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To combat the permanent exposure to potential pathogens every organism relies on an immune system. Important factors in innate immunity are antimicrobial peptides (AMPs) that are structurally highly diverse. Some AMPs are known to belong to the saposin-like proteins (SAPLIPs), a group of polypeptides with a broad functional spectrum. The model organism Dictyostelium discoideum possesses a remarkably large arsenal of potential SAPLIPs, which are termed amoebapore-like peptides (Apls), but the knowledge about these proteins is very limited. Here, we report about the biochemical characterization of AplE1, AplE2, AplK1, and AplK2, which are derived from the two precursor proteins AplE and AplK, thereby resembling prosaposins of vertebrates. We produced these Apls as recombinant polypeptides in Escherichia coli using a self-splicing intein to remove an affinity tag used for purification. All recombinant Apls exhibited pore-forming activity in a pH-dependent manner, as evidenced by liposome depolarization, showing higher activities the more acidic the setting was. Lipid preference was detected for negatively charged phospholipids and in particular for cardiolipin. Antimicrobial activity against various bacteria was found to be inferior in classical microdilution assays. However, all of the Apls studied permeabilized the cytoplasmic membrane of live Bacillus subtilis . Collectively, we assume that the selected Apls interact by their cationic charge with negatively charged bacterial membranes in acidic environments such as phagolysosomes and eventually lyse the target cells by pore formation.

Pluripotent stem cells can differentiate into distinct cell types but the intracellular pathways controlling cell fate choice are not well understood. The social amoeba Dictyostelium discoideum is a simplified system to study choice preference as proliferating amoebae enter a developmental cycle upon starvation and differentiate into two major cell types, stalk and spores, organised in a multicellular fruiting body. Factors such as acidic vesicle pH predispose amoebae to one fate. Here we show that the mechanistic target of rapamycin complex 1 (mTORC1) pathway has a role in cell fate bias in Dictyostelium. Inhibiting the mTORC1 pathway activity by disruption of Rheb (activator Ras homolog enriched in brain), or treatment with the mTORC1 inhibitor rapamycin prior to development, biases cells to a spore cell fate. Conversely activation of the pathway favours stalk cell differentiation. The Set1 histone methyltransferase, responsible for histone H3 lysine4 methylation, in Dictyostelium cells regulates transcription at the onset of development. Disruption of Set1 leads to high mTORC1 pathway activity and stalk cell predisposition. The ability of the mTORC1 pathway to regulate cell fate bias of cells undergoing differentiation offers a potential target to increase the efficiency of stem cell differentiation into a particular cell type., (© 2024. The Author(s).)

Macropinocytosis mediates the non-selective bulk uptake of extracellular fluid, enabling cells to survey the environment and obtain nutrients. A conserved set of signaling proteins orchestrates the actin dynamics that lead to membrane ruffling and macropinosome formation across various eukaryotic organisms. At the center of this signaling network are Ras GTPases, whose activation potently stimulates macropinocytosis. However, how Ras signaling is initiated and spatiotemporally regulated during macropinocytosis is not well understood. By using the model system Dictyostelium and a proteomics-based approach to identify regulators of macropinocytosis, we uncovered Leep2, consisting of Leep2A and Leep2B, as a RasGAP complex. The Leep2 complex specifically localizes to emerging macropinocytic cups and nascent macropinosomes, where it modulates macropinosome formation by regulating the activities of three Ras family small GTPases. Deletion or overexpression of the complex, as well as disruption or sustained activation of the target Ras GTPases, impairs macropinocytic activity. Our data reveal the critical role of fine-tuning Ras activity in directing macropinosome formation., (© 2024 Chao et al.)

The social amoeba Dictyostelium discoideum has been studied for close to a century to better understand conserved cellular and developmental processes. The life cycle of this model eukaryote is composed of a unicellular growth phase and a multicellular developmental phase that is induced by starvation. When starved, individual cells undergo chemotactic aggregation to form multicellular mounds that develop into slugs. Terminal differentiation of cells within slugs forms fruiting bodies, each composed of a stalk that supports a mass of viable spores that germinate and restart the life cycle when nutrients become available. Calcium-dependent cell adhesion protein A (CadA) and countin (CtnA) are two proteins that regulate adhesion and aggregation, respectively, during the early stages of D. discoideum development. While the functions of these proteins have been well-studied, the mechanisms regulating their trafficking are not fully understood. In this study, we reveal pathways and cellular components that regulate the intracellular and extracellular amounts of CadA and CtnA during aggregation. During growth and starvation, CtnA localizes to cytoplasmic vesicles and punctae. We show that CtnA is glycosylated and this post-translational modification is required for its secretion. Upon autophagy induction, a signal peptide for secretion facilitates the release of CtnA from cells via a pathway involving the μ subunit of the AP3 complex (Apm3) and the WASP and SCAR homolog, WshA. Additionally, CtnA secretion is negatively regulated by the D. discoideum orthologs of the human non-selective cation channel mucolipin-1 (Mcln) and sorting receptor sortilin (Sort1). As for CadA, it localizes to the cell periphery in growth-phase and starved cells. The intracellular and extracellular amounts of CadA are modulated by autophagy genes (atg1, atg9), Apm3, WshA, and Mcln. We integrate these data with previously published findings to generate a comprehensive model summarizing the trafficking of CadA and CtnA in D. discoideum. Overall, this study enhances our understanding of protein trafficking during D. discoideum aggregation, and more broadly, provides insight into the multiple pathways that regulate protein trafficking and secretion in all eukaryotes., Competing Interests: Declaration of competing interest The authors have no interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Phagocytic cells of the mammalian innate immune system play a critical role in protecting the body from bacterial infections. The multiple facets of this encounter (chemotaxis, phagocytosis, destruction, evasion and pathogenicity) are largely recapitulated in the phagocytic amoeba Dictyostelium discoideum. Here we identified a new chemical compound (K14; ZINC19168591) which inhibited intracellular destruction of ingested K. pneumoniae in D. discoideum cells. Concomitantly, K14 reduced proteolytic activity in D. discoideum phagosomes. In kil1 KO cells, K14 lost its ability to inhibit phagosomal proteolysis and to inhibit intra-phagosomal bacterial destruction, suggesting that K14 inhibits a Kil1-dependent protease involved in bacterial destruction. These observations stress the key role that proteases play in bacterial destruction. They also reveal an unsuspected link between Kil1 and phagosomal proteases. K14 can be used in the future as a tool to probe the role of different proteases in phagosomal physiology and in the destruction of ingested bacteria., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ifrid et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Hermansky-Pudlak syndrome (HPS) is an inherited disorder of intracellular vesicle trafficking affecting the function of lysosome-related organelles (LROs). At least 11 genes underlie the disease, encoding four protein complexes, of which biogenesis of lysosome-related organelles complex-2 (BLOC-2) is the last whose molecular action is unknown. We find that the unicellular eukaryote Dictyostelium unexpectedly contains a complete BLOC-2, comprising orthologs of the mammalian subunits HPS3, -5, and -6, and a fourth subunit, an ortholog of the Drosophila LRO-biogenesis gene, Claret. Lysosomes from Dictyostelium BLOC-2 mutants fail to mature, similar to LROs from HPS patients, but for all endolysosomes rather than a specialized subset. They also strongly resemble lysosomes from WASH mutants. Dictyostelium BLOC-2 localizes to the same compartments as WASH, and in BLOC-2 mutants, WASH is inefficiently recruited, accounting for their impaired lysosomal maturation. BLOC-2 is recruited to endolysosomes via its HPS3 subunit. Structural modeling suggests that all four subunits are proto-coatomer proteins, with important implications for BLOC-2's molecular function. The discovery of Dictyostelium BLOC-2 permits identification of orthologs throughout eukaryotes. BLOC-2 and lysosome-related organelles, therefore, pre-date the evolution of Metazoa and have broader and more conserved functions than previously thought., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ras small GTPases act as molecular switches in various cellular signaling pathways, including cell migration, proliferation, and differentiation. Three Rap proteins are present in Dictyostelium; RapA, RapB, and RapC. RapA and RapC have been reported to have opposing functions in the control of cell adhesion and migration. Here, we investigated the role of RapB, a member of the Ras GTPase subfamily in Dictyostelium, focusing on its involvement in cell adhesion, migration, and developmental processes. This study revealed that RapB, similar to RapA, played a crucial role in regulating cell morphology, adhesion, and migration. rapB null cells, which were generated by CRISPR/Cas9 gene editing, displayed altered cell size, reduced cell-substrate adhesion, and increased migration speed during chemotaxis. These phenotypes of rapB null cells were restored by the expression of RapB and RapA, but not RapC. Consistent with these results, RapB, similar to RapA, failed to rescue the phenotypes of rapC null cells, spread morphology, increased cell adhesion, and decreased migration speed during chemotaxis. Multicellular development of rapB null cells remained unaffected. These results suggest that RapB is involved in controlling cell morphology and cell adhesion. Importantly, RapB appears to play an inhibitory role in regulating the migration speed during chemotaxis, possibly by controlling cell-substrate adhesion, resembling the functions of RapA. These findings contribute to the understanding of the functional relationships among Ras subfamily proteins., (© 2024. The Author(s), under exclusive licence to Microbiological Society of Korea.)

