Ax-2 Cells Research Articles

Proteasomes of Autophagy-Deficient Cells Exhibit Alterations in Regulatory Proteins and a Marked Reduction in Activity.

Autophagy and the ubiquitin proteasome system are the two major processes for the clearance and recycling of proteins and organelles in eukaryotic cells. Evidence is accumulating that there is extensive crosstalk between the two pathways, but the underlying mechanisms are still unclear. We previously found that autophagy 9 (ATG9) and 16 (ATG16) proteins are crucial for full proteasomal activity in the unicellular amoeba Dictyostelium discoideum. In comparison to AX2 wild-type cells, ATG9-and ATG16- cells displayed a 60%, and ATG9-/16- cells a 90%, decrease in proteasomal activity. Mutant cells also showed a significant increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates. Here, we focus on possible reasons for these results. Reanalysis of published tandem mass tag-based quantitative proteomic results of AX2, ATG9-, ATG16-, and ATG9-/16- cells revealed no change in the abundance of proteasomal subunits. To identify possible differences in proteasome-associated proteins, we generated AX2 wild-type and ATG16- cells expressing the 20S proteasomal subunit PSMA4 as GFP-tagged fusion protein, and performed co-immunoprecipitation experiments followed by mass spectrometric analysis. The results revealed no significant differences in the abundance of proteasomes between the two strains. However, we found enrichment as well as depletion of proteasomal regulators and differences in the ubiquitination of associated proteins for ATG16-, as compared to AX2 cells. Recently, proteaphagy has been described as a means to replace non-functional proteasomes. We propose that autophagy-deficient D. discoideum mutants suffer from inefficient proteaphagy, which results in the accumulation of modified, less-active, and also of inactive, proteasomes. As a consequence, these cells exhibit a dramatic decrease in proteasomal activity and deranged protein homeostasis.

Novel micropatterning technique reveals dependence of cell-substrate adhesion and migration of social amoebas on parental strain, development, and fluorescent markers.

Cell-substrate adhesion of the social amoeba Dictyostelium discoideum, a model organism often used for the study of chemotaxis, is non-specific and does not involve focal adhesion complexes. Therefore, micropatterned substrates where adherent Dictyostelium cells are constrained to designated microscopic regions are difficult to make. Here we present a micropatterning technique for Dictyostelium cells that relies on coating the substrate with an ∼1μm thick layer of polyethylene glycol (PEG) gel. We show that, when plated on a substrate with narrow parallel stripes of PEG-gel and glass, Dictyostelium cells nearly exclusive adhere to and migrate along the glass stripes, thus providing a model system to study one-dimensional migration of amoeboid cells. Surprisingly, we find substantial differences in the adhesion to PEG-gel and glass stripes between vegetative and developed cells and between two different axenic laboratory strains of Dictyostelium, AX2 and AX4. Even more surprisingly, we find that the distribution of Dictyostelium cells between PEG-gel and glass stripes is significantly affected by the expression of several fluorescent protein markers of the cytoskeleton. We carry out atomic force microscopy based single cell force spectroscopy measurements that confirm that the force of adhesion to PEG-gel substrate can be significantly different between vegetative and developed cells, AX2 and AX4 cells, and cells with and without fluorescent markers. Thus, the choice of parental background, the degree of development, and the expression of fluorescent protein markers can all have a profound effect on cell-substrate adhesion and should be considered when comparing migration of cells and when designing micropatterned substrates.

Proteomic and Transcriptomic Profiling Identifies Early Developmentally Regulated Proteins in Dictyostelium Discoideum.

