A Mutant of Dictyostelium discoideum, KI-Cell, as a Model of Collective Cell Migration Independent of Chemotaxis.

Collective cell migration occurs when the orientation of cell polarity is aligned with each other in a group of cells. Such collective polarization depends on a reciprocal process between cell intrinsic mechanisms such as cell-cell adhesion and extracellular guidance mechanism such as wound healing and chemotaxis. As part of its development life cycle, individual single cells of Dictyostelium discoideum exhibit chemotaxis toward cAMP, which is secreted from a certain population of cells. During the formation of multicellular body by chemotaxis-dependent cell aggregation, D. discoideum is also known to relay on multiple cell-cell adhesion mechanisms. In particular, tail-following behavior at the contact site, called contact following of locomotion (CFL), plays a pivotal role on the formation of the multicellular body. However, whether and how CFL alone can lead to a formation of collective behavior was not well understood. KI cell is a mutant of D. discoideum that lacks all chemotactic activity. Yet, it can exhibit the CFL activity and show nontrivial collective cell migration. This mutant provides an excellent model system to analyze the mechanism of the CFL and the macroscopic phenomena brought by the CFL. This chapter describes protocols for using KI cell to understand the biophysics and cell biology behind the collective cell migration induced by CFL.

Peptide nucleic acid as a template for Taq DNA polymerase.

Peptide nucleic acid (PNA), an artificial DNA analog, comprises a purine or pyrimidine base and a pseudo-peptide backbone instead of deoxyribose-phosphate. PNA has been found to have stronger adhesion and higher stability in binding to its complementary DNA than deoxyribose-phosphate. Thus, it could serve as an agent for gene modulation, demonstrating potential in antisense therapy, molecular diagnostics, and nanotechnology. However, the applications of PNA remain limited because its biological activities are not fully known. Here, I demonstrate that a thermostable DNA polymerase, Thermus aquaticus (Taq) polymerase, exhibits transcriptase activity when a PNA oligomer is used as a template and that genetic information of the oligomer can be amplified by PCR using DNA primers. Furthermore, the insertion of a glutamine peptide stretch in the middle part of the PNA template did not interfere with transcription; it was transcribed into a guanosine or adenosine stretch. Intriguingly, this amino acid-to-DNA transcription did not occur when glycine residues were inserted. A synthetic PNA oligomer can, therefore, function as a template for a DNA polymerase, and polyglutamine peptides can be transcribed into guanosine or adenosine. These findings provide a cornerstone to reveal all amino acid genetic codes and transcription activity in the future.

The Life Cycle of Dictyostelium discoideum Is Accelerated via MAP Kinase Cascade by a Culture Extract Produced by a Synthetic Microbial Consortium.

A cellular slime mold, Dictyostelium discoideum, is an amoeboid organism that has a unique life cycle consisting of distinctly separated vegetative and developmental phases. Thus, this organism presents a rare opportunity in which to examine the effects of bioactive substances on separate cellular activities. In this research, we investigated the effect of a culture extract, termed EMXG, produced by a synthetic microbial consortium. EMXG promoted proliferative response of amoeba cells. It further accelerated the developmental phase, leading to the preferred fruiting body formation from fewer cells. Furthermore, EMXG modulated biological rhythm of this organism, that is, interval of oscillation of cAMP level observed in suspension starvation was significantly shortened. Concomitantly, the level of ERKB, a MAP kinase, was found to oscillate in a similar fashion to that of cAMP. Additionally, ErkB-deficient mutant amoeboid cells did not respond to proliferative stimulation by EMXG. These lines of evidence point to a likelihood that MAP kinase cascade is involved and further that ErkB could be the molecular target of EMXG.

Some chemotactic mutants can be progress through development in chimeric populations.

