In Dictyostelium, the intracellular cAMP-specific phosphodiesterase RegA is a negative regulator of cAMP-dependent protein kinase (PKA), a key determinant in the timing of developmental morphogenesis and spore formation. To assess the role of protein kinases in the regulation of RegA function, this study identified phosphorylation sites on RegA and characterized the role of these modifications through the analysis of phospho-mimetic and phospho-ablative mutations. Mutations affecting residue T676 of RegA, a presumed target of the atypical MAP kinase Erk2, altered the rate of development and impacted cell distribution in chimeric organisms suggesting that phosphorylation of this residue reduces RegA function and regulates cell localization during multicellular development. Mutations affecting the residue S142 of RegA also impacted the rate developmental morphogenesis but in a manner opposite of changes at T676 suggesting the phosphorylation of the S142 residue increases RegA function. Mutations affecting residue S413 residue altered aggregate sizes and delayed developmental progression suggesting that PKA operates in a negative feedback mechanism to increase RegA function. These results suggest that the phosphorylation of different residues on RegA can lead to increased or decreased RegA function and therefore in turn regulate developmental processes such as aggregate formation, cell distribution, and the kinetics of developmental morphogenesis.

Mutations in CLN3 cause a juvenile form of neuronal ceroid lipofuscinosis (NCL). This devastating neurological disorder, commonly known as Batten disease, is currently untreatable due to a lack of understanding of the physiological role of the protein. Recently, work in the social amoeba Dictyostelium discoideum has provided valuable new insight into the function of CLN3 in the cell. More specifically, research has linked the Dictyostelium homolog (gene: cln3, protein: Cln3) to protein secretion, adhesion, and aggregation during starvation, which initiates multicellular development. In this study, we used comparative transcriptomics to explore the mechanisms underlying the aberrant response of cln3- cells to starvation. During starvation, 1153 genes were differentially expressed in cln3- cells compared to WT. Among the differentially expressed genes were homologs of other human NCL genes including TPP1/CLN2, CLN5, CTSD/CLN10, PGRN/CLN11, and CTSF/CLN13. STRING and GO term analyses revealed an enrichment of genes linked to metabolic, biosynthetic, and catalytic processes. We then coupled the findings from the RNA-seq analysis to biochemical assays, specifically showing that loss of cln3 affects the expression and activity of lysosomal enzymes, increases endo-lysosomal pH, and alters nitric oxide homeostasis. Finally, we show that cln3- cells accumulate autofluorescent storage bodies during starvation and provide evidence linking the function of Cln3 to Tpp1 and CtsD activity. In total, this study enhances our knowledge of the molecular mechanisms underlying Cln3 function in Dictyostelium.

Dictyostelium discoideum cells normally exist as individual amoebae, but will enter a period of multicellular development upon starvation. The initial stages of development involve the aggregation of individual cells, using cAMP as a chemoattractant. Chemotaxis is initiated when cAMP binds to its receptor, cAR1, and activates the associated G protein, Gα2βγ. However, chemotaxis will not occur unless there is a high density of starving cells present, as measured by high levels of the secreted quorum sensing molecule, CMF. We previously demonstrated that cells lacking PldB bypass the need for CMF and can aggregate at low cell density, whereas cells overexpressing pldB do not aggregate even at high cell density. Here, we found that PldB controlled both cAMP chemotaxis and cell sorting. PldB was also required by CMF to regulate G protein signaling. Specifically, CMF used PldB, to regulate the dissociation of Gα2 from Gβγ. Using fluorescence resonance energy transfer (FRET), we found that along with cAMP, CMF increased the dissociation of the G protein. In fact, CMF augmented the dissociation induced by cAMP. This augmentation was lost in cells lacking PldB. PldB appears to mediate the CMF signal through the production of phosphatidic acid, as exogenously added phosphatidic acid phenocopies overexpression of pldB. These results suggest that phospholipase D activity is required for CMF to alter the kinetics of cAMP-induced G protein signaling.

