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Self-organized and excitable signaling activities play important roles in a wide range of cellular functions in eukaryotic and prokaryotic cells. Cells require signaling networks to communicate amongst themselves, but also for response to environmental cues. Such signals involve complex spatial and temporal loops that may propagate as oscillations or waves. When Dictyostelium become starved for nutrients, cells within a localized space begin to secrete cAMP. Starved cells also become chemotactic to cAMP. cAMP signals propagate as outwardly moving waves that oscillate at ∼6 min intervals, which creates a focused territorial region for centralized cell aggregation. Proximal cells move inwardly toward the cAMP source and relay cAMP outwardly to recruit additional cells. To ensure directed inward movement and outward cAMP relay, cells go through adapted and de-adapted states for both cAMP synthesis/degradation and for directional cell movement. Although many immediate components that regulate cAMP signaling (including receptors, G proteins, an adenylyl cyclase, phosphodiesterases, and protein kinases) are known, others are only inferred. Here, using biochemical experiments coupled with gene inactivation studies, we model an integrated large, multi-component kinetic pathway involving activation, inactivation (adaptation), re-activation (re-sensitization), feed-forward, and feed-back controls to generate developmental cAMP oscillations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jaiswal, Meena, Chang, Liao, Kim and Kimmel.)

Perilipin 2 (Plin2) binds to the surface of hepatic lipid droplets (LDs) with expression levels that correlate with triacylglyceride (TAG) content. We investigated if Plin2 is important for hepatic LD storage in fasted or high-fat diet-induced obese Plin2 +/+ and Plin2 -/- mice. Plin2 -/- mice had comparable body weights, metabolic phenotype, glucose tolerance, and circulating TAG and total cholesterol levels compared with Plin2 +/+ mice, regardless of the dietary regime. Both fasted and high-fat fed Plin2 -/- mice stored reduced levels of hepatic TAG compared with Plin2 +/+ mice. Fasted Plin2 -/- mice stored fewer but larger hepatic LDs compared with Plin2 +/+ mice. Detailed hepatic lipid analysis showed substantial reductions in accumulated TAG species in fasted Plin2 -/- mice compared with Plin2 +/+ mice, whereas cholesteryl esters and phosphatidylcholines were increased. RNA-Seq revealed minor differences in hepatic gene expression between fed Plin2 +/+ and Plin2 -/- mice, in contrast to marked differences in gene expression between fasted Plin2 +/+ and Plin2 -/- mice. Our findings demonstrate that Plin2 is required to regulate hepatic LD size and storage of neutral lipid species in the fasted state, while its role in obesity-induced steatosis is less clear., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)