Cell migration in response to morphogen gradients affects morphogenesis. Chemotaxis towards adenosine 3', 5'-monophosphate (cAMP) is essential for the early stage of morphogenesis in the slime mold Dictyostelium discoideum. Here, we show that D. discoideum completes morphogenesis without cAMP-chemotaxis-dependent cell migration. The extracellular cAMP gradient is believed to cause cells to form a slug-shaped multicellular structure and fruiting body. The cAMP receptor, cAR1, was not expressed at the cell surface during these stages, correlating with reduced chemotactic activity. Gß-null cells expressing temperature sensitive Gß are unable to generate extracellular cAMP (Jin et al., 1998) and thus unable to aggregate and exhibit proper morphogenesis under restrictive temperature. However, when mixed with wild type cells ts-Gß expressing gß-null cells normally aggregated and exhibited normal morphogenesis under restrictive temperature. Furthermore, cells migrated after aggregation in a mixture containing wild-type cells. KI-5 cells, which do not show aggregation or morphogenesis, spontaneously migrated to a transplanted wild-type tip and underwent normal morphogenesis and cell differentiation; this was not observed in cells lacking tgrB1and tgrC1 cells adhesion molecules. Thus, cAMP gradient-dependent cell migration may not be required for multicellular pattern formation in late Dictyostelium development.

Chemoattractant receptors activate, recruit and capture G proteins for wide range chemotaxis.

The wide range sensing of extracellular signals is a common feature of various sensory cells. Eukaryotic chemotactic cells driven by GPCRs and their cognate G proteins are one example. This system endows the cells directional motility towards their destination over long distances. There are several mechanisms to achieve the long dynamic range, including negative regulation of the receptors upon ligand interaction and spatial regulation of G proteins, as we found recently. However, these mechanisms are insufficient to explain the 105-fold range of chemotaxis seen in Dictyostelium. Here, we reveal that the receptor-mediated activation, recruitment, and capturing of G proteins mediate chemotactic signaling at the lower, middle and higher concentration ranges, respectively. These multiple mechanisms of G protein dynamics can successfully cover distinct ranges of ligand concentrations, resulting in seamless and broad chemotaxis. Furthermore, single-molecule imaging analysis showed that the activated Gα subunit forms an unconventional complex with the agonist-bound receptor. This complex formation of GPCR-Gα increased the membrane-binding time of individual Gα molecules and therefore resulted in the local accumulation of Gα. Our findings provide an additional chemotactic dynamic range mechanism in which multiple G protein dynamics positively contribute to the production of gradient information.

Evidence that differentiation-inducing factor-1 controls chemotaxis and cell differentiation, at least in part, via mitochondria in D. discoideum.

Differentiation-inducing factor-1 [1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one (DIF-1)] is an important regulator of cell differentiation and chemotaxis in the development of the cellular slime mold Dictyostelium discoideum However, the entire signaling pathways downstream of DIF-1 remain to be elucidated. To characterize DIF-1 and its potential receptor(s), we synthesized two fluorescent derivatives of DIF-1, boron-dipyrromethene (BODIPY)-conjugated DIF-1 (DIF-1-BODIPY) and nitrobenzoxadiazole (NBD)-conjugated DIF-1 (DIF-1-NBD), and investigated their biological activities and cellular localization. DIF-1-BODIPY (5 µM) and DIF-1 (2 nM) induced stalk cell differentiation in the DIF-deficient strain HM44 in the presence of cyclic adenosine monosphosphate (cAMP), whereas DIF-1-NBD (5 µM) hardly induced stalk cell differentiation under the same conditions. Microscopic analyses revealed that the biologically active derivative, DIF-1-BODIPY, was incorporated by stalk cells at late stages of differentiation and was localized to mitochondria. The mitochondrial uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), at 25-50 nM, and dinitrophenol (DNP), at 2.5-5 µM, induced partial stalk cell differentiation in HM44 in the presence of cAMP. DIF-1-BODIPY (1-2 µM) and DIF-1 (10 nM), as well as CCCP and DNP, suppressed chemotaxis in the wild-type strain Ax2 in shallow cAMP gradients. These results suggest that DIF-1-BODIPY and DIF-1 induce stalk cell differentiation and modulate chemotaxis, at least in part, by disturbing mitochondrial activity.