Ceroid lipofuscinosis neuronal 5 (CLN5) is a member of a family of proteins that are linked to neuronal ceroid lipofuscinosis (NCL). This devastating neurological disorder, known commonly as Batten disease, affects all ages and ethnicities and is currently incurable. The precise function of CLN5, like many of the NCL proteins, remains to be elucidated. In this study, we report the localization, molecular function, and interactome of Cln5, the CLN5 homolog in the social amoeba Dictyostelium discoideum. Residues that are glycosylated in human CLN5 are conserved in the Dictyostelium homolog as are residues that are mutated in patients with CLN5 disease. Dictyostelium Cln5 contains a putative signal peptide for secretion and we show that the protein is secreted during growth and starvation. We also reveal that both Dictyostelium Cln5 and human CLN5 are glycoside hydrolases, providing the first evidence in any system linking a molecular function to CLN5. Finally, immunoprecipitation coupled with mass spectrometry identified 61 proteins that interact with Cln5 in Dictyostelium. Of the 61 proteins, 67% localize to the extracellular space, 28% to intracellular vesicles, and 20% to lysosomes. A GO term enrichment analysis revealed that a majority of the interacting proteins are involved in metabolism, catabolism, proteolysis, and hydrolysis, and include other NCL-like proteins (e.g., Tpp1/Cln2, cathepsin D/Cln10, cathepsin F/Cln13) as well as proteins linked to Cln3 function in Dictyostelium (e.g., AprA, CfaD, CadA). In total, this work reveals a CLN5 homolog in Dictyostelium and further establishes this organism as a complementary model system for studying the functions of proteins linked to NCL in humans.

Neuronal ceroid lipofuscinosis (NCL), also referred to as Batten disease, is the most common form of childhood neurodegeneration. Mutations in CLN3 cause the most prevalent subtype of the disease, which manifests during early childhood and is currently untreatable. The precise function of the CLN3 protein is still not known, which has inhibited the development of targeted therapies. In the social amoeba Dictyostelium discoideum, loss of the CLN3 homolog, Cln3, reduces adhesion during early development, which delays streaming and aggregation. The results of the present study indicate that this phenotype may be at least partly due to aberrant protein secretion in cln3- cells. It is well-established that Cln3 localizes primarily to the contractile vacuole (CV) system in Dictyostelium, and to a lesser extent, compartments of the endocytic pathway. Intriguingly, the CV system has been linked to the secretion of proteins that do not contain a signal peptide for secretion (i.e., unconventional protein secretion). Proteins that do contain a signal peptide are secreted via a conventional mechanism involving the endoplasmic reticulum, transport through the Golgi, and secretion via vesicle release. In this study, Cln3 was observed to co-localize with the Golgi marker wheat germ agglutinin suggesting that Cln3 participates in both secretion mechanisms. Chimeras of wild-type (WT) and cln3- cells displayed delayed streaming and aggregation, and interestingly, cln3- cells starved in conditioned media (CM) harvested from starving WT cells showed near normal timing of streaming and aggregation suggesting aberrant protein secretion in Cln3-deficient cells. Based on these observations, LC-MS/MS was used to reveal the protein content of CM from starved cells (mass spectrometry data are available via ProteomeXchange with identifier PXD004897). A total of 450 proteins were detected in WT and cln3- CM, of which 3 were absent in cln3- CM. Moreover, 12 proteins that were present in cln3- CM were absent in WT CM. Label-free quantification identified 42 proteins that were present in significantly higher amounts in cln3- CM compared to WT, and 3 proteins that were present in significantly reduced amounts. A GO term enrichment analysis showed that a majority of the affected proteins are linked to endocytosis, vesicle-mediated transport, proteolysis, and metabolism. In total, the results of this study indicate that Cln3 functions in both conventional and unconventional protein secretion and that loss of Cln3 results in deregulated secretion during early development. Importantly, this is the first evidence in any system linking CLN3 function to protein secretion.