Cholesteryl esters (CEs) are the water-insoluble transport and storage form of cholesterol. Steroidogenic cells primarily store CEs in cytoplasmic lipid droplet (LD) organelles, as contrasted to the majority of mammalian cell types that predominantly store triacylglycerol (TAG) in LDs. The LD-binding Plin2 binds to both CE- and TAG-rich LDs, and although Plin2 is known to regulate degradation of TAG-rich LDs, its role for regulation of CE-rich LDs is unclear. To investigate the role of Plin2 in the regulation of CE-rich LDs, we performed histological and molecular characterization of adrenal glands from Plin2 +/+ and Plin2 -/- mice. Adrenal glands of Plin2 -/- mice had significantly enlarged organ size, increased size and numbers of CE-rich LDs in cortical cells, elevated cellular unesterified cholesterol levels, and increased expression of macrophage markers and genes facilitating reverse cholesterol transport. Despite altered LD storage, mobilization of adrenal LDs and secretion of corticosterone induced by adrenocorticotropic hormone stimulation or starvation were similar in Plin2 +/+ and Plin2 -/- mice. Plin2 -/- adrenals accumulated ceroid-like structures rich in multilamellar bodies in the adrenal cortex-medulla boundary, which increased with age, particularly in females. Finally, Plin2 -/- mice displayed unexpectedly high levels of phosphatidylglycerols, which directly paralleled the accumulation of these ceroid-like structures. Our findings demonstrate an important role of Plin2 for regulation of CE-rich LDs and cellular cholesterol balance in the adrenal cortex., Competing Interests: Conflict of interest C. A. D. is a founder, shareholder, board member, and consultant in Vitas Ltd. T. B. is an employee and a shareholder of Vitas Ltd. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Cellular functions can be regulated by cell-cell interactions that are influenced by extra-cellular, density-dependent signaling factors. Dictyostelium grow as individual cells in nutrient-rich sources, but, as nutrients become depleted, they initiate a multi-cell developmental program that is dependent upon a cell-density threshold. We hypothesized that novel secreted proteins may serve as density-sensing factors to promote multi-cell developmental fate decisions at a specific cell-density threshold, and use Dictyostelium in the identification of such a factor., Results: We show that multi-cell developmental aggregation in Dictyostelium is lost upon minimal (2-fold) reduction in local cell density. Remarkably, developmental aggregation response at non-permissive cell densities is rescued by addition of conditioned media from high-density, developmentally competent cells. Using rescued aggregation of low-density cells as an assay, we purified a single, 150-kDa extra-cellular protein with density aggregation activity. MS/MS peptide sequence analysis identified the gene sequence, and cells that overexpress the full-length protein accumulate higher levels of a development promoting factor (DPF) activity than parental cells, allowing cells to aggregate at lower cell densities; cells deficient for this DPF gene lack density-dependent developmental aggregation activity and require higher cell density for cell aggregation compared to WT. Density aggregation activity co-purifies with tagged versions of DPF and tag-affinity-purified DPF possesses density aggregation activity. In mixed development with WT, cells that overexpress DPF preferentially localize at centers for multi-cell aggregation and define cell-fate choice during cytodifferentiation. Finally, we show that DPF is synthesized as a larger precursor, single-pass transmembrane protein, with the p150 fragment released by proteolytic cleavage and ectodomain shedding. The TM/cytoplasmic domain of DPF possesses cell-autonomous activity for cell-substratum adhesion and for cellular growth., Conclusions: We have purified a novel secreted protein, DPF, that acts as a density-sensing factor for development and functions to define local collective thresholds for Dictyostelium development and to facilitate cell-cell communication and multi-cell formation. Regions of high DPF expression are enriched at centers for cell-cell signal-response, multi-cell formation, and cell-fate determination. Additionally, DPF has separate cell-autonomous functions for regulation of cellular adhesion and growth.

Background: Kinases mTORC1 and AMPK act as energy sensors, controlling nutrient responses and cellular growth. Changes in nutrient levels affect diverse transcriptional networks, making it challenging to identify downstream paths that regulate cellular growth or a switch to development via nutrient variation. The life cycle of Dictyostelium presents an excellent model to study the mTORC1 signaling function for growth and development. Dictyostelium grow as single cells in nutrient-rich media, but, upon nutrient withdrawal, growth ceases and cells enter a program for multi-cell development. While nearly half the genome shows gene expression changes upon nutrient removal, we hypothesized that not all of these genes are required for the switch to program development. Through manipulation of mTORC1 activity alone, without nutrient removal, we focused on a core network of genes that are required for switching between growth and development for regulation of cell fate decisions., Results: To identify developmentally essential genes, we sought ways to promote development in the absence of nutrient loss. We first examined the activities of mTORC1 and AMPK in Dictyostelium during phases of rapid growth and starvation-induced development and showed they exhibited reciprocal patterns of regulation under various conditions. Using these as initial readouts, we identified rich media conditions that promoted rapid cell growth but, upon mTORC1 inactivation by rapamycin, led to a growth/development switch. Examination of gene expression during cell fate switching showed that changes in expression of most starvation-regulated genes were not required for developmental induction. Approximately 1000 genes which become downregulated upon rapamycin treatment comprise a cellular growth network involving ribosome biogenesis, protein synthesis, and cell cycle processes. Conversely, the upregulation of ~ 500 genes by rapamycin treatment defines essential signaling pathways for developmental induction, and ~ 135 of their protein products intersect through the well-defined cAMP/PKA network. Many of the rapamycin-induced genes we found are currently unclassified, and mutation analyses of 5 such genes suggest a novel gene class essential for developmental regulation., Conclusions: We show that manipulating activities of mTORC1/AMPK in the absence of nutrient withdrawal is sufficient for a growth-to-developmental fate switch in Dictyostelium, providing a means to identify transcriptional networks and signaling pathways essential for early development.