Cyclic AMP acts as a secondary messenger involving different cellular functions in eukaryotes. Here, proteomic and transcriptomic profiling has been combined to identify novel early developmentally regulated proteins in eukaryote cells. These proteomic and transcriptomic experiments were performed in Dictyostelium discoideum given the unique advantages that this organism offers as a eukaryotic model for cell motility and as a nonmammalian model of human disease. By comparing whole-cell proteome analysis of developed (cAMP-pulsed) wild-type AX2 cells and an independent transcriptomic analysis of developed wild-type AX4 cells, our results show that up to 70% of the identified proteins overlap in the two independent studies. Among them, we have found 26 proteins previously related to cAMP signaling and identified 110 novel proteins involved in calcium signaling, adhesion, actin cytoskeleton, the ubiquitin-proteasome pathway, metabolism, and proteins that previously lacked any annotation. Our study validates previous findings, mostly for the canonical cAMP-pathway, and also generates further insight into the complexity of the transcriptomic changes during early development. This article also compares proteomic data between parental and cells lacking glkA, a GSK-3 kinase implicated in substrate adhesion and chemotaxis in Dictyostelium. This analysis reveals a set of proteins that show differences in expression in the two strains as well as overlapping protein level changes independent of GlkA.

The second mesomegaloprepid damselfly (Odonata: Zygoptera) from mid-Cretaceous Burmese amber

The mesomegaloprepids are some of the most abundant fossil damselflies in mid-Cretaceous Burmese amber characterized by large size, brown colour and dense wing venation. Here we described a new damselfly, Cretamegaloprepus zhouae Zheng, Nel and Wang, gen. et sp. nov., representing the second known genus and species of Mesomegaloprepidae Huang et al. (2017). Cretamegaloprepus Zheng, Nel and Wang, gen. nov. greatly differs from Mesomegaloprepus Huang et al. (2017) in having no secondary antenodal and antesubnodal crossveins, the Arc being aligned with Ax2, free discoidal and subdiscoidal cells, base of IR2 being very far distal of Sn, shorter CuA ending on posterior wing margin before RP2 base, and a net of irregular cells near base of RP2. The diagnostic characters of Mesomegaloprepidae and Mesomegaloprepus are revised based on the new damselfly.

Dictyostelium AMPKα regulates aggregate size and cell-type patterning.

Starved Dictyostelium cells aggregate into groups of nearly 105 cells. AMPK is a highly conserved serine/threonine protein kinase consisting of a catalytic and two regulatory subunits. As multi-cellular development in Dictyostelium is initiated upon starvation, we explored the role of the energy sensor, AMPK, which shows significant similarity to human AMPK and is expressed throughout development. Deletion of the ampkα gene results in the formation of numerous small-sized aggregates that develop asynchronously to form few fruiting bodies with small sori and long stalks. On the other hand, ampkαOE cells form fruiting bodies with small stalks and large sori when compared with wild-type, Ax2. A minimum of 5% ampkα− cells in a chimaera with Ax2 cells was sufficient to reduce the aggregate size. Also, the conditioned media collected from ampkα− cells triggered Ax2 cells to form smaller aggregates. The starved ampkα− cells showed low glucose levels and formed large aggregates when glucose was supplied exogenously. Interestingly, ampkα− cells exhibit abnormal cell-type patterning with increased prestalk region and a concomitant reduction of prespore region. In addition, there was a loss of distinct prestalk/prespore boundary in the slugs.

Disruption of homeobox containing gene, hbx9 results in the deregulation of prestalk cell patterning in Dictyostelium discoideum

We have earlier classified the homeobox containing genes from Dictyostelium discoideum and identified 5 genes belonging to the TALE class. Here, we have characterized hbx9, a member of TALE class. In situ hybridization results show it to be preferentially expressed in the prestalkA (pstA) cells but not in the prestalkO (pstO) cells. Disruption of the hbx9 gene in Ax2 cells delay initiation of development and also slows cell proliferation. There was decreased cadA expression during the development of hbx9- cells, which results in inappropriate cell contacts, altered cell motility and patterning properties. ecmA and ecmB were mis-expressed in the pstO and pstA regions resulting in an effectively smaller pstO region. Surprisingly, the total ecmAO expression in pstAO cells was comparable to the wild type, suggesting that in hbx9- cells, ecmA was expressed in the pstO region. In hbx9-, proportioning and patterning was altered in favour of pstA cell-type. Moreover, precocious expression of ecmB and pspA was also observed in the vegetative cells. Expression profiling of components of cAMP signalling suggest that Hbx9 controls cell proportioning of prestalk cells by modulating cAMP signalling during growth-to-development transition.