Differentiation-inducing factor 2 modulates chemotaxis via the histidine kinase DhkC-dependent pathway in Dictyostelium discoideum.

Differentiation-inducing factor 1(DIF-1) and DIF-2 are signaling molecules that control chemotaxis in Dictyostelium discoideum. Whereas DIF-1 suppresses chemotaxis in shallow cAMP gradients, DIF-2 enhances chemotaxis under the same conditions via a phosphodiesterase, response regulator A (RegA), which is a part of the DhkC-RdeA-RegA two-component signaling system. In this study, to investigate the mechanism of the chemotaxis regulation by DIF-2, we examined the effects of DIF-2 (and DIF-1) on chemotaxis in rdeA(-) and dhkC(-) mutant strains. In the parental wild-type strains, chemotactic cell movement was suppressed with DIF-1 and enhanced with DIF-2 in shallow cAMP gradients. In contrast, in both rdeA(-) and dhkC(-) strains, chemotaxis was suppressed with DIF-1 but unaffected by DIF-2. The results suggest that DIF-2 modulates chemotaxis via the DhkC-RdeA-RegA signaling system.

Biological soliton in multicellular movement.

Solitons have been observed in various physical phenomena. Here, we show that the distinct characteristics of solitons are present in the mass cell movement of non-chemotactic mutants of the cellular slime mould Dictyostelium discoideum. During starvation, D. discoideum forms multicellular structures that differentiate into spore or stalk cells and, eventually, a fruiting body. Non-chemotactic mutant cells do not form multicellular structures; however, they do undergo mass cell movement in the form of a pulsatile soliton-like structure (SLS). We also found that SLS induction is mediated by adhesive cell-cell interactions. These observations provide novel insights into the mechanisms of biological solitons in multicellular movement.

Arachidonic acid enhances caffeine-induced cell death via caspase-independent cell death.

Caffeine is a globally consumed psychostimulant but can be fatal to cells at overdose exposures. Although caspase-dependent apoptosis plays a role in caffeine-induced cell death, the responsible intracellular signalling cascade remains incompletely understood. The cellular slime mould, Dictyostelium discoideum, does not possess caspase-dependent apoptotic machinery. Here, we observed that ablation of D. discoideumplaA, which encodes a phospholipase A2 (PLA2) homolog, leads to a decreased rate of cell death under high caffeine concentrations and to enhanced cell death with the addition of arachidonic acid. Moreover, the inhibition of PLA2 activity lead to a recovery of the survival rate in caspase-inhibited Hela cervical carcinoma cells under high caffeine concentrations, indicating that caffeine-induced cell death is enhanced via PLA2-dependent signalling. Our results indicate that arachidonic acid may be a general second messenger that negatively regulates caffeine tolerance via a caspase-independent cell death cascade, which leads to multiple effects in eukaryotic cells.

Developmental significance of cyanide-resistant respiration under stressed conditions: experiments in Dictyostelium cells.

We have previously reported that benzohydroxamic acid (BHAM), a potent inhibitor of cyanide (CN)-resistant respiration mediated by alternative oxidase (AOX), induces formation of unique cell masses (i.e., stalk-like cells with a large vacuole and thick cell wall) in starved Dictyostelium cells. Unexpectedly, however, aox-null cells prepared by homologous recombination exhibited normal development under normal culture conditions on agar, indicating that BHAM-induced stalk formation is not solely attributable to inhibition of CN-resistant respiration. This also suggests that a series of pharmacological approaches in the field of life science has serious limitations. Under stress (e.g., in submerged culture), starved aox-null cells exhibited slightly delayed aggregation compared with parental Ax-2 cells; most cells remained as loose aggregates even after prolonged incubation. Also, the developmental defects of aox-null cells became more marked upon incubation for 30 min just after starvation in the presence of ≥ 1.75 mmol/L H(2)O(2). This seems to indicate that CN-resistant respiration could mitigate cellular damage through reactive oxygen species (ROS), because AOX has a potential role in reduction of ROS production. Starved aox-null cells did not develop in the presence of 5 mmol/L KCN (which completely inhibited the conventional cytochrome-mediated respiration) and remained as non-aggregated single cells on agar even after prolonged incubation. Somewhat surprisingly, however, parental Ax-2 cells were found to develop normally, forming fruiting bodies even in the presence of 10 mmol/L KCN. Taken together, these results suggest that CN-resistant respiration might compensate for the production of adenosine tri-phosphate via oxidative phosphorylation.