Myocardial dysfunction is commonly associated with accumulation of cardiac lipid droplets (LDs). Perilipin 2 (Plin2) is a LD protein that is involved in LD formation, stability and trafficking events within the cell. Even though Plin2 is highly expressed in the heart, little is known about its role in myocardial lipid storage. A recent report shows that cardiac overexpression of Plin2 result in massive myocardial steatosis suggesting that Plin2 stabilizes LDs. In this study, we hypothesized that deficiency in Plin2 would result in reduced myocardial lipid storage. In contrast to our hypothesis, we found increased accumulation of triglycerides in hearts, and specifically in cardiomyocytes, from Plin2 -/- mice. Although Plin2 -/- mice had markedly enhanced lipid levels in the heart, they had normal heart function under baseline conditions and under mild stress. However, after an induced myocardial infarction, stroke volume and cardiac output were reduced in Plin2 -/- mice compared with Plin2 +/+ mice. We further demonstrated that the increased triglyceride accumulation in Plin2-deficient hearts was caused by altered lipophagy. Together, our data show that Plin2 is important for proper hydrolysis of LDs.

In this issue of Developmental Cell, Nichols et al. (2019) establish the MAP kinase ErkB as a critical component for chemotaxis signaling in Dictyostelium. Using phosphoproteomics, they identify a chemoattractant-dependent ErkB targeted core set of signal transduction proteins, which collectively suggest an added mechanistic pathway for chemotactic regulation., (Copyright © 2019. Published by Elsevier Inc.)

Beige adipocytes can dissipate energy as heat. Elaborate communication between metabolism and gene expression is important in the regulation of beige adipocytes. Although lipid droplet (LD) binding proteins play important roles in adipose tissue biology, it remains unknown whether perilipin 3 ( Plin3 ) is involved in the regulation of beige adipocyte formation and thermogenic activities. In this study, we demonstrate that Plin3 ablation stimulates beige adipocytes and thermogenic gene expression in inguinal white adipose tissue (iWAT). Compared with wild-type mice, Plin3 knockout mice were cold tolerant and displayed enhanced basal and stimulated lipolysis in iWAT, inducing peroxisome proliferator-activated receptor α ( PPARα ) activation. In adipocytes, Plin3 deficiency promoted PPARα target gene and uncoupling protein 1 expression and multilocular LD formation upon cold stimulus. Moreover, fibroblast growth factor 21 expression and secretion were upregulated, which was attributable to activated PPARα in Plin3 -deficient adipocytes. These data suggest that Plin3 acts as an intrinsic protective factor preventing futile beige adipocyte formation by limiting lipid metabolism and thermogenic gene expression., (© 2018 by the American Diabetes Association.)

Initial immunological defense mechanisms to pathogen invasion rely on innate pathways of chemotaxis and phagocytosis, original to ancient phagocytes. Although chemotaxis has been well-studied in mammalian and model systems using purified chemoattractants in defined conditions, directed movement toward live bacteria has been more difficult to assess. Dictyostelium discoideum is a professional phagocyte that chemotaxes toward bacteria during growth-phase in a process to locate nutrient sources. Using Dictyostelium as a model, we have developed a system that is able to quantify chemotaxis to very high sensitivity. Here, Dictyostelium can detect various chemoattractants at concentrations <1 nM. Given this exceedingly sensitive signal response, Dictyostelium will migrate directionally toward live gram positive and gram negative bacteria, in a highly quantifiable manner, and dependent upon bacterially-secreted chemoattractants. Additionally, we have developed a real-time, quantitative assay for phagocytosis of live gram positive and gram negative bacteria. To extend the analyses of endocytic functions, we further modified the system to quantify cellular uptake via macropinocytosis of smaller (<100 kDa) molecules. These various approaches provide novel means to dissect potential for identification of novel chemoattractants and mechanistic factors that are essential for chemotaxis, phagocytosis, and/or macropinocytosis and for more detailed understanding in host-pathogen interactive defenses.