Little is known about eukaryotic chemorepulsion. The enzymes phosphatase and tensin homolog (PTEN) and CnrN dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Dictyostelium discoideum cells require both PTEN and CnrN to induce chemorepulsion of cells away from the secreted chemorepellent protein AprA. How D. discoideum cells utilize two proteins with redundant phosphatase activities in response to AprA is unclear. Here, we show that D. discoideum cells require both PTEN and CnrN to locally inhibit Ras activation, decrease basal levels of PI(3,4,5)P3 and increase basal numbers of macropinosomes, and AprA prevents this increase. AprA requires both PTEN and CnrN to increase PI(4,5)P2 levels, decrease PI(3,4,5)P3 levels, inhibit proliferation, decrease myosin II phosphorylation and increase filopod sizes. PTEN, but not CnrN, decreases basal levels of PI(4,5)P2, and AprA requires PTEN, but not CnrN, to induce cell roundness. Together, our results suggest that CnrN and PTEN play unique roles in AprA-induced chemorepulsion., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Autophagy is a degradative recycling process central to the maintenance of homeostasis in all eukaryotes. By ensuring the degradation of damaged mitochondria, it plays a key role in maintaining mitochondrial health and function. Of the highly conserved autophagy proteins, autophagy-related protein 1 (Atg1) is essential to the process. The involvement of these proteins in intracellular signalling pathways, including those involving mitochondrial function, are still being elucidated. Here the role of Atg1 was investigated in the simple model organism Dictyostelium discoideum using an atg1 null mutant and mutants overexpressing or antisense-inhibiting atg1 . When evaluated against the well-characterised outcomes of mitochondrial dysfunction in this model, altered atg1 expression resulted in an unconventional set of phenotypic outcomes in growth, endocytosis, multicellular development, and mitochondrial homeostasis. The findings here show that Atg1 is involved in a tightly regulated signal transduction pathway coordinating energy-consuming processes such as cell growth and multicellular development, along with nutrient status and energy production. Furthermore, Atg1's effects on energy homeostasis indicate a peripheral ancillary role in the mitochondrial signalling network, with effects on energy balance rather than direct effects on electron transport chain function. Further research is required to tease out these complex networks. Nevertheless, this study adds further evidence to the theory that autophagy and mitochondrial signalling are not opposing but rather linked, yet strictly controlled, homeostatic mechanisms.

Ras has been extensively studied as a promoter of cell proliferation, whereas few studies have explored its role in migration. To investigate the direct and immediate effects of Ras activity on cell motility or polarity, we focused on RasGAPs, C2GAPB in Dictyostelium amoebae and RASAL3 in HL-60 neutrophils and macrophages. In both cellular systems, optically recruiting the respective RasGAP to the cell front extinguished pre-existing protrusions and changed migration direction. However, when these respective RasGAPs were recruited uniformly to the membrane, cells polarized and moved more rapidly, whereas targeting to the back exaggerated these effects. These unexpected outcomes of attenuating Ras activity naturally had strong, context-dependent consequences for chemotaxis. The RasGAP-mediated polarization depended critically on myosin II activity and commenced with contraction at the cell rear, followed by sustained mTORC2-dependent actin polymerization at the front. These experimental results were captured by computational simulations in which Ras levels control front- and back-promoting feedback loops. The discovery that inhibiting Ras activity can produce counterintuitive effects on cell migration has important implications for future drug-design strategies targeting oncogenic Ras., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested the involvement of the Switch I (SWI) effector domain of Ras in binding mTORC2 components, the regulation of the Ras-mTORC2 pathway is not entirely understood. In Dictyostelium, mTORC2 is selectively activated by the Ras protein RasC, and the RasC-mTORC2 pathway then mediates chemotaxis to cAMP and cellular aggregation by regulating the actin cytoskeleton and promoting cAMP signal relay. Here, we investigated the role of specific residues in RasC's SWI, C-terminal allosteric domain, and hypervariable region (HVR) related to mTORC2 activation. Interestingly, our results suggest that RasC SWI residue A31, which was previously implicated in RasC-mediated aggregation, regulates RasC's specific activation by the Aimless RasGEF. On the other hand, our investigation identified a crucial role for RasC SWI residue T36, with secondary contributions from E38 and allosteric domain residues. Finally, we found that conserved basic residues and the adjacent prenylation site in the HVR, which are crucial for RasC's membrane localization, are essential for RasC-mTORC2 pathway activation by allowing for both RasC's own cAMP-induced activation and its subsequent activation of mTORC2. Therefore, our findings revealed new determinants of RasC-mTORC2 pathway specificity in Dictyostelium, contributing to a deeper understanding of Ras signaling regulation in eukaryotic cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The patterns of Formin B and of the Arp2/3 complex formed during mitosis were studied in a mutant of Dictyostelium discoideum that produces multinucleate cells, which divide by the ingression of unilateral cleavage furrows. During cytokinesis the cells of this mutant remain spread on a glass surface where they generate a planar pattern based on the sorting-out of actin-binding proteins. During anaphase, Formin B and Arp2/3 became localized to the regions of microtubule asters around the centrosomes; Formin B in particular in the form of round, quite uniformly covered areas. These areas have been shown to be depleted of myosin II and the actin-filament crosslinker cortexillin, and to be avoided by cleavage furrows on their path into the cell., (© 2024. The Author(s).)