Differentiation-inducing factor-1 and -2 function also as modulators for Dictyostelium chemotaxis.

BACKGROUND: In the early stages of development of the cellular slime mold Dictyostelium discoideum, chemotaxis toward cAMP plays a pivotal role in organizing discrete cells into a multicellular structure. In this process, a series of signaling molecules, such as G-protein-coupled cell surface receptors for cAMP, phosphatidylinositol metabolites, and cyclic nucleotides, function as the signal transducers for controlling dynamics of cytoskeleton. Differentiation-inducing factor-1 and -2 (DIF-1 and DIF-2) were originally identified as the factors (chlorinated alkylphenones) that induce Dictyostelium stalk cell differentiation, but it remained unknown whether the DIFs had any other physiologic functions. METHODOLOGY/PRINCIPAL FINDINGS: To further elucidate the functions of DIFs, in the present study we investigated their effects on chemotaxis under various conditions. Quite interestingly, in shallow cAMP gradients, DIF-1 suppressed chemotaxis whereas DIF-2 promoted it greatly. Analyses with various mutants revealed that DIF-1 may inhibit chemotaxis, at least in part, via GbpB (a phosphodiesterase) and a decrease in the intracellular cGMP concentration ([cGMP](i)). DIF-2, by contrast, may enhance chemotaxis, at least in part, via RegA (another phosphodiesterase) and an increase in [cGMP](i). Using null mutants for DimA and DimB, the transcription factors that are required for DIF-dependent prestalk differentiation, we also showed that the mechanisms for the modulation of chemotaxis by DIFs differ from those for the induction of cell differentiation by DIFs, at least in part. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that DIF-1 and DIF-2 function as negative and positive modulators for Dictyostelium chemotaxis, respectively. To our knowledge, this is the first report in any organism of physiologic modulators (small molecules) for chemotaxis having differentiation-inducing activity.

A stress response kinase, KrsA, controls cAMP relay during the early development of Dictyostelium discoideum.

Solitary amoebae of Dictyostelium discoideum are frequently exposed to stressful conditions in nature, and their multicellular development is one response to environmental stress. Here we analyzed an aggregation stage abundant gene, krsA, homologous to human krs1 (kinase responsive to stress 1) to understand the mechanisms for the initiation of development and cell fate determination. The krsA- cells exhibited reduced viability under hyperosmotic conditions. They produced smaller aggregates on membrane filters and did not form aggregation streams on a plastic surface under submerged starvation conditions, but were normal in sexual development. During early asexual development, the expression of cAMP-related genes peaked earlier in the knockout mutants. Neither cAMP oscillation in starved cells nor an increase in the cAMP level following osmotic stress was observed in krsA-. The nuclear export signal, as well as the kinase domain, in KrsA was necessary for stream formation. These results strongly suggest that krsA is involved in cAMP relay, and that signaling pathways for multicellular development have evolved in unison with the stress response.

Single-molecule analysis of chemoattractant-stimulated membrane recruitment of a PH-domain-containing protein.