Lipid droplet (LD) coating proteins are essential for the formation and stability of intracellular LDs. Plin2 is an abundant LD coating protein in skeletal muscle, but its importance for muscle function is unclear. We show that myotubes established from Plin2 -/- mice contain reduced content of LDs and accumulate less oleic acid (OA) in triacylglycerol (TAG) due to elevated LD hydrolysis in comparison with Plin2 +/+ myotubes. The reduced ability to store TAG in LDs in Plin2 -/- myotubes is accompanied by a shift in energy metabolism. Plin2 -/- myotubes are characterized by increased oxidation of OA, lower glycogen synthesis, and reduced glucose oxidation in comparison with Plin2 +/+ myotubes, perhaps reflecting competition between FAs and glucose as part of the Randle cycle. In accord with these metabolic changes, Plin2 -/- myotubes have elevated expression of Ppara and Ppargc1a , transcription factors that stimulate expression of genes important for FA oxidation, whereas genes involved in glucose uptake and oxidation are suppressed. Loss of Plin2 had no impact on insulin-stimulated Akt phosphorylation. Our results suggest that Plin2 is essential for protecting the pool of skeletal muscle LDs to avoid an uncontrolled hydrolysis of stored TAG and to balance skeletal muscle energy metabolism., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Aspects of innate immunity derive from characteristics inherent to phagocytes, including chemotaxis toward and engulfment of unicellular organisms or cell debris. Ligand chemotaxis has been biochemically investigated using mammalian and model systems, but precision of chemotaxis towards ligands being actively secreted by live bacteria is not well studied, nor has there been systematic analyses of interrelationships between chemotaxis and phagocytosis. The genetic/molecular model Dictyostelium and mammalian phagocytes share mechanistic pathways for chemotaxis and phagocytosis; Dictyostelium chemotax toward bacteria and phagocytose them as food sources. We quantified Dictyostelium chemotaxis towards live gram positive and gram negative bacteria and demonstrate high sensitivity to multiple bacterially-secreted chemoattractants. Additive/competitive assays indicate that intracellular signaling-networks for multiple ligands utilize independent upstream adaptive mechanisms, but common downstream targets, thus amplifying detection at low signal propagation, but strengthening discrimination of multiple inputs. Finally, analyses of signaling-networks for chemotaxis and phagocytosis indicate that chemoattractant receptor-signaling is not essential for bacterial phagocytosis.

Nucleosome placement and repositioning can direct transcription of individual genes; however, the precise interactions of these events are complex and largely unresolved at the whole-genome level. The Chromodomain-Helicase-DNA binding (CHD) Type III proteins are a subfamily of SWI2/SNF2 proteins that control nucleosome positioning and are associated with several complex human disorders, including CHARGE syndrome and autism. Type III CHDs are required for multicellular development of animals and Dictyostelium but are absent in plants and yeast. These CHDs can mediate nucleosome translocation in vitro, but their in vivo mechanism is unknown. Here, we use genome-wide analysis of nucleosome positioning and transcription profiling to investigate the in vivo relationship between nucleosome positioning and gene expression during development of wild-type (WT) Dictyostelium and mutant cells lacking ChdC, a Type III CHD protein ortholog. We demonstrate major nucleosome positional changes associated with developmental gene regulation in WT. Loss of chdC caused an increase of intragenic nucleosome spacing and misregulation of gene expression, affecting ∼50% of the genes that are repositioned during WT development. These analyses demonstrate active nucleosome repositioning during Dictyostelium multicellular development, establish an in vivo function of CHD Type III chromatin remodeling proteins in this process, and reveal the detailed relationship between nucleosome positioning and gene regulation, as cells transition between developmental states., (© 2017 Platt et al.; Published by Cold Spring Harbor Laboratory Press.)