Greenbeard genetic elements encode rare perceptible signals, signal recognition ability, and altruism towards others that display the same signal. Putative greenbeards have been described in various organisms but direct evidence for all the properties in one system is scarce. The tgrB1-tgrC1 allorecognition system of Dictyostelium discoideum encodes two polymorphic membrane proteins which protect cells from chimerism-associated perils. During development, TgrC1 functions as a ligand-signal and TgrB1 as its receptor, but evidence for altruism has been indirect. Here, we show that mixing wild-type and activated tgrB1 cells increases wild-type spore production and relegates the mutants to the altruistic stalk, whereas mixing wild-type and tgrB1-null cells increases mutant spore production and wild-type stalk production. The tgrB1-null cells cheat only on partners that carry the same tgrC1-allotype. Therefore, TgrB1 activation confers altruism whereas TgrB1 inactivation causes allotype-specific cheating, supporting the greenbeard concept and providing insight into the relationship between allorecognition, altruism, and exploitation., (© 2024. The Author(s).)

Background Information: Two pore channels (TPCs) are voltage-gated ion channel superfamily members that release Ca 2+ from acidic intracellular stores and are ubiquitously present in both animals and plants. Starvation initiates multicellular development in Dictyostelium discoideum. Increased intracellular calcium levels bias Dictyostelium cells towards the stalk pathway and thus we decided to analyze the role of TPC2 in development, differentiation, and autophagy., Results: We showed TPC2 protein localizes in lysosome-like acidic vesicles and the in situ data showed stalk cell biasness. Deletion of tpc2 showed defective and delayed development with formation of multi-tipped structures attached to a common base, while tpc2 OE cells showed faster development with numerous small-sized aggregates and wiry fruiting bodies. The tpc2 OE cells showed higher intracellular cAMP levels as compared to the tpc2 - cells while pinocytosis was found to be higher in the tpc2 - cells. Also, TPC2 regulates cell-substrate adhesion and cellular morphology. Under nutrient starvation, deletion of tpc2 reduced autophagic flux as compared to Ax2. During chimera formation, tpc2 - cells showed a bias towards the prestalk/stalk region while tpc2 OE cells showed a bias towards the prespore/spore region. tpc2 deficient strain exhibits aberrant cell-type patterning and loss of distinct boundary between the prestalk/prespore regions., Conclusion: TPC2 is required for effective development and differentiation in Dictyostelium and supports autophagic cell death and cell-type patterning., Significance: Decreased calcium due to deletion of tpc2 inhibit autophagic flux., (© 2024 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.)

The large-scale proteomic analysis of Dictyostelium discoideum has contributed to our understanding of intracellular as well as secreted proteins in this versatile model eukaryote. Mass spectrometry-based proteomic analysis is a robust, sensitive, and rapid analytical method for identification and characterization of proteins extracted from tissues, cells, cell fractions, or pull-down assays. The availability of core facilities which make proteomics inexpensive and easy to do has facilitated a wide range of research projects. In this chapter, we present a simple standard methodology to extract proteins and prepare samples from D. discoideum for mass spectrometry and methods to analyze the identified proteins., (© 2024. The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The social amoeba Dictyostelium discoideum is a versatile model for understanding many different cellular processes involving cell motility including chemotaxis, phagocytosis, and cytokinesis. Cytokinesis, in particular, is a model cell-shaped change process in which a cell separates into two daughter cells. D. discoideum has been used extensively to identify players in cytokinesis and understand how they comprise the mechanosensory and biochemical pathways of cytokinesis. In this chapter, we describe how we use cDNA library complementation with D. discoideum to discover potential regulators of cytokinesis. Once identified, these regulators are further analyzed through live cell imaging, immunofluorescence imaging, fluorescence correlation and cross-correlation spectroscopy, micropipette aspiration, and fluorescence recovery after photobleaching. Collectively, these methods aid in detailing the mechanisms and signaling pathways that comprise cell division., (© 2024. The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Ras and Rap small GTPases of the Ras superfamily act as molecular switches to control diverse cellular processes as part of different signaling pathways. Dictyostelium expresses several Ras and Rap proteins, and their study has and continues to greatly contribute to our understanding of their role in eukaryote biology. To study the activity of Ras and Rap proteins in Dictyostelium, several assays based on their interaction with the Ras binding domain of known eukaryotic Ras/Rap effectors have been developed and proved extremely useful to study their regulation and cellular roles. Here, we describe methods to assess Ras/Rap activity biochemically using a pull-down assay and through live-cell imaging using fluorescent reporters., (© 2024. The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Autophagy is an intracellular clearance and recycling pathway that delivers different types of cargos to lysosomes for degradation. In recent years, autophagy has attracted considerable medical interest, and many different techniques are being developed to study this process in experimental models such as Dictyostelium. Here we describe the use of different autophagic markers in confocal microscopy, in vivo and also in fixed cells. In particular, we describe the use of the GFP-Atg8-RFP-Atg8ΔG marker and the optimization of the GFP-PgkA cleavage assay to detect small differences in autophagy flux., (© 2024. The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Biochemical assays are described to analyze signal transduction by the second messenger cGMP in Dictyostelium. The methods include enzyme assays to measure the activity and regulation of cGMP synthesizing guanylyl cyclases and cGMP-degrading phosphodiesterases. In addition, several methods are described to quantify cGMP levels. The target of cGMP in Dictyostelium is the large protein GbpC that has multiple domains including a Roc domain, a kinase domain, and a cGMP-stimulated Ras-GEF domain. A cGMP-binding assay is described to detect and quantify GbpC., (© 2024. The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Largely due to its simplicity, while being more like human cells compared to other experimental models, Dictyostelium continues to be of great use to discover basic molecular mechanisms and signaling pathways underlying evolutionarily conserved biological processes. However, the identification of new protein interactions implicated in signaling pathways can be particularly challenging in Dictyostelium due to its extremely fast signaling kinetics coupled with the dynamic nature of signaling protein interactions. Recently, the proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells was shown to allow the detection of weak and/or transient protein interactions and also to obtain spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in Dictyostelium. Coupled with the identification of the labeled proteins by mass spectrometry, this method expands Dictyostelium's proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in Dictyostelium., (© 2024. The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lamins are nuclear intermediate filament proteins that are ubiquitously found in metazoan cells, where they contribute to nuclear morphology, stability, and gene expression. Lamin-like sequences have recently been identified in distantly related eukaryotes, but it remains unclear whether these proteins share conserved functions with the lamins found in metazoans. Here, we investigate conserved features between metazoan and amoebozoan lamins using a genetic complementation system to express the Dictyostelium discoideum lamin-like protein NE81 in mammalian cells lacking either specific lamins or all endogenous lamins. We report that NE81 localizes to the nucleus in cells lacking Lamin A/C, and that NE81 expression improves nuclear circularity, reduces nuclear deformability, and prevents nuclear envelope rupture in these cells. However, NE81 did not completely rescue loss of Lamin A/C, and was unable to restore normal distribution of metazoan lamin interactors, such as emerin and nuclear pore complexes, which are frequently displaced in Lamin A/C deficient cells. Collectively, our results indicate that the ability of lamins to modulate the morphology and mechanical properties of nuclei may have been a feature present in the common ancestor of Dictyostelium and animals, whereas other, more specialized interactions may have evolved more recently in metazoan lineages.

