximately 13 nm wide and up to 0.5 micron in length--dimensions appropriate for myosin thick filaments. If ATP was added to cytoskeletons containing such filaments, the cytoskeletons contracted and the ring of filaments disappeared. ATP-induced contraction of cytoskeletons was also visualized by indirect immunofluorescence by using monoclonal antibodies to Dictyostelium myosin. All data were consistent with the identification of the protein ring seen by electron microscopy as cortical myosin. Its location and organization were appropriate for the production of cortical contraction through a sliding filament mechanism.     

Cyclic nucleotide-dependent phosphorylation in Dictyostelium discoideum amoebae

Stimulation of Dictyostelium discoideum amoebae with cAMP was found to induce the specific phosphorylation of a 47,000 molecular weight protein (pP47). This cellular response to cAMP was developmentally regulated. It was first detected in 3 1/2-h starved cells and appeared to persist throughout the aggregation phase of the cells' life cycle. pP47 phosphorylation was specifically induced by cAMP in that amoebae did not respond to stimulation with 5'-AMP, folic acid, Ca2+, and/or the Ca2+ ionophore A23187. cGMP could elicit pP47 phosphorylation but only at high concentrations. Phosphorylation of pP47 in response to cAMP occurred rapidly (within 5 s). The length of time for which it remained phosphorylated depended upon the concentration of the stimulus. With 10(-6) M cAMP, pP47 was phosphorylated for less than 4 min. If amoebae were stimulated with 10(-4) M cAMP, over 30 min were necessary before pP47 was dephosphorylated. Once dephosphorylated, pP47 could again be phosphorylated upon reapplication of the cAMP stimulus.     

Cyclic AMP waves during aggregation of Dictyostelium amoebae

During the aggregation phase of their life cycle, Dictyostelium discoideum amoebae communicate with each other by traveling waves of cyclic AMP. These waves are generated by an interplay between random diffusion of cyclic AMP in the extracellular milieu and the signal-reception/signal/relaying capabilities of individual amoebae. Kinetic properties of the enzymes, transport proteins and cell-surface receptor proteins involved in the cyclic AMP signaling system have been painstakingly worked out over the past fifteen years in many laboratories. Recently Martiel & Goldbeter (1987) incorporated this biochemical information into a unified mathematical model of communication among Dictyostelium amoebae. Numerical simulations of the mathematical model, carried out by Tyson et al. (1989), agree in quantitative detail with experimental observations of cyclic AMP traveling waves in Dictyostelium cultures. Such mathematical modeling and numerical experimentation provide a necessary link between detailed studies of the molecular control mechanism and experimental observations of the intact developmental system.     

Signal transduction for chemotaxis in Dictyostelium amoebae

The signal for chemotaxis in D. discoideum is cyclic AMP. This molecule binds to cell surface receptors and triggers the production of inositol (1,4,5)trisphosphate which releases Ca2+ from non-mitochondrial stores. The subsequent chain of signal transduction events brings about the polymerization of cytoskeletal actin (associated with pseudopodium formation) within five seconds and the formation of a peak of cyclic GMP within 10 s. Evidence from streamer F mutants indicates that the cyclic GMP regulates the association of myosin with the cytoskeleton that occurs at 25-50 s and that this phenomenon is concerned with elongation of the amoebae during chemotactic movement.     

Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae

The natural hosts of the bacterial pathogen Legionella pneumophila are amoebae and protozoa. In these hosts, as in human macrophages, the pathogen enters the cell through phagocytosis, then rapidly modifies the phagosome to create a compartment that supports its replication. We have examined L. pneumophila entry and behaviour during early stages of the infection of Dictyostelium discoideum amoebae. Bacteria were labelled with a red fluorescent marker, and selected proteins and organelles in the host were labelled with GFP, allowing the dynamics and interactions of L. pneumophila -containing phagosomes to be tracked in living cells. These studies demonstrated that entry of L. pneumophila is an actin-mediated process, that the actin-binding protein coronin surrounds the nascent phagosome but dissociates immediately after internalization, that ER membrane is not incorporated into a phagosome during uptake, that the newly internalized phagosome is rapidly transported about the cell on microtubules, that association of ER markers with the phagosome occurs in two steps that correlate with distinct changes in phagosome movement, and that the vacuolar H(+)-ATPase does not associate with mature replication vacuoles. These studies have clarified certain aspects of the infection process and provided new insights into the dynamic interactions between the pathogen and its host.     