Calmodulin is an essential protein in the model organism Dictyostelium discoideum. As in other organisms, this small, calcium-regulated protein mediates a diversity of cellular events including chemotaxis, spore germination, and fertilization. Calmodulin works in a calcium-dependent or -independent manner by binding to and regulating the activity of target proteins called calmodulin-binding proteins. Profiling suggests that Dictyostelium has 60 or more calmodulin-binding proteins with specific subcellular localizations. In spite of the central importance of calmodulin, the study of these target proteins is still in its infancy. Here we critically review the history and state of the art of research into all of the identified and presumptive calmodulin-binding proteins of Dictyostelium detailing what is known about each one with suggestions for future research. Two individual calmodulin-binding proteins, the classic enzyme calcineurin A (CNA; protein phosphatase 2B) and the nuclear protein nucleomorphin (NumA), which is a regulator of nuclear number, have been particularly well studied. Research on the role of calmodulin in the function and regulation of the various myosins of Dictyostelium, especially during motility and chemotaxis, suggests that this is an area in which future active study would be particularly valuable. A general, hypothetical model for the role of calmodulin in myosin regulation is proposed.

CyrA is a novel cysteine-rich protein with four EGFL repeats that was isolated using the calmodulin (CaM) binding overlay technique (CaMBOT), suggesting it is a CaM-binding protein (CaMBP). The full-length 63kDa cyrA is cleaved into two major C-terminal fragments, cyrA-C45 and cyrA-C40. A putative CaM-binding domain was detected and both CaM-agarose binding and CaM immunoprecipitation verified that cyrA-C45 and cyrA-C40 each bind to CaM in both a Ca(2+)-dependent and -independent manner. cyrA-C45 was present continuously throughout growth and development but was secreted at high levels during the multicellular slug stage of Dictyostelium development. At this time, cyrA localizes to the extracellular matrix (ECM). ECM purification verified the presence of cyrA-C45. An 18 amino acid peptide (DdEGFL1) from the first EGFL repeat sequence of cyrA (EGFL1) that is present in both cyrA-C45 and -C40 enhances both random cell motility and cAMP-mediated chemotaxis. Here we reveal that the dose-dependent enhancement of motility by DdEGFL1 is related to the time of cell starvation. Addition of DdEGFL1 also inhibits cyrA proteolysis. The status of cyrA as an extracellular CaMBP was further clarified by the demonstration that CaM is secreted during development. Antagonism of CaM with W7 resulted in enhanced cyrA proteolysis suggesting a functional role for extracellular CaM in protecting CaMBPs from proteolysis. cyrA is the first extracellular CaMBP identified in Dictyostelium and since it is an ECM protein with EGF-like repeats that enhance cell motility and it likely also represents the first matricellular protein identified in a lower eukaryote.

The neuronal ceroid lipofuscinoses (NCLs) are a family of neurodegenerative diseases that affect people of all ages and ethnicities, yet many of the associated genes/proteins are not well characterized. Mutations in MFSD8 (major facilitator superfamily domain-containing 8) cause an infantile form of NCL referred to as CLN7 disease. In this study, we revealed the localization and binding partners of an ortholog of human MFSD8 (Mfsd8) in the social amoeba Dictyostelium discoideum. Putative lysosomal targeting motifs are conserved in Dictyostelium Mfsd8, as are several residues mutated in CLN7 disease patients. Mfsd8 tagged with GFP localizes to endocytic compartments, which includes acidic intracellular vesicles and late endosomes. We pulled-down GFP-Mfsd8 and used mass spectrometry to reveal the Mfsd8 interactome during Dictyostelium growth and starvation. Among the identified hits were the Dictyostelium ortholog of human cathepsin D (CtsD), as well as proteins linked to the functions of the CLN3 (Cln3) and CLN5 (Cln5) orthologs in Dictyostelium. To study the function of Mfsd8, we validated a publically available mfsd8- cell line (GWDI Project) and then used this knockout cell line to show that Mfsd8 influences the secretion of Cln5 and CtsD. This information is then integrated into an emerging model describing the molecular networking of NCL proteins in Dictyostelium. In total, this study identifies Dictyostelium as a new model system for studying CLN7 disease.