Glutathione S-transferase 4 is a putative DIF-binding protein that regulates the size of fruiting bodies in Dictyostelium discoideum

In the development of the cellular slime mold Dictyostelium discoideum, two chlorinated compounds, the differentiation-inducing factors DIF-1 and DIF-2, play important roles in the regulation of both cell differentiation and chemotactic cell movement. However, the receptors of DIFs and the components of DIF signaling systems have not previously been elucidated. To identify the receptors for DIF-1 and DIF-2, we here performed DIF-conjugated affinity gel chromatography and liquid chromatography–tandem mass spectrometry and identified the glutathione S-transferase GST4 as a major DIF-binding protein. Knockout and overexpression mutants of gst4 (gst4– and gst4OE, respectively) formed fruiting bodies, but the fruiting bodies of gst4– cells were smaller than those of wild-type Ax2 cells, and those of gst4OE cells were larger than those of Ax2 cells. Both chemotaxis regulation and in vitro stalk cell formation by DIFs in the gst4 mutants were similar to those of Ax2 cells. These results suggest that GST4 is a DIF-binding protein that regulates the sizes of cell aggregates and fruiting bodies in D. discoideum.

Stalk cell differentiation without polyketides in the cellular slime mold.

Polyketides induce prestalk cell differentiation in Dictyostelium. In the double-knockout mutant of the SteelyA and B polyketide synthases, most of the pstA cells-the major part of the prestalk cells-are lost, and we show by whole mount in situ hybridization that expression of prestalk genes is also reduced. Treatment of the double-knockout mutant with the PKS inhibitor cerulenin gave a further reduction, but some pstA cells still remained in the tip region, suggesting the existence of a polyketide-independent subtype of pstA cells. The double-knockout mutant and cerulenin-treated parental Ax2 cells form fruiting bodies with fragile, single-cell layered stalks after cerulenin treatment. Our results indicate that most pstA cells are induced by polyketides, but the pstA cells at the very tip of the slug are induced in some other way. In addition, a fruiting body with a single-cell layered, vacuolated stalk can form without polyketides.

Functional Characterization of a Novel Aquaporin from Dictyostelium discoideum Amoebae Implies a Unique Gating Mechanism

The social amoeba Dictyostelium discoideum is a widely used model organism for studying basic functions of protozoan and metazoan cells, such as osmoregulation and cell motility. There is evidence from other species that cellular water channels, aquaporins (AQP), are central to both processes. Yet, data on D. discoideum AQPs is almost absent. Despite cloning of two putative D. discoideum AQPs, WacA, and AqpA, water permeability has not been shown. Further, WacA and AqpA are expressed at the late multicellular stage and in spores but not in amoebae. We cloned a novel AQP, AqpB, from amoeboidal D. discoideum cells. Wild-type AqpB was impermeable to water, glycerol, and urea when expressed in Xenopus laevis oocytes. Neither stepwise truncation of the N terminus nor selected point mutations activated the water channel. However, mutational truncation by 12 amino acids of an extraordinary long intracellular loop induced water permeability of AqpB, hinting at a novel gating mechanism. This AqpB mutant was inhibited by mercuric chloride, confirming the presence of a cysteine residue in the selectivity filter as predicted by our structure model. We detected AqpB by Western blot analysis in a glycosylated and a non-glycosylated form throughout all developmental stages. When expressed in D. discoideum amoebae, AqpB-GFP fusion constructs localized to vacuolar structures, to the plasma membrane, and to lamellipodia-like membrane protrusions. We conclude that the localization pattern in conjunction with channel gating may be indicative of AqpB functions in osmoregulation as well as cell motility of D. discoideum.