Chemotaxis and cell migration are fundamental, universal eukaryotic processes essential for biological functions such as embryogenesis, immunity, cell renewal, and wound healing, as well as for pathogenesis of many diseases including cancer metastasis and chronic inflammation. To identify novel chemotaxis inhibitors as probes for mechanistic studies and leads for development of new therapeutics, we developed a unique, unbiased phenotypic chemotaxis-dependent Dictyostelium aggregation assay for high-throughput screening using rapid, laser-scanning cytometry. Under defined conditions, individual Dictyostelium secrete chemoattractants, migrate, and aggregate. Chemotaxis is quantified by laser-scanning cytometry with a GFP marker expressed only in cells after chemotaxis/multi-cell aggregation. We applied the assay to screen 1,280 known compounds in a 1536-well plate format and identified two chemotaxis inhibitors. The chemotaxis inhibitory activities of both compounds were confirmed in both Dictyostelium and in human neutrophils in a directed EZ-TAXIscan chemotaxis assay. The compounds were also shown to inhibit migration of two human cancer cell lines in monolayer scratch assays. This test screen demonstrated that the miniaturized assay is extremely suited for high-throughput screening of very large libraries of small molecules to identify novel classes of chemotaxis/migratory inhibitors for drug development and research tools for targeting chemotactic pathways universal to humans and other systems.

Mutations in either of two presenilin genes can cause familial Alzheimer's disease. Presenilins have both proteolysis-dependent functions, as components of the γ-secretase complex, and proteolysis-independent functions in signalling. In this study, we investigate a conserved function of human presenilins in the development of the simple model organism Dictyostelium discoideum. We show that the block in Dictyostelium development caused by the ablation of both Dictyostelium presenilins is rescued by the expression of human presenilin 1, restoring the terminal differentiation of multiple cell types. This developmental role is independent of proteolytic activity, because the mutation of both catalytic aspartates does not affect presenilin ability to rescue development, and the ablation of nicastrin, a γ-secretase component that is crucial for proteolytic activity, does not block development. The role of presenilins during Dictyostelium development is therefore independent of their proteolytic activity. However, presenilin loss in Dictyostelium results in elevated cyclic AMP (cAMP) levels and enhanced stimulation-induced calcium release, suggesting that presenilins regulate these intracellular signalling pathways. Our data suggest that presenilin proteins perform an ancient non-proteolytic role in regulating intracellular signalling and development, and that Dictyostelium is a useful model for analysing human presenilin function.

Control of chromatin structure is crucial for multicellular development and regulation of cell differentiation. The CHD (chromodomain-helicase-DNA binding) protein family is one of the major ATP-dependent, chromatin remodeling factors that regulate nucleosome positioning and access of transcription factors and RNA polymerase to the eukaryotic genome. There are three mammalian CHD subfamilies and their impaired functions are associated with several human diseases. Here, we identify three CHD orthologs (ChdA, ChdB and ChdC) in Dictyostelium discoideum. These CHDs are expressed throughout development, but with unique patterns. Null mutants lacking each CHD have distinct phenotypes that reflect their expression patterns and suggest functional specificity. Accordingly, using genome-wide (RNA-seq) transcriptome profiling for each null strain, we show that the different CHDs regulate distinct gene sets during both growth and development. ChdC is an apparent ortholog of the mammalian Class III CHD group that is associated with the human CHARGE syndrome, and GO analyses of aberrant gene expression in chdC nulls suggest defects in both cell-autonomous and non-autonomous signaling, which have been confirmed through analyses of chdC nulls developed in pure populations or with low levels of wild-type cells. This study provides novel insight into the broad function of CHDs in the regulation development and disease, through chromatin-mediated changes in directed gene expression.