During developmental and immune responses, cells move towards or away from some signals. Although much is known about chemoattraction, chemorepulsion (the movement of cells away from a stimulus) remains poorly understood. Proliferating Dictyostelium discoideum cells secrete a chemorepellent protein called AprA. Examining existing knockout strains, we previously identified proteins required for AprA-induced chemorepulsion, and a genetic screen suggested that the enzyme phosphatidylinositol phosphate kinase A (PIPkinA, also known as Pik6) might also be needed for chemorepulsion. Here, we show that cells lacking PIPkinA are not repelled by AprA, and that this phenotype is rescued by expression of PIPkinA. To bias cell movement, AprA inhibits Ras activation at the side of the cell closest to the source of AprA, and we find that PIPkinA is required for AprA to inhibit Ras activation. PIPkinA decreases levels of phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], and possibly because of these effects, potentiates phagocytosis and inhibits cell proliferation. Cells lacking PIPkinA show normal AprA binding, suggesting that PIPkinA regulates chemorepulsion at a step between the AprA receptor and AprA inhibition of Ras activation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

DydA plays an important role in chemotaxis, development, and cell growth as an adaptor protein that connects Ras signaling and cytoskeletal rearrangement. DydA is a downstream effector of RasG and is involved in controlling cell polarity and pseudopodia formation during chemoattractant-directed cell migration. To understand the mechanism by which DydA functions on the cell migration, we investigated the dynamic subcellular localization of DydA in response to chemoattractant stimulation and found that DydA rapidly and transiently translocated to the cell cortex through the RA domain and the PRM region in DydA in response to chemoattractant stimulation. The PRM region appears to play a primary role in the translocation of DydA to the cell cortex and in its localization to the actin foci at the bottom of cells. Colocalization experiments of GFP-PRM with RFP-coronin indicated that GFP-PRM preceded GFP-coronin by 2-3 s in response to chemoattractant stimulation. These results suggest that the PRM region plays an indispensable role in relaying upstream regulators, such as RasG, to downstream effectors by mediating the localization of DydA to the cell cortex upon chemoattractant stimulation., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The actin-rich cortex plays a fundamental role in many cellular processes. Its architecture and molecular composition vary across cell types and physiological states. The full complement of actin assembly factors driving cortex formation and how their activities are spatiotemporally regulated remain to be fully elucidated. Using Dictyostelium as a model for polarized and rapidly migrating cells, we show that GxcM, a RhoGEF localized specifically in the rear of migrating cells, functions together with F-BAR protein Fbp17, a small GTPase RacC, and the actin nucleation-promoting factor WASP to coordinately promote Arp2/3 complex-mediated cortical actin assembly. Overactivation of this signaling cascade leads to excessive actin polymerization in the rear cortex, whereas its disruption causes defects in cortical integrity and function. Therefore, apart from its well-defined role in the formation of the protrusions at the cell front, the Arp2/3 complex-based actin carries out a previously unappreciated function in building the rear cortical subcompartment in rapidly migrating cells., (© 2023 Li et al.)

STATa is a pivotal transcription factor for Dictyostelium development. dutA is the most abundant RNA transcribed by RNA polymerase II in Dictyostelium, and its functional interplay with STATa has been suggested. This study demonstrates that dutA RNA molecules are distributed as spot-like structures in the cytoplasm, and that its cell type-specific expression changes dramatically during development. dutA RNA was exclusively detectable in the prespore region of slugs and then predominantly localized in prestalk cells, including the organizer region, at the Mexican hat stage before most dutA transcripts, excluding those in prestalk O cells, disappeared as culmination proceeded. dutA RNA was not translated into small peptides from any potential open reading frame, which confirmed that it is a cytoplasmic lncRNA. Ectopic expression of dutA RNA in the organizer region of slugs caused a prolonged slug migration period. In addition, buffered suspension-cultured cells of the strain displayed reduced STATa nuclear translocation and phosphorylation on Tyr702. Analysis of gene expression in various dutA mutants revealed changes in the levels of several STATa-regulated genes, such as the transcription factors mybC and gtaG, which might affect the phenotype. dutA RNA may regulate several mRNA species, thereby playing an indirect role in STATa activation., (© 2022 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Cellular functions, such as division and migration, require cells to undergo robust shape changes. Through their contractility machinery, cells also sense, respond, and adapt to their physical surroundings. In the cytoplasm, the contractility machinery organizes into higher order assemblies termed contractility kits (CKs). Using Dictyostelium discoideum, we previously identified Discoidin I (DscI), a classic secreted lectin, as a CK component through its physical interactions with the actin crosslinker Cortexillin I (CortI) and the scaffolding protein IQGAP2. Here, we find that DscI ensures robust cytokinesis through regulating intracellular components of the contractile machinery. Specifically, DscI is necessary for normal cytokinesis, cortical tension, membrane-cortex connections, and cortical distribution and mechanoresponsiveness of CortI. The dscI deletion mutants also have complex genetic epistatic relationships with CK components, acting as a genetic suppressor of cortI and iqgap1, but as an enhancer of iqgap2. This work underscores the fact that proteins like DiscI contribute in diverse ways to the activities necessary for optimal cell function., (© 2022 Nguyen and Robinson.)

Mitochondrial Complex II is composed of four core subunits and mutations to any of the subunits result in lowered Complex II activity. Surprisingly, although mutations in any of the subunits can yield similar clinical outcomes, there are distinct differences in the patterns of clinical disease most commonly associated with mutations in different subunits. Thus, mutations to the SdhA subunit most often result in mitochondrial disease phenotypes, whilst mutations to the other subunits SdhB-D more commonly result in tumour formation. The reason the clinical outcomes are so different is unknown. Here, we individually antisense-inhibited three of the Complex II subunits, SdhA, SdhB or SdhC, in the simple model organism Dictyostelium discoideum . Whilst SdhB and SdhC knockdown resulted in growth defects on bacterial lawns, antisense inhibition of SdhA expression resulted in a different pattern of phenotypic defects, including impairments of growth in liquid medium, enhanced intracellular proliferation of the bacterial pathogen Legionella pneumophila and phagocytosis. Knockdown of the individual subunits also produced different abnormalities in mitochondrial function with only SdhA knockdown resulting in broad mitochondrial dysfunction. Furthermore, these defects were shown to be mediated by the chronic activation of the cellular energy sensor AMP-activated protein kinase. Our results are in agreement with a role for loss of function of SdhA but not the other Complex II subunits in impairing mitochondrial oxidative phosphorylation and they suggest a role for AMP-activated protein kinase in mediating the cytopathological outcomes.