Molecular mechanisms of chemotactic response are highly conserved among many eukaryotic cells including human leukocytes and Dictyostelium discoideum cells. The cells can sense the differences in chemoattractant concentration across the cell body and respond by extending pseudopods from the cell side facing to a higher concentration. Pseudopod formation is regulated by binding of pleckstrin homology (PH)-domain-containing proteins to phosphatidylinositol 3,4,5-trisphosphates [PtdIns(3,4,5)P3] localized at the leading edge of chemotaxing cells. However, molecular mechanisms underlying dynamic features of a pseudopod have not been fully explained by the known properties of PH-domain-containing proteins. To investigate the mechanisms, we visualized single molecules of green fluorescent protein tagged to Crac (Crac-GFP), a PH-domain-containing protein in D. discoideum cells. Whereas populations of Crac molecules exhibited a stable steady-state localization at pseudopods, individual molecules bound transiently to PtdIns(3,4,5)P3 for approximately 120 milliseconds, indicating dynamic properties of the PH-domain-containing protein. Receptor stimulation did not alter the binding stability but regulated the number of bound PH-domain molecules by metabolism of PtdIns(3,4,5)P3. These results demonstrate that the steady-state localization of PH-domain-containing proteins at the leading edge of chemotaxing cells is dynamically maintained by rapid recycling of individual PH-domain-containing proteins. The short interaction between PH domains and PtdIns(3,4,5)P3 contributes to accurate and sensitive chemotactic movements through the dynamic redistributions. These dynamic properties might be a common feature of signaling components involved in chemotaxis.

Periodic signaling controlled by an oscillatory circuit that includes protein kinases ERK2 and PKA.

Self-regulating systems often use robust oscillatory circuits. One such system controls the chemotactic signaling mechanism of Dictyostelium, where pulses of adenosine 3',5'-monophosphate (cAMP) are generated with a periodicity of 7 minutes. We have observed spontaneous oscillations in activation of the mitogen-activated protein (MAP) kinase ERK2 that occur in phase with peaks of cAMP, and we show that ERK2 modulates cAMP levels through the phosphodiesterase RegA. Computer modeling and simulations of the underlying circuit faithfully account for the ability of the cells to spontaneously generate periodic pulses during specific stages of development. Similar oscillatory processes may occur in cells of many different species.

Defect in peroxisomal multifunctional enzyme MFE1 affects cAMP relay in Dictyostelium.

We have previously reported that cells of Dictyostelium discoideum lacking the fatty acid oxidation enzyme MFE1 accumulate excess cyclopropane fatty acids from ingested bacteria. Cells in which mfeA(-) is disrupted fail to develop when grown in association with bacteria but form normal fruiting bodies when grown in axenic media. Bacterially grown mfeA(-) cells express the genes for the cyclic AMP (cAMP) receptor (carA) and adenylyl cyclase (acaA) but fail to respond to a cAMP pulse by synthesis of additional cAMP which normally relays the signal. Moreover, they do not accumulate the adhesion protein, gp80, which is encoded by the cAMP-induced gene, csaA. As a consequence, they do not acquire developmentally regulated EDTA-resistant cell-cell adhesion. When mutant cells are mixed with wild-type cells and allowed to develop together, they co-aggregate and differentiate into both spores and stalk cells. Thus, most of the developmental consequences of excess cyclopropane fatty acids appear to result from impaired cAMP relay.

A putative serpentine receptor gene tasA required for normal morphogenesis of primary stalk and branch structure in Polysphondylium pallidum.

The fruiting body of Polysphondylium pallidum is composed of whorls of branches along the axis of a primary stalk. In the course of fruiting body formation, the interval between neighboring whorls and the number and the spacing of branches in a whorl are highly regulated. In this study, using restriction enzyme mediated integration mutagenesis, we have obtained a mutant (strain M6226) with thicker and aberrant primary stalk. The gene responsible for the mutant phenotype, confirmed by homologous recombination, encodes an open reading frame with 383 aa residues (46.3 kDa) and was named thick and aberrant stalk A (tasA). TasA is highly homologous to Dictyostelium discoideum cyclic adenosine 3',5'-monophosphate receptors. A tasA transcript is expressed strictly at the late aggregation stage. Cells expressing a tasA::gfp fusion DNA are localized at the posterior region of the primary sorogen where secondary sorogens and branches originate. This result indicates the existence of 'prebranch' and 'pretrunk' regions in P. pallidum instead of the prespore and prestalk regions in D. discoideum. The analyzes of the gene disruptant and chimeric fruiting bodies also suggests that TasA affects the normal morphogenesis of the primary stalk and the process of cell differentiation into prebranch cells, but not into spore or stalk cells directly.