Migratory cells, including mammalian leukocytes and Dictyostelium, use G-protein-coupled receptor (GPCR) signaling to regulate MAPK/ERK, PI3K, TORC2/AKT, adenylyl cyclase and actin polymerization, which collectively direct chemotaxis. Upon ligand binding, mammalian GPCRs are phosphorylated at cytoplasmic residues, uncoupling G-protein pathways, but activating other pathways. However, connections between GPCR phosphorylation and chemotaxis are unclear. In developing Dictyostelium, secreted cAMP serves as a chemoattractant, with extracellular cAMP propagated as oscillating waves to ensure directional migratory signals. cAMP oscillations derive from transient excitatory responses of adenylyl cyclase, which then rapidly adapts. We have studied chemotactic signaling in Dictyostelium that express non-phosphorylatable cAMP receptors and show through chemotaxis modeling, single-cell FRET imaging, pure and chimeric population wavelet quantification, biochemical analyses and TIRF microscopy, that receptor phosphorylation is required to regulate adenylyl cyclase adaptation, long-range oscillatory cAMP wave production and cytoskeletal actin response. Phosphorylation defects thus promote hyperactive actin polymerization at the cell periphery, misdirected pseudopodia and the loss of directional chemotaxis. Our data indicate that chemoattractant receptor phosphorylation is required to co-regulate essential pathways for migratory cell polarization and chemotaxis. Our results significantly extend the understanding of the function of GPCR phosphorylation, providing strong evidence that this evolutionarily conserved mechanism is required in a signal attenuation pathway that is necessary to maintain persistent directional movement of Dictyostelium, neutrophils and other migratory cells.

Global stimulation of Dictyostelium with different chemoattractants elicits multiple transient signaling responses, including synthesis of cAMP and cGMP, actin polymerization, activation of kinases ERK2, TORC2, and phosphatidylinositide 3-kinase, and Ras-GTP accumulation. Mechanisms that down-regulate these responses are poorly understood. Here we examine transient activation of TORC2 in response to chemically distinct chemoattractants, cAMP and folate, and suggest that TORC2 is regulated by adaptive, desensitizing responses to stimulatory ligands that are independent of downstream, feedback, or feedforward circuits. Cells with acquired insensitivity to either folate or cAMP remain fully responsive to TORC2 activation if stimulated with the other ligand. Thus TORC2 responses to cAMP or folate are not cross-inhibitory. Using a series of signaling mutants, we show that folate and cAMP activate TORC2 through an identical GEF/Ras pathway but separate receptors and G protein couplings. Because the common GEF/Ras pathway also remains fully responsive to one chemoattractant after desensitization to the other, GEF/Ras must act downstream and independent of adaptation to persistent ligand stimulation. When initial chemoattractant concentrations are immediately diluted, cells rapidly regain full responsiveness. We suggest that ligand adaptation functions in upstream inhibitory pathways that involve chemoattractant-specific receptor/G protein complexes and regulate multiple response pathways.

Perilipin family proteins (Plins) coat the surface of intracellular neutral lipid storage droplets in various cell types. Studies across diverse species demonstrate that Plins regulate lipid storage metabolism through recruitment of lipases and other regulatory proteins to lipid droplet surfaces. Mammalian genomes have distinct Plin gene members and additional protein forms derived from specific mRNA splice variants. However, it is not known if the different Plins have distinct functional properties. Using biochemical, cellular imaging and flow cytometric analyses, we now show that within individual cells of various types, the different Plin proteins preferentially sequester to separate pools of lipid storage droplets. By examining ectopically expressed GFP fusions and all endogenous Plin protein forms, we demonstrate that different Plins sequester to different types of lipid droplets that are composed of either triacylcerides or cholesterol esters. Furthermore, Plins with strong association preferences to triacylceride (or cholesterol ester) droplets can re-direct the relative intracellular triacylceride-cholesterol ester balance toward the targeted lipid. Our data suggest diversity of Plin function, alter previous assumptions about shared collective actions of the Plins, and indicate that each Plin can have separate and unique functions.

The TOR protein kinase functions in two distinct complexes, TOR complex 1 (TORC1) and 2 (TORC2). TORC1 is required for growth in response to growth factors, nutrients and the cellular energy state; TORC2 regulates AKT signaling, which can modulate cytoskeletal polarization. In its ecological niche, Dictyostelium engulf bacteria and yeast for nutrient capture. Despite the essential role of TORC1 in control of cellular growth, we show that nutrient particle capture (phagocytosis) in Dictyostelium is independent of TORC1-mediated nutrient sensing and growth regulation. However, loss of Dictyostelium TORC2 components Rictor/Pia, SIN1/RIP3 and Lst8 promotes nutrient particle uptake; inactivation of TORC2 leads to increased efficiency and speed of phagocytosis. In contrast to phagocytosis, we show that macropinocytosis, an AKT-dependent process for cellular uptake of fluid phase nutrients, is not regulated by either of the TOR complexes. The integrated and balanced regulation of TORC1 and TORC2 might be crucial in Dictyostelium to coordinate growth and energy needs with other essential TOR-regulated processes.