The ability of cells to sense chemical gradients is essential during development, morphogenesis, and immune responses. Although much is known about chemoattraction, chemorepulsion remains poorly understood. Proliferating Dictyostelium cells secrete a chemorepellent protein called AprA. AprA prevents pseudopod formation at the region of the cell closest to the source of AprA, causing the random movement of cells to be biased away from the AprA. Activation of Ras proteins in a localized sector of a cell cortex helps to induce pseudopod formation, and Ras proteins are needed for AprA chemorepulsion. Here we show that AprA locally inhibits Ras cortical activation through the G protein-coupled receptor GrlH, the G protein subunits Gβ and Gα8, Ras protein RasG, protein kinase B, the p21-activated kinase PakD, and the extracellular signal-regulated kinase Erk1. Diffusion calculations and experiments indicate that in a colony of cells, high extracellular concentrations of AprA in the center can globally inhibit Ras activation, while a gradient of AprA that naturally forms at the edge of the colony allows cells to activate Ras at sectors of the cell other than the sector of the cell closest to the center of the colony, effectively inducing both repulsion from the colony and cell differentiation. Together, these results suggest that a pathway that inhibits local Ras activation can mediate chemorepulsion.

The lamellipodia and pseudopodia of migrating cells are produced and maintained by the Scar/WAVE complex. Thus, actin-based cell migration is largely controlled through regulation of Scar/WAVE. Here, we report that the Abi subunit-but not Scar-is phosphorylated in response to extracellular signalling in Dictyostelium cells. Like Scar, Abi is phosphorylated after the complex has been activated, implying that Abi phosphorylation modulates pseudopodia, rather than causing new ones to be made. Consistent with this, Scar complex mutants that cannot bind Rac are also not phosphorylated. Several environmental cues also affect Abi phosphorylation-cell-substrate adhesion promotes it and increased extracellular osmolarity diminishes it. Both unphosphorylatable and phosphomimetic Abi efficiently rescue the chemotaxis of Abi KO cells and pseudopodia formation, confirming that Abi phosphorylation is not required for activation or inactivation of the Scar/WAVE complex. However, pseudopodia and Scar patches in the cells with unphosphorylatable Abi protrude for longer, altering pseudopod dynamics and cell speed. Dictyostelium , in which Scar and Abi are both unphosphorylatable, can still form pseudopods, but migrate substantially faster. We conclude that extracellular signals and environmental responses modulate cell migration by tuning the behaviour of the Scar/WAVE complex after it has been activated.

Post-transcriptional processes mediated by mRNA binding proteins represent important control points in gene expression. In eukaryotes, mRNAs containing specific AU-rich motifs are regulated by binding of tristetraprolin (TTP) family tandem zinc finger proteins, which promote mRNA deadenylation and decay, partly through interaction of a conserved C-terminal CNOT1 binding (CNB) domain with CCR4-NOT protein complexes. The social ameba Dictyostelium discoideum shared a common ancestor with humans more than a billion years ago, and expresses only one TTP family protein, TtpA, in contrast to three members expressed in humans. Evaluation of ttpA null-mutants identified six transcripts that were consistently upregulated compared to WT during growth and early development. The 3'-untranslated regions (3'-UTRs) of all six 'TtpA-target' mRNAs contained multiple TTP binding motifs (UUAUUUAUU), and one 3'-UTR conferred TtpA post-transcriptional stability regulation to a heterologous mRNA that was abrogated by mutations in the core TTP-binding motifs. All six target transcripts were upregulated to similar extents in a C-terminal truncation mutant, in contrast to less severe effects of analogous mutants in mice. All six target transcripts encoded probable membrane proteins. In Dictyostelium, TtpA may control an 'RNA regulon', where a single RNA binding protein, TtpA, post-transcriptionally co-regulates expression of several functionally related proteins., (Published by Oxford University Press on behalf of Nucleic Acids Research 2021.)

Some G protein alpha subunits contain a mitogen-activated protein kinase (MAPK) docking motif (D-motif) near the amino terminus that can impact cellular responses to external signals. The Dictyostelium Gα2 G protein subunit is required for chemotaxis to cAMP during the onset of multicellular development and this subunit contains a putative D-motif near the amino terminus. The Gα2 subunit D-motif was altered to examine its potential role in chemotaxis and multicellular development. In gα2 - cells the expression of the D-motif mutant (Gα2 D- ) or wild-type subunit from high copy number vectors rescued cell aggregation but blocked the transition of mounds into slugs. This phenotype was also observed in parental strains with a wild-type gα2 locus indicating that the heterologous Gα2 subunit expression interferes with multicellular morphogenesis. Expression of the Gα2 D- subunit from a low copy number vectors in gα2 - cells did not rescue aggregation whereas the wild-type Gα2 subunit rescued aggregation efficiently and allowed wild-type morphological development. The Gα2 D- and Gα2 subunit were both capable of restoring comparable levels of cAMP stimulated motility and the ability to co-aggregate with wild-type cells implying that the aggregation defect of Gα2 D- expressing cells is due to insufficient intercellular signaling. Expression of the Gα2 subunit but not the Gα2 D- subunit fully restored the ability of cAMP to stimulate the translocation of the GtaC transcription factor suggesting the D-motif is important for transcription factor regulation. These results suggest that the D-motif of Gα2 plays a role in aggregation and other developmental responses involved with cAMP signaling., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Poly (ADP-ribose) polymerase-1 (PARP-1) is a multifunctional protein that is associated with various biological processes like chromatin remodeling, DNA damage, cell death etc. In Dictyostelium discoideum, PARP-1 has also been implicated in cellular differentiation and development. However, its interacting proteins during multicellular development are not yet explored. Hence, the present study aims to identify PARP-1 interacting proteins during multicellular development of D. discoideum. BRCA1 C-terminus (BRCT) domain of PARP-1, which is mainly involved in protein-protein interactions was cloned in pGEX4T1 vector and developmental interactome of PARP-1 were analyzed by affinity purification-mass spectrometry. These interactions were further confirmed by in-silico protein-protein docking analysis, which led to identification of the proteins that show high affinity for BRCT domain. Initially, the protein structures were modeled on SWISS MODEL and PHYRE2 servers, refined by 3Drefine and validated by PROCHECK. Further, interaction sites of BRCT and the conserved regions in all interacting proteins were predicted using cons-PPISP and ConSurf, respectively. Finally, protein-protein docking analysis was done by HADDOCK. Our results identified 19 possible BRCT interacting proteins during D. discoideum development. Furthermore, interacting residues involved in the interactions and functional regions were explored. This is the first report where PARP-1's developmental interactome in D. discoideum is well established. The current findings demonstrate PARP-1's developmental interactome in D. discoideum and provide the groundwork to understand its regulated functions in developmental biology which would undoubtedly extend our perception towards developmental diseases in higher complex organisms and their treatment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The Dictyostelium centrosome is a nucleus-associated body with a diameter of approx. 500 nm. It contains no centrioles but consists of a cylindrical layered core structure surrounded by a microtubule-nucleating corona. At the onset of mitosis, the corona disassembles and the core structure duplicates through growth, splitting, and reorganization of the outer core layers. During the last decades our research group has characterized the majority of the 42 known centrosomal proteins. In this work we focus on the conserved, previously uncharacterized Cep192 protein. We use superresolution expansion microscopy (ExM) to show that Cep192 is a component of the outer core layers. Furthermore, ExM with centrosomal marker proteins nicely mirrored all ultrastructurally known centrosomal substructures. Furthermore, we improved the proximity-dependent biotin identification assay (BioID) by adapting the biotinylase BioID2 for expression in Dictyostelium and applying a knock-in strategy for the expression of BioID2-tagged centrosomal fusion proteins. Thus, we were able to identify various centrosomal Cep192 interaction partners, including CDK5RAP2, which was previously allocated to the inner corona structure, and several core components. Studies employing overexpression of GFP-Cep192 as well as depletion of endogenous Cep192 revealed that Cep192 is a key protein for the recruitment of corona components during centrosome biogenesis and is required to maintain a stable corona structure.