Cell‐cycle checkpoint for transition from cell division to differentiation

In general, growth and differentiation are mutually exclusive, but they are cooperatively regulated during the course of development. Thus, the process of a cell's transition from growth to differentiation is of general importance for the development of organisms, and terminally differentiated cells such as nerve cells never divide. Meanwhile, the growth rate speeds up when cells turn malignant. The cellular slime mold Dictyostelium discoideum grows and multiplies as long as nutrients are supplied, and its differentiation is triggered by starvation. A critical checkpoint (growth/differentiation transition or GDT point), from which cells start differentiating in response to starvation, has been precisely specified in the cell cycle of D. discoideum Ax-2 cells. Accordingly, integration of GDT point-specific events with starvation-induced events is needed to understand the mechanism regulating GDTs. A variety of intercellular and intracellular signals are involved positively or negatively in the initiation of differentiation, making a series of cross-talks. As was expected from the presence of the GDT point, the cell's positioning in cell masses and subsequent cell-type choices occur depending on the cell's phase in the cell cycle at the onset of starvation. Since novel and multiple functions of mitochondria in various respects of development including the initiation of differentiation have been directly realized in Dictyostelium cells, they are also reviewed in this article.

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.

Expression of Actin-interacting Protein 1 Suppresses Impaired Chemotaxis ofDictyosteliumCells Lacking the Na+-H+Exchanger NHE1

Increased intracellular pH is an evolutionarily conserved signal necessary for directed cell migration. We reported previously that in Dictyostelium cells lacking H(+) efflux by a Na(+)-H(+) exchanger (NHE; Ddnhe1(-)), chemotaxis is impaired and the assembly of filamentous actin (F-actin) is attenuated. We now describe a modifier screen that reveals the C-terminal fragment of actin-interacting protein 1 (Aip1) enhances the chemotaxis defect of Ddnhe1(-) cells but has no effect in wild-type Ax2 cells. However, expression of full-length Aip1 mostly suppresses chemotaxis defects of Ddnhe1(-) cells and restores F-actin assembly. Aip1 functions to promote cofilin-dependent actin remodeling, and we found that although full-length Aip1 binds cofilin and F-actin, the C-terminal fragment binds cofilin but not F-actin. Because pH-dependent cofilin activity is attenuated in mammalian cells lacking H(+) efflux by NHE1, our current data suggest that full-length Aip1 facilitates F-actin assembly when cofilin activity is limited. We predict the C-terminus of Aip1 enhances defective chemotaxis of Ddnhe1(-) cells by sequestering the limited amount of active cofilin without promoting F-actin assembly. Our findings indicate a cooperative role of Aip1 and cofilin in pH-dependent cell migration, and they suggest defective chemotaxis in Ddnhe1(-) cells is determined primarily by loss of cofilin-dependent actin dynamics.

Dictyostelium Myosin-5b Is a Conditional Processive Motor

Dictyostelium myosin-5b is the gene product of myoJ and one of two closely related myosin-5 isoenzymes produced in Dictyostelium discoideum. Here we report a detailed investigation of the kinetic and functional properties of the protein. In standard assay buffer conditions, Dictyostelium myosin-5b displays high actin affinity in the presence of ADP, fast ATP hydrolysis, and a high steady-state ATPase activity in the presence of actin that is rate limited by ADP release. These properties are typical for a processive motor that can move over long distances along actin filaments without dissociating. Our results show that a physiological decrease in the concentration of free Mg(2+)-ions leads to an increased rate of ADP release and shortening of the fraction of time the motor spends in the strong actin binding states. Consistently, the ability of the motor to efficiently translocate actin filaments at very low surface densities decreases with decreasing concentrations of free Mg(2+)-ions. In addition, we provide evidence that the observed changes in Dd myosin-5b motor activity are of physiological relevance and propose a mechanism by which this molecular motor can switch between processive and non-processive movement.