In Dictyostelium, the interaction of secreted cAMP with specific cell surface receptors regulates the activation/de-activation of GSK3, which mediates developmental cell patterning. In addition, Dictyostelium cells polarize in response to extracellular cAMP, although a potential role for GSK3 in this pathway has not been investigated. Previously, we had shown that ZAK1 was an activating tyrosine kinase for GSK3 function in Dictyostelium and we now identify ZAK2 as the other tyrosine kinase in the cAMP-activation pathway for GSK3; no additional family members exist. We also now show that tyrosine phosphorylation/activation of GSK3 by ZAK2 and ZAK1 separately regulate GSK3 in distinct differentiated cell populations, and that ZAK2 acts in both autonomous and non-autonomous pathways to regulate these cell-type differentiations. Finally, we demonstrate that efficient polarization of Dictyostelium towards cAMP depends on ZAK1-mediated tyrosine phosphorylation of GSK3. Combinatorial regulation of GSK3 by ZAK kinases in Dictyostelium guides cell polarity, directional cell migration and cell differentiation, pathways that extend the complexity of GSK3 signaling throughout the development of Dictyostelium.

Presenilin (PS) is the catalytic moiety of the gamma-secretase complex. PS and other gamma-secretase components are well conserved among metazoa, but their presence and function in more-distant species are not resolved. Because inappropriate gamma-secretase processing of amyloid precursor protein (APP) in humans is associated with familial Alzheimer's disease, understanding essential elements within each gamma-secretase component is crucial to functional studies. Diverged proteins have been identified in primitive plants but experiments have failed to demonstrate gamma-secretase activity. We have identified highly diverged orthologs for each gamma-secretase component in the ancient eukaryote Dictyostelium, which lacks equivalents of APP, Notch and other characterized PS/gamma-secretase substrates. We show that wild-type (WT) Dictyostelium is capable of amyloidogenic processing of ectopically expressed human APP to generate amyloid-beta peptides Abeta(40) and Abeta(42); strains deficient in gamma-secretase cannot produce Abeta peptides but accumulate processed intermediates of APP that co-migrate with the C-terminal fragments alpha- and beta-CTF of APP that are found in mammalian cells. We further demonstrate that Dictyostelium requires PS for phagocytosis and cell-fate specification in a cell-autonomous manner, and show that regulation of phagocytosis requires an active gamma-secretase, a pathway suggested, but not proven, to occur in mammalian and Drosophila cells. Our results indicate that PS signaling is an ancient process that arose prior to metazoan radiation, perhaps independently of Notch. Dictyostelium might serve to identify novel PS/gamma-secretase signaling targets and provide a unique system for high-throughput screening of small-molecule libraries to select new therapeutic targets for diseases associated with this pathway.

Protein kinases AKT and PKBR1 of Dictyostelium belong to the AGC protein kinase superfamily. AKT and PKBR1 are phosphorylated at similar sites by phosphoinositide-dependent kinase 1 (PDK1) and TORC2 kinases; however, they have different subcellular localizing domains. AKT has a phosphoinositide 3-kinase (PI3K)/phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)]-regulated PH (pleckstrin homology) domain whereas PKBR1 is myristoylated and persistently membrane localized. Using strains defective for PI3K/PtdIns(3,4,5)P(3)-, PDK1- and TORC2-signaling or strains that express phospho-site mutants of AKT and PKBR1, we dissect the different roles of PI3K/PtdIns(3,4,5)P(3), PDK1 and TORC2. We show that activation of AKT and PKBR1 requires PDK1-site phosphorylation, but that phosphorylation by TORC2 is insufficient for AKT or PKBR1 activation. However, PDK1-site phosphorylation is dependent on phosphorylation by TORC2, which suggests that there is regulatory coordination among PDK1, TORC2 and their phospho-site targets. This defines a separate input for signaling in control of chemotaxis and dependency on PDK1 function. We also demonstrate that PDK1 in Dictyostelium functions independently of PI3K/PtdIns(3,4,5)P(3). Finally, we show that AKT and PKBR1 exhibit substrate selectivity and identify two novel lipid-interacting proteins preferentially phosphorylated by AKT. Despite certain similarities, AKT and PKBR1 have distinct regulatory paths that impact activation and effector targeting, with PDK1 serving a central role.