Rap small GTPases are involved in diverse signaling pathways associated with cell growth, proliferation, and cell migration. There are three Rap proteins in Dictyostelium, RapA, RapB, and RapC. RapA is a key regulator in the control of cell adhesion and migration. Recently RapA and RapC have been reported to have opposite functions in the regulation of cellular processes. In this study, we demonstrate that the C-terminus of RapC, which is not found in RapA, is essential for the opposite functions of RapC and is able to reverse the functions of RapA when fused to the tail of RapA. Cells lacking RapC displayed several defective phenotypes, including spread morphology, strong adhesion, and decreased cell migration compared to wild-type cells. These phenotypes were rescued by full-length RapC, but not by RapC missing the C-terminus. Furthermore, recombinant RapA fused with the C-terminus of RapC completely recovered the phenotypes of rapC null cells, indicating that the functions of RapA were modified to become similar to those of RapC by the C-terminus of RapC with respect to cell morphology, cell adhesion and migration, cytokinesis, and development. These results suggest that the C-terminal residues of RapC are able to suppress and change the functions of other Ras proteins in Ras oncogenic signaling pathways., (© 2021. The Microbiological Society of Korea.)

Mitochondrial dysfunction has been implicated in the pathology of Parkinson's disease (PD). In Dictyostelium discoideum , strains with mitochondrial dysfunction present consistent, AMPK-dependent phenotypes. This provides an opportunity to investigate if the loss of function of specific PD-associated genes produces cellular pathology by causing mitochondrial dysfunction with AMPK-mediated consequences. DJ-1 is a PD-associated, cytosolic protein with a conserved oxidizable cysteine residue that is important for the protein's ability to protect cells from the pathological consequences of oxidative stress. Dictyostelium DJ-1 (encoded by the gene deeJ ) is located in the cytosol from where it indirectly inhibits mitochondrial respiration and also exerts a positive, nonmitochondrial role in endocytosis (particularly phagocytosis). Its loss in unstressed cells impairs endocytosis and causes correspondingly slower growth, while also stimulating mitochondrial respiration. We report here that oxidative stress in Dictyostelium cells inhibits mitochondrial respiration and impairs phagocytosis in an AMPK-dependent manner. This adds to the separate impairment of phagocytosis caused by DJ-1 knockdown. Oxidative stress also combines with DJ-1 loss in an AMPK-dependent manner to impair or exacerbate defects in phototaxis, morphogenesis and growth. It thereby phenocopies mitochondrial dysfunction. These results support a model in which the oxidized but not the reduced form of DJ-1 inhibits AMPK in the cytosol, thereby protecting cells from the adverse consequences of oxidative stress, mitochondrial dysfunction and the resulting AMPK hyperactivity.

Little is known about how eukaryotic cells can sense their number or spatial density and stop proliferating when the local density reaches a set value. We previously found that Dictyostelium discoideum accumulates extracellular polyphosphate to inhibit its proliferation, and this requires the G protein-coupled receptor GrlD and the small GTPase RasC. Here, we show that cells lacking the G protein component Gβ, the Ras guanine nucleotide exchange factor GefA, phosphatase and tensin homolog (PTEN), phospholipase C (PLC), inositol 1,4,5-trisphosphate (IP3) receptor-like protein A (IplA), polyphosphate kinase 1 (Ppk1), or the TOR complex 2 component PiaA have significantly reduced sensitivity to polyphosphate-induced proliferation inhibition. Polyphosphate upregulates IP3, and this requires GrlD, GefA, PTEN, PLC, and PiaA. Polyphosphate also upregulates cytosolic Ca 2+ , and this requires GrlD, Gβ, GefA, RasC, PLC, IplA, Ppk1, and PiaA. Together, these data suggest that polyphosphate uses signal transduction pathways including IP3/Ca 2+ to inhibit the proliferation of D. discoideum. IMPORTANCE Many mammalian tissues such as the liver have the remarkable ability to regulate their size and have their cells stop proliferating when the tissue reaches the correct size. One possible mechanism involves the cells secreting a signal that they all sense, and a high level of the signal tells the cells that there are enough of them and to stop proliferating. Although regulating such mechanisms could be useful to regulate tissue size to control cancer or birth defects, little is known about such systems. Here, we use a microbial system to study such a mechanism, and we find that key elements of the mechanism have similarities to human proteins. This then suggests the possibility that we may eventually be able to regulate the proliferation of selected cell types in humans and animals.

The dynein-dynactin nanomachine transports cargoes along microtubules in cells. Why dynactin interacts separately with the dynein motor and also with microtubules is hotly debated. Here we disrupted these interactions in a targeted manner on phagosomes extracted from cells, followed by optical trapping to interrogate native dynein-dynactin teams on single phagosomes. Perturbing the dynactin-dynein interaction reduced dynein's on rate to microtubules. In contrast, perturbing the dynactin-microtubule interaction increased dynein's off rate markedly when dynein was generating force against the optical trap. The dynactin-microtubule link is therefore required for persistence against load, a finding of importance because disease-relevant mutations in dynein-dynactin are known to interfere with "high-load" functions of dynein in cells. Our findings call attention to a less studied property of dynein-dynactin, namely, its detachment against load, in understanding dynein dysfunction., Competing Interests: The authors declare no competing interest.