Exploitation of other social amoebae by Dictyostelium caveatum

Dictyostelium amoebae faced with starvation trigger a developmental program during which many cells aggregate and form fruiting bodies that consist of a ball of spores held aloft by a thin stalk. This developmental strategy is open to several forms of exploitation, including the remarkable case of Dictyostelium caveatum, which, even when it constitutes 1/10(3) of the cells in an aggregate, can inhibit the development of the host and eventually devour it. We show that it accomplishes this feat by inhibiting a region of cells, called the tip, which organizes the development of the aggregate into a fruiting body. We use live-cell microscopy to define the D. caveatum developmental cycle and to show that D. caveatum amoebae have the capacity to ingest amoebae of other Dictyostelid species, but do not attack each other. The block in development induced by D. caveatum does not affect the expression of specific markers of prespore cell or prestalk cell differentiation, but does stop the coordinated cell movement leading to tip formation. The inhibition mechanism involves the constitutive secretion of a small molecule by D. caveatum and is reversible. Four Dictyostelid species were inhibited in their development, while D. caveatum is not inhibited by its own compound(s). D. caveatum has evolved a predation strategy to exploit other members of its genus, including mechanisms of developmental inhibition and specific phagocytosis.     

Kinetics of fluid-phase pinocytosis in Dictyostelium discoideum amoebae

Kinetics of pinocytosis in Dictyostelium discoideum were investigated over an extended period of time (up to 6 hours) using fluorescein isothiocyanate (FITC)-dextran as a fluid-phase marker. FITC-dextran added to the medium accumulated rapidly inside the cells with a rate of influx equivalent to 9 microns3 of fluid/cell x min. After a period of about 90 min of uptake, the intracellular FITC-dextran level reached a plateau which corresponded to a strict balance between pinocytosis and exocytosis as shown both by efflux measurements and pulse experiments with (3H) dextran. At equilibrium, the amount of internalized marker reached a value equivalent to 790 microns3 of fluid taken up per amoeba, i.e. a volume paradoxically higher than the total aqueous space of the cell (520 microns3 ). FITC-dextran was thus markedly concentrated intracellularly. The endocytic compartment in which the intracellular FITC-dextran was concentrated could be completely washed out when FITC-dextran was removed from the external medium.     

Contact alters cAMP metabolism in aggregation-competent Dictyostelium amoebae

cAMP and cell-cell contact are involved in the coordination of differentiation and morphogenesis in Dictyostelium discoideum. The experiments described in this paper establish a relationship between cAMP and cell-cell contact. Contact between Enterobacter aerogenes and aggregation-competent Dictyostelium amoebae and contact between Dictyostelium amoebae themselves results in the transient secretion of cAMP and an alteration in the amount of cAMP secreted in response to subsequent stimulation by cAMP, i.e., an alteration in magnitude of a cAMP relay response. The subsequent cAMP relay response can be enhanced or diminished depending upon the number of contacts formed and the concentration of cAMP present at the time of contact. Latex beads are capable of evoking cAMP secretion. However, the bead/amoebal contact is unable to alter the magnitude of a subsequent response to cAMP. This suggests that a nonspecific interaction via cell-cell contact elicits transient cAMP secretion in aggregation-competent Dictyostelium amoebae. The two responses to cell-cell contact are distinct from each other and distinct from the cAMP relay response. 1) The dose-response curves for the responses to Enterobacter contact are clearly different. 2) Contact with latex beads can elicit cAMP secretion but not alter the magnitude of a subsequent cAMP relay response. 3) The temperature dependences of the contact-induced responses and the cAMP relay response show that only the contact-induced cAMP secretion is inhibited at 12 and 15 degrees C, while only the cAMP relay response is inhibited at 28 degrees C. A 4-second application of cAMP at the time that contact is initiated enhances both contact-induced responses. Whether the relationship between these two developmental regulators is important for the regulation of Dictyostelium development has yet to be established.     

The cAMP signal from Dictyostelium discoideum amoebae.