DdEGFL1, a synthetic epidermal growth factor-like (EGFL) peptide based on the first EGFL repeat of the extracellular matrix, cysteine-rich, calmodulin-binding protein CyrA, has previously been shown to sustain the threonine phosphorylation of a 210kDa protein during the starvation of Dictyostelium cells. Immunoprecipitation coupled with a LC/MS/MS analysis identified the 210kDa protein as vinculin B (VinB). VinB shares sequence similarity with mammalian vinculin, a protein that links the actin cytoskeleton to the plasma membrane. Both threonine phosphorylated VinB (P-VinB) and VinB-GFP localized to the cytoplasm and cytoskeleton of Dictyostelium amoebae. VinB-GFP was also shown to be threonine phosphorylated and co-immunoprecipitated with established vinculin-binding cytoskeletal proteins (e.g. myosin II heavy chain, actin, alpha-actinin, talin). P-VinB and VinB-GFP were detected in DdEGFL1 pull-down assays, which also identified a 135kDa phosphothreonine protein and two phosphotyrosine proteins (35 and 32kDa) as potential components of the DdEGFL1 signaling pathway. DdEGFL1-enhanced cell movement required the cytoskeletal proteins talin B and paxillin B and tyrosine kinase activity mediated by PKA signaling, however VinB threonine phosphorylation was shown to be independent of PI3K/PLA2 signaling and PI3K and PKA kinase activity. Finally, VinB-GFP over-expression suppressed DdEGFL1-enhanced random cell movement, but not folic acid-mediated chemotaxis. Together, this study provides the first evidence for VinB function plus new insight into the signaling pathway(s) mediating EGFL repeat/peptide-enhanced cell movement in Dictyostelium. This information is integrated into an emerging model that summarizes existing knowledge.

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-QSer431Ala 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.

Amoebas survive environmental stress by differentiating into encapsulated cysts. As cysts, pathogenic amoebas resist antibiotics, which particularly counteracts treatment of vision-destroying Acanthamoeba keratitis. Limited genetic tractability of amoeba pathogens has left their encystation mechanisms unexplored. The social amoeba Dictyostelium discoideum forms spores in multicellular fruiting bodies to survive starvation, while other dictyostelids, such as Polysphondylium pallidum can additionally encyst as single cells. Sporulation is induced by cAMP acting on PKA, with the cAMP phosphodiesterase RegA critically regulating cAMP levels. We show here that RegA is deeply conserved in social and pathogenic amoebas and that deletion of the RegA gene in P. pallidum causes precocious encystation and prevents cyst germination. We heterologously expressed and characterized Acanthamoeba RegA and performed a compound screen to identify RegA inhibitors. Two effective inhibitors increased cAMP levels and triggered Acanthamoeba encystation. Our results show that RegA critically regulates Amoebozoan encystation and that components of the cAMP signalling pathway could be effective targets for therapeutic intervention with encystation.

Human Sin1 (SAPK-interacting protein 1) is a member of a conserved family of orthologous proteins that have an essential role in signal transduction in yeast and Dictyostelium. This study demonstrates that most Sin1 orthologues contain both a Raf-like Ras-binding domain (RBD) and a pleckstrin homology (PH) domain. These domains are functional in the human Sin1 protein, with the PH domain involved in lipid and membrane binding by Sin1, and the RBD able to bind activated H-and K-Ras. Sin1 and Ras co-immunoprecipitated and co-localised, showing that these proteins associate with each other in vivo. Overexpression of Sin1 inhibited the activation of ERK, Akt and JNK signalling pathways by Ras. In contrast, siRNA knockdown of endogenous Sin1 protein expression in HEK293 cells enhanced the activation of ERK1/2 by Ras. These data suggest that Sin1 is a mammalian Ras-inhibitor.