The phospholipid phosphatidylserine (PS) is a key signaling molecule and binding partner for many intracellular proteins. PS is normally found on the inner surface of the cell membrane, but PS can be flipped to the outer surface in a process called PS exposure. PS exposure is important in many cell functions, yet the mechanisms that control PS exposure have not been extensively studied. Copines (Cpn), found in most eukaryotic organisms, make up a family of calcium-dependent phospholipid binding proteins. In Dictyostelium, which has six copine genes, CpnA strongly binds to PS and translocates from the cytosol to the plasma membrane in response to a rise in calcium. Cells lacking the cpnA gene (cpnA-) have defects in adhesion, chemotaxis, membrane trafficking, and cytokinesis. In this study we used both flow cytometry and fluorescent microscopy to show that cpnA- cells have increased adhesion to beads and bacteria and that the increased adhesion was not due to changes in the actin cytoskeleton or cell surface proteins. We found that cpnA- cells bound higher amounts of Annexin V, a PS binding protein, than parental cells and showed that unlabeled Annexin V reduced the increased cell adhesion property of cpnA- cells. We also found that cpnA- cells were more sensitive to Polybia-MP1, which binds to external PS and induces cell lysis. Overall, this suggests that cpnA- cells have increased PS exposure and this property contributes to the increased cell adhesion of cpnA- cells. We conclude that CpnA has a role in the regulation of plasma membrane lipid composition and may act as a negative regulator of PS exposure., Competing Interests: The authors have declared that no competing interests exist.

Filopodia are thin, actin-based structures that cells use to interact with their environments. Filopodia initiation requires a suite of conserved proteins but the mechanism remains poorly understood. The actin polymerase VASP and a MyTH-FERM (MF) myosin, DdMyo7 in amoeba, are essential for filopodia initiation. DdMyo7 is localized to dynamic regions of the actin-rich cortex. Analysis of VASP mutants and treatment of cells with anti-actin drugs shows that myosin recruitment and activation in Dictyostelium requires localized VASP-dependent actin polymerization. Targeting of DdMyo7 to the cortex alone is not sufficient for filopodia initiation; VASP activity is also required. The actin regulator locally produces a cortical actin network that activates myosin and together they shape the actin network to promote extension of parallel bundles of actin during filopodia formation. This work reveals how filopodia initiation requires close collaboration between an actin-binding protein, the state of the actin cytoskeleton and MF myosin activity., Competing Interests: AA, AC, AH, MT No competing interests declared, (© 2021, Arthur et al.)

Wound repair of cell membranes is essential for cell survival. Myosin II contributes to wound pore closure by interacting with actin filaments in larger cells; however, its role in smaller cells is unclear. In this study, we observed wound repair in dividing cells for the first time. The cell membrane in the cleavage furrow, where myosin II localized, was wounded by laserporation. Upon wounding, actin transiently accumulated, and myosin II transiently disappeared from the wound site. Ca 2+ influx from the external medium triggered both actin and myosin II dynamics. Inhibition of calmodulin reduced both actin and myosin II dynamics. The wound closure time in myosin II-null cells was the same as that in wild-type cells, suggesting that myosin II is not essential for wound repair. We also found that disassembly of myosin II filaments by phosphorylation did not contribute to their disappearance, indicating a novel mechanism for myosin II delocalization from the cortex. Furthermore, we observed that several furrow-localizing proteins such as GAPA, PakA, myosin heavy chain kinase C, PTEN, and dynamin disappeared upon wounding. Herein, we discuss the possible mechanisms of myosin dynamics during wound repair.

Dictyostelium discoideum Sey1 is the single ortholog of mammalian atlastin 1-3 (ATL1-3), which are large homodimeric GTPases mediating homotypic fusion of endoplasmic reticulum (ER) tubules. In this study, we generated a D. discoideum mutant strain lacking the sey1 gene and found that amoebae deleted for sey1 are enlarged, but grow and develop similarly to the parental strain. The ∆sey1 mutant amoebae showed an altered ER architecture, and the tubular ER network was partially disrupted without any major consequences for other organelles or the architecture of the secretory and endocytic pathways. Macropinocytic and phagocytic functions were preserved; however, the mutant amoebae exhibited cumulative defects in lysosomal enzymes exocytosis, intracellular proteolysis, and cell motility, resulting in impaired growth on bacterial lawns. Moreover, ∆sey1 mutant cells showed a constitutive activation of the unfolded protein response pathway (UPR), but they still readily adapted to moderate levels of ER stress, while unable to cope with prolonged stress. In D. discoideum ∆sey1 the formation of the ER-associated compartment harbouring the bacterial pathogen Legionella pneumophila was also impaired. In the mutant amoebae, the ER was less efficiently recruited to the "Legionella-containing vacuole" (LCV), the expansion of the pathogen vacuole was inhibited at early stages of infection and intracellular bacterial growth was reduced. In summary, our study establishes a role of D. discoideum Sey1 in ER architecture, proteolysis, cell motility and intracellular replication of L. pneumophila., (© 2021 John Wiley & Sons Ltd.)

The slime mold Dictyostelium discoideum's life cycle includes different unicellular and multicellular stages that provide a convenient model for research concerning intracellular and intercellular mechanisms influencing mitochondria's structure and function. We aim to determine the differences between the mitochondria isolated from the slime mold regarding its early developmental stages induced by starvation, namely the unicellular (U), aggregation (A) and streams (S) stages, at the bioenergetic and proteome levels. We measured the oxygen consumption of intact cells using the Clarke electrode and observed a distinct decrease in mitochondrial coupling capacity for stage S cells and a decrease in mitochondrial coupling efficiency for stage A and S cells. We also found changes in spare respiratory capacity. We performed a wide comparative proteomic study. During the transition from the unicellular stage to the multicellular stage, important proteomic differences occurred in stages A and S relating to the proteins of the main mitochondrial functional groups, showing characteristic tendencies that could be associated with their ongoing adaptation to starvation following cell reprogramming during the switch to gluconeogenesis. We suggest that the main mitochondrial processes are downregulated during the early developmental stages, although this needs to be verified by extending analogous studies to the next slime mold life cycle stages.

Calmodulin (CaM) is an essential calcium-binding protein within eukaryotes. CaM binds to calmodulin-binding proteins (CaMBPs) and influences a variety of cellular and developmental processes. In this study, we used immunoprecipitation coupled with mass spectrometry (LC-MS/MS) to reveal over 500 putative CaM interactors in the model organism Dictyostelium discoideum . Our analysis revealed several known CaMBPs in Dictyostelium and mammalian cells (e.g., myosin, calcineurin), as well as many novel interactors (e.g., cathepsin D). Gene ontology (GO) term enrichment and Search Tool for the Retrieval of Interacting proteins (STRING) analyses linked the CaM interactors to several cellular and developmental processes in Dictyostelium including cytokinesis, gene expression, endocytosis, and metabolism. The primary localizations of the CaM interactors include the nucleus, ribosomes, vesicles, mitochondria, cytoskeleton, and extracellular space. These findings are not only consistent with previous work on CaM and CaMBPs in Dictyostelium , but they also provide new insight on their diverse cellular and developmental roles in this model organism. In total, this study provides the first in vivo catalogue of putative CaM interactors in Dictyostelium and sheds additional light on the essential roles of CaM and CaMBPs in eukaryotes.