Involvements of a novel protein, DIA2, in cAMP signaling and spore differentiation during Dictyostelium development

The novel gene dia2 (differentiation-associated gene 2) was originally isolated as a gene expressed specifically in response to initial differentiation of Dictyostelium discoideum Ax-2 cells. Using dia2 AS cells in which the dia2 expression was inactivated by the antisense RNA method, DIA2 protein was found to be required for cAMP signaling during cell aggregation. During late development, the DIA2 protein changed its location from the endoplasmic reticulum (ER) to prespore-specific vacuoles (PSVs) that are specifically present in prespore cells of the slug. In differentiating prestalk cells, however, DIA2 was found to be nearly lost from the cells. Importantly, exocytosis of PSVs from prespore cells and the subsequent spore differentiation were almost completely impaired in dia2 AS cells. In addition, spore induction by externally applied 8-bromo cAMP was significantly suppressed in dia2 AS cells. Taken together, these results strongly suggested that DIA2 might be closely involved in cAMP signaling and spore differentiation as well as in the initiation of differentiation during Dictyostelium development.

Fluctuation Theorem Applied to Dictyostelium discoideum System

Directional cell movement is a fundamental phenomenon exhibited by many biological processes. It has been known that an electric field exists on the surfaces of the tissues of organisms and that it acts as a directional cue for the type of the cell migration known as electrotaxis. Electrotaxis is thought to play important roles in various physiological processes including embryogenesis and wound healing, and the underlying molecular mechanisms of electrotaxis are now extensively studied. 1) In order to elucidate the mechanisms of the electrotactic responses of cells, the cellular slime mold Dictyostelium discoideum [see Fig. 1 (left)] is a suitable organism to study, because of its high motility and strong electrotactic response. With well-established genetic engineering techniques and advanced microscopic techniques, the input–output relationship in the electrotactic response of Dictyostelium cells has been investigated to elucidate the stochastic processes involved in the signaling systems responsible for cell motility and their regulations. 2) In this paper, we analyze the electrotactic movement of Dictyostelium discoideum from the viewpoint of non-equilibrium statistical mechanics. Because we can observe fluctuating behavior of cellular trajectories, we analyze the probability distribution of the trajectories with the aid of the fluctuation theorem. Recently, the validity of the fluctuation theorem was verified in a colloidal system, 3) and it has also been applied to granular systems, 4) turbulent systems, 5) and chemical oscillatory waves 6) to investigate some of their statistical properties that are not yet completely understood. Noting that the fluctuation theorem is potentially applicable to cellular electrotaxis, here we employ it to help us obtain a phenomenological model of this biological system. System. In this study, Dictyostelium discoideum, Ax2 cells (wild type) were starved for up to 4 h, with a pulse of 100 nM cAMP applied every 6 min. The cell suspension was injected into the chamber for electrotactic assay, and the cells were allowed to spread over the coverslip for 20 min at T ¼ 294 K. Direct current was applied to the chamber as illustrated in the paper of Sato et al. 2) The cells in the chamber were observed with a microscope capable of producing differential interference contrast optics. Data acquisition started 5 min after the electric field was first applied, and the electric field remained at a constant strength, E, throughout experiments. Under these conditions, we considered the system to be in a steady state. To analyze the motile activities of the cells under the electric field, cell images were processed automatically with a time resolution of 5 s and converted into binary images by selecting an optimal value of the brightness threshold. In this way, the trajectory of the center position of the observed cellular region, ðxðtÞ; yðtÞÞ, was determined. Because the gradient of the electric field is non-zero only along the x direction, we particularly investigate the trajectories in the x direction for the case that E ¼ 10 V/cm, which is sufficiently large for the response saturation of the cell. 2) Fluctuation theorem. When the electric field is turned on, Dictyostelium cells begin to migrate toward the cathode (the þx direction). In Fig. 1 (right), we plot example trajectories, xðt Þ� xð0Þ and yðt Þ� yð0Þ, as functions of time in the case E ¼ 10 V/cm. The fluctuating behavior of the cell movement was observed. Then, using these fluctuating trajectories

Import-associated translational inhibition: novel in vivo evidence for cotranslational protein import into Dictyostelium discoideum mitochondria.