Dictyostelium discoideum cells were allowed to differentiate on agar for 600 min at room temperature. All of the cells were then competent to relay or amplify a cAMP signal, but none to produce a cAMP signal autonomously. The cells were stimulated with cAMP concentrations ranging from 10^?9 to 3.5 × 10^?7M. Populations of 10⁶ cells could amplify an initial cAMP concentration of 2.5 × 10^?9M with a low probability, while an initial cAMP concentration of 5 × 10^?8M always induced a response. An initial cAMP concentration of 1.2 × 10^?7M induced the maximum cellular release of cAMP observed; this corresponded to 3 × 10⁷ molecules per cell. No cellular release of cAMP was detected for initial cAMP concentrations of 3 × 10^?7M or more. The amplification of a 10^?7M cAMP stimulus was complete within 8 sec, indicating the pulsatile nature of the cellular release of cAMP. The phosphodiesterase (PDE) activities of D. discoideum cells were measured over a wide range of cell densities. At densities above 7.5 × 10⁴ cells/cm², both cell-bound and extracellular (ePDE) activities declined, per cell, as cell density increased. These results are compared to ePDE activities derived from critical density measurements. We found that PDE activities were in the range of 10^?13–10^?14 moles of cAMP converted/cell/min under culture conditions consistent with normal aggregation.     

Investigating the macropinocytic proteome of Dictyostelium amoebae by high-resolution mass spectrometry

The cellular slime mold Dictyostelium discoideum is a soil-living eukaryote, which feeds on microorganisms engulfed by phagocytosis. Axenic laboratory strains have been produced that are able to use liquid growth medium internalized by macropinocytosis as the source of food. To better define the macropinocytosis process, we established the inventory of proteins associated with this pathway using mass spectrometry-based proteomics. Using a magnetic purification procedure and high-performance LC-MS/MS proteome analysis, a list of 2108 non-redundant proteins was established, of which 24% featured membrane-spanning domains. Bioinformatics analyses indicated that the most abundant proteins were linked to signaling, vesicular trafficking and the cytoskeleton. The present repertoire validates our purification method and paves the way for a future proteomics approach to study the dynamics of macropinocytosis.     

Structure and behavior of contractile vacuoles inChlamydomonas reinhardtii

Summary The contractile vacuole (CV) cycle ofChlamydomonas reinhardtii has been investigated by videomicroscopy and electron microscopy. Correlation of the two kinds of observation indicates that the total cycle (15 s under the hypo-osmotic conditions used for videomicroscopy) can be divided into early, middle, and late stages. In the early stage (early diastole, about 3 s long) numerous small vesicles about 70–120 nm in diameter are present. In the middle stage (mid-diastole, about 6 s long), the vesicles appear to fuse with one another to form the contractile vacuole proper. In the late stage (late diastole, also about 6 s long), the CV increases in diameter by the continued fusion of small vesicles with the vacuole, and makes contact with the plasma membrane. The CV then rapidly decreases in size (systole, about 0.2 s). In isosmotic media, CVs do not appear to be functioning; under these conditions, the CV regions contain numerous small vesicles typical of the earliest stage of diastole. Fine structure observations have provided no evidence for a two-component CV system such as has been observed in some other cell types. Electron microscopy of cryofixed and freeze-substituted cells suggests that the irregularity of the profiles of larger vesicles and vacuoles and some other morphological details seen in conventionally fixed cells may be shrinkage artefacts. This study thus defines some of the membrane events in the normal contractile vacuole cycle ofChlamydomonas, and provides a morphological and temporal basis for the study of membrane fusion and fluid transport across membranes in a cell favorable for genetic analysis.Abbrevations CV contractile vacuole - PM plasma membrane     

Effects of pteridines on the filopodia of Dictyostelium discoideum vegetative amoebae

Living vegetative amoebae of NC-4H Dictyostelium discoideum were studied to determine if a variety of pteridines had any effect on the filopodia. We observed that production, elongation, and branching of these filopodia were stimulated by pteridines that are chemoattractants for cells of this strain. This stimulation occurs at chemotactically effective concentrations and is observed before motility is evident. A relationship between filopodia and chemoattractant signal processing is discussed.