The ability of Salmonella to survive and replicate within mammalian host cells involves the generation of a membranous compartment known as the Salmonella-containing vacuole (SCV). Salmonella employs a number of effector proteins that are injected into host cells for SCV formation using its type-3 secretion systems encoded in SPI-1 and SPI-2 (T3SS-1 and T3SS-2, respectively). Recently, we reported that S. Typhimurium requires T3SS-1 and T3SS-2 to survive in the model amoeba Dictyostelium discoideum. Despite these findings, the involved effector proteins have not been identified yet. Therefore, we evaluated the role of two major S. Typhimurium effectors SopB and SifA during D. discoideum intracellular niche formation. First, we established that S. Typhimurium resides in a vacuolar compartment within D. discoideum. Next, we isolated SCVs from amoebae infected with wild type or the ΔsopB and ΔsifA mutant strains of S. Typhimurium, and we characterised the composition of this compartment by quantitative proteomics. This comparative analysis suggests that S. Typhimurium requires SopB and SifA to modify the SCV proteome in order to generate a suitable intracellular niche in D. discoideum. Accordingly, we observed that SopB and SifA are needed for intracellular survival of S. Typhimurium in this organism. Thus, our results provide insight into the mechanisms employed by Salmonella to survive intracellularly in phagocytic amoebae., (© 2020 John Wiley & Sons Ltd.)

Eukaryotic phagocytes locate microorganisms via chemotaxis and consume them through phagocytosis. The social amoeba Dictyostelium discoideum is a stereotypical phagocyte and a well-established model to study both processes. Recent studies show that a G-protein-coupled receptor (fAR1) mediate a signaling network to control reorganization of the actin cytoskeleton leading both the directional cell movement and the engulfment of bacteria. Many live cell imaging methods have been developed and applied to monitor these signaling events. In this chapter, we will introduce how to measure GPCR-mediated signaling events for cell migration and phagocytosis in Dictyostelium.

Macropinocytosis and phagocytosis are the processes by which eukaryotic cells use their plasma membrane to engulf liquid or a large particle and give rise to an internal compartment called the macropinosomes or phagosome, respectively. Dictyostelium discoideum provides a powerful system to understand the molecular mechanism of these two fundamental cellular processes that impact human health and disease. Recent developments in fluorescence microscopy allow direct visualization of intracellular signaling events with high temporal and spatial resolution. Here, we describe methods to visualize temporospatial activation or localization of key signaling components that are crucial for macropinocytosis and phagocytosis using confocal fluorescence microscopy.

Poly (ADP-ribose) polymerase-1 (PARP1) is a DNA damage sensor that gets activated in proportion to the damage, helping cells to determine whether to repair the damage or initiate cell death processes. We have previously shown PARP1's significance in the developmental processes of Dictyostelium discoideum in addition to its role in oxidative stress and UV-C stress induced cell death. In this study, we show the significance of ROS in PARP1 mediated responses of D. discoideum under different stress conditions. Interestingly, our results suggest differential kinetics of PARP1 activation and implications of ROS in starvation and cadmium induced cell death events. Increased accumulation of Poly (ADP-ribose), a product of PARP activation, could be detected within minutes post cadmium stress, whereas PARP1 activation was only a later event with starvation. Starvation induced PARP1 activation was supported by the depletion of ATP and NAD + , while PARP inhibitor confers protective effect during starvation. During starvation, cell death is induced in two phases, a primary ROS driven PARP1 independent early necrotic phase followed by a PARP1 driven ROS dependent paraptotic phase; both of which comprise mitochondrial changes. Cadmium (Cd) exerted a dose-dependent effect on cell death; a low dose of 0.2 mM Cd led to paraptosis and a higher dose of 0.5 mM Cd led to necrosis in D. discoideum cells within 24 h. Interestingly, glutathione (GSH) exposure could rescue cells from Cd stress mediated cell death. Besides unicellular cell death, the developmental arrest induced by cadmium and oxidative stress could be rescued by reinstating the redox equilibrium using GSH. In conclusion, we underscore the significant link between PARP1 and ROS in regulating the process of cell death and development in D. discoideum., (Copyright © 2020. Published by Elsevier Inc.)

In animals, the response to chronic hypoxia is mediated by prolyl hydroxylases (PHDs) that regulate the levels of hypoxia-inducible transcription factor α (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the cellular slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii , both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-phase kinase-associated protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full-length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies, TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, Trichoplax adhaerens , and prokaryotes, informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles., Competing Interests: Conflict of interest—The authors declare no competing financial interests., (© 2020 Liu et al.)

The evolutionarily-conserved 14-3-3 proteins regulate many cellular processes through binding to various phosphorylated targets in eukaryotes. It first appears in Dictyostelium, however its role in this organism is poorly understood. Here we show that down-regulation of the 14-3-3 impairs chemotaxis and causes multiple-tip formation in Dictyostelium. Mechanistically, the 14-3-3 is a critical component of cyclic AMP (cAMP) signaling and binds to nearly a hundred of proteins in Dictyostelium, including a number of evolutionarily-conserved proteins. 14-3-3 - interaction with its targets is up-regulated in response to developmental cues/regulators including starvation, osmotic stress and cAMP. cAMP stimulates 14-3-3 - binding to phospho-Ser431 on a guanine nucleotide exchange factor Gef-Q. Interestingly, overexpression of Gef-Q Ser431Ala mutant but not wild-type Gef-Q protein causes a multiple-tip phenotype in Dictyostelium, which partially resembles phenotypes of the 14-3-3 - deficient mutant. Collectively, these data demonstrate that the 14-3-3 plays an important role in Dictyostelium and may help to deepen our understanding of the evolution of 14-3-3 - interactomes in eukaryotes., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The CRISPR/Cas9 system is a powerful method of editing genes by randomly introducing errors into the target sites. Here, we describe a CRISPR-based test for gene essentiality (CRISPR-E test) that allows the identification of essential genes. Specifically, we use sgRNA-mediated CRISPR/Cas9 to target the open reading frame of a gene in the genome and analyze the in-frame (3n) and frameshift (3n + 1 and 3n + 2) mutations in the targeted region of the gene in surviving cells. If the gene is non-essential, the cells would carry both in-frame (3n) and frameshift (3n + 1 and 3n + 2) mutations. In contrast, the cells would carry only in-frame (3n) mutations if the targeted gene is essential, and this selective elimination of frameshift (3n + 1 and 3n + 2) mutations of the gene indicate its essentiality. As a proof of concept, we have used this CRISPR-E test in the model organism Dictyostelium discoideum to demonstrate that Dync1li1 is an essential gene while KIF1A and fAR1 are not. We further propose a simple method for quantifying the essentiality of a gene using the CRISPR-E test.