To investigate protein import into the mitochondria of Dictyostelium discoideum, green fluorescent protein (GFP) was fused as a reporter protein either to variable lengths of the N-terminal region of chaperonin 60 (the first 23, 40, 80, 97, and 150 amino acids) or to the mitochondrial targeting sequence of DNA topoisomerase II. The fusion proteins were expressed in AX2 cells under the actin-15 promoter. Fluorescence images of GFP transformants confirmed that Dictyostelium chaperonin 60 is a mitochondrial protein. The level of the mitochondrially targeted GFP fusion proteins was unexpectedly much lower than the nontargeted (cytoplasmic) forms. The distinction between targeted and nontargeted protein activities was investigated at both the transcriptional and translational levels in vivo. We found that targeting GFP to the mitochondria results in reduced levels of the fusion protein even though transcription of the fusion gene and the stability of the protein are unaffected. [(35)S]methionine labeling and GFP immunoprecipitation confirmed that mitochondrially targeted GFP is translated at much slower rates than nontargeted GFP. The results indicate a novel phenomenon, import-associated translational inhibition, whereby protein import into the mitochondria limits the rate of translation. The simplest explanation for this is that import of the GFP fusion proteins occurs cotranslationally, i.e., protein synthesis and import into mitochondria are coupled events. Consistent with cotranslational import, Northern analysis showed that the GFP mRNA is associated with isolated mitochondria. This association occurred regardless of whether the GFP was fused to a mitochondrial leader peptide. However, the presence of an import-competent leader peptide stabilized the mRNA-mitochondria association, rendering it more resistant to extensive EDTA washing. In contrast with GFP, the mRNA of another test protein, aequorin, did not associate with the mitochondria, and its translation was unaffected by import of the encoded polypeptide into the mitochondria.

A Metabotropic Glutamate Receptor Family Gene in Dictyostelium discoideum

Metabotropic glutamate receptors (mGluRs) are a class of G-protein-coupled receptors that possess a seven transmembrane region involved in the modulation of excitatory synaptic transmission in the nervous system. mGluR orthologs have been identified in Drosophila, Caenorhabditis elegans, and higher organisms. Drosophila possesses two mGluR genes, DmGluRA and DmXR. We screened the Dictyostelium genome data base using the ligand binding domain of rat mGluR1 as bait, and identified a new receptor, DdmGluPR, belonging to the mGluR family. Similar to Drosophila DmXR, the residues of mGluRs involved in the binding of the alpha-carboxylic and alpha-amino groups of glutamate were well conserved in DdmGluPR, but the residues interacting with the gamma-carboxylic group of glutamate were not. The phylogenetic analysis suggests that DdmGluPR diverged after the mGluR family-GABA(B) receptors split but before mGluR family divergence. DdmGluPR mRNA was expressed in vegetative cells and throughout starvation-induced development, but the level of the expression was relatively high until 4 h after starvation. DdmGluPR was localized to the plasma membrane of axenically grown Ax-2 cells expressed as a green fluorescent protein fusion protein. DdmGluPR-null cells grew faster at high cell density and reached higher densities than wild-type cells. DdmGluPR-null cells exhibited delayed aggregates formation upon starvation and impaired chemotaxis toward cAMP. Although expressions of cAR1 and aca, cAMP-signaling components, were rapidly induced and peaked at 2-4 h in wild-type cells, DdmGluPR-null cells displayed sustained and peaked at 8 h of the expressions of these genes. Our findings suggest the involvement of DdmGluPR in the early development of Dictyostelium discoideum.

Functional analysis of a novel gene, DD3–3, from Dictyostelium discoideum

A novel gene, DD3–3, from Dictyostelium discoideum has been isolated by an mRNA differential display between a wild-type strain AX2 and a mutant HG794 which is defective in O-glycosylation. Functional analysis of the novel gene, DD3–3, was conducted by preparing a knockout mutant, DD3–3KO, and a GST:DD3–3 fusion protein. The mutant DD3–3KO cells were allowed to develop about 1.5 h earlier than the wild-type strain AX2 cells. Northern blotting analysis of the knockout mutant cells showed a remarkable downregulation of Reg A, cAMP-dependent phosphodiesterase, and overexpression of protein tyrosine kinase (PTK) during early development and its shutdown during late development. The relationship between O-glycosylation and phosphorylation involving Reg A gene is discussed.

Involvement of the TRAP-1 homologue, Dd-TRAP1, in spore differentiation during Dictyostelium development

Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a member of the molecular chaperone HSP90 (90-kDa heat shock protein) family. We have previously demonstrated that Dictyostelium discoideum TRAP1 (Dd-TRAP1) synthesized at the vegetative growth phase is retained during the whole course of D. discoideum development, and that at the multicellular slug stage, it is located in prespore-specific vacuoles (PSVs) of prespore cells as well as in the cell membrane and mitochondria. Thereupon, we examined the function of Dd-TRAP1 in prepore and spore differentiation, using Dd-TRAP1-knockdown cells (TRAP1-RNAi cells) produced by the RNA interference method. As was expected, Dd-TRAP1 contained in the PSV was found to be exocytosed during sporulation to constitute the outer-most layer of the spore cell wall. In the TRAP1-RNAi cells, PSV formation and therefore prespore differentiation were significantly impaired, particularly under a heat stress condition. Although the TRAP1-RNAi cells formed apparently normal-shaped spores with a cellulosic wall, the spores were less resistant to heat and detergent treatments, as compared with those of parental MB35 cells derived from Ax-2 cells. These findings strongly suggest that Dd-TRAP1 may be closely involved in late development including spore differentiation, as well as in early development as realized by its induction of prestarvation response.

Changes in spatial and temporal localization of Dictyostelium homologues of TRAP1 and GRP94 revealed by immunoelectron microscopy

TRAP1 (tumor necrosis factor receptor-associated protein 1) is a member of the molecular chaperone HSP90 (90-kDa heat shock protein) family. In this study, we mainly examined the behavior of Dictyostelium TRAP1 homologue, Dd-TRAP1, during Dictyostelium development by immunoelectron microscopy. In vegetatively growing D. discoideum Ax-2 cells, Dd-TRAP1 locates in nucleolus and vesicles in addition to the cell cortex including cell membrane. Many of Dd-TRAP1 molecules moved to the mitochondrial matrix in response to differentiation, although Dd-TRAP1 on the cell membrane seems to be retained. Some Dd-TRAP1 was also found to be secreted to locate outside the cell membrane in Ax-2 cells starved for 6 h. At the multicellular slug stage, Dd-TRAP1 was primarily located in mitochondria and cell membrane in both prestalk and prespore cells. More importantly, in differentiating prespore cells, a significant number of Dd-TRAP1 locates in the PSV (prespore-specific vacuole) that is a sole cell type-specific organelle and essential for spore wall formation, whereas some Dd-TRAP1 in the cell cortical region of prestalk cells. These findings strongly suggest the importance of Dd-TRAP1 regulated temporally and spatially during Dictyostelium development. Incidentally, we also have certified that the glucose-regulated protein 94 (Dd-GRP94) is predominantly located in Golgi vesicles and cisternae, followed by its colocalization with Dd-TRAP1 in the PSV.