The PAT family of proteins has been identified in eukaryotic species as diverse as vertebrates, insects, and amebazoa. These proteins share a highly conserved sequence organization and avidity for the surfaces of intracellular, neutral lipid storage droplets. The current nomenclature of the various members lacks consistency and precision, deriving more from historic context than from recognition of evolutionary relationship and shared function. In consultation with the Mouse Genomic Nomenclature Committee, the Human Genome Organization Genomic Nomenclature Committee, and conferees at the 2007 FASEB Conference on Lipid Droplets: Metabolic Consequences of the Storage of Neutral Lipids, we have established a unifying nomenclature for the gene and protein family members. Each gene member will incorporate the root term PERILIPIN (PLIN), the founding gene of the PAT family, with the different genes/proteins numbered sequentially.

In this issue of Molecular Cell, Lee et al. (2010) show that the retinoic acid receptor-related orphan receptor alpha (RORalpha) can suppress the Wnt/beta-catenin signaling pathway in a Wnt5a- and PKC-dependent manner that does not alter beta-catenin stabilization or the recruitment of beta-catenin to Wnt target promoters., (Copyright 2010 Elsevier Inc. All rights reserved.)

The complex cytopathology of mitochondrial diseases is usually attributed to insufficient ATP. AMP-activated protein kinase (AMPK) is a highly sensitive cellular energy sensor that is stimulated by ATP-depleting stresses. By antisense-inhibiting chaperonin 60 expression, we produced mitochondrially diseased strains with gene dose-dependent defects in phototaxis, growth, and multicellular morphogenesis. Mitochondrial disease was phenocopied in a gene dose-dependent manner by overexpressing a constitutively active AMPK alpha subunit (AMPKalphaT). The aberrant phenotypes in mitochondrially diseased strains were suppressed completely by antisense-inhibiting AMPKalpha expression. Phagocytosis and macropinocytosis, although energy consuming, were unaffected by mitochondrial disease and AMPKalpha expression levels. Consistent with the role of AMPK in energy homeostasis, mitochondrial "mass" and ATP levels were reduced by AMPKalpha antisense inhibition and increased by AMPKalphaT overexpression, but they were near normal in mitochondrially diseased cells. We also found that 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, a pharmacological AMPK activator in mammalian cells, mimics mitochondrial disease in impairing Dictyostelium phototaxis and that AMPKalpha antisense-inhibited cells were resistant to this effect. The results show that diverse cytopathologies in Dictyostelium mitochondrial disease are caused by chronic AMPK signaling not by insufficient ATP.

Ectopic accumulation of lipid droplets in non-adipose tissues correlates with the degree of insulin resistance in these tissues. Emerging evidence indicates that lipid droplets are specialized organelles that participate in lipid metabolism and intracellular trafficking. These properties are thought to derive from the lipid droplet-associated PAT protein family (perilipin, ADFP, and Tip47). The functions of the ubiquitously distributed adipose differentiation-related protein (ADFP) and Tip47 remain unknown. To evaluate the roles of ADFP and Tip47 in lipid biogenesis and metabolism, ADFP null and wild type (wt) clonal cell lines were established from ADFP null and wt mice, respectively. In ADFP null cells, Tip47 was identified as the sole lipid droplet-associated protein from the PAT family by mass spectroscopy, which was further confirmed by immunoblotting and immunocytochemistry. Following incubation with oleic acid, ADFP null cells were able to form lipid droplets to the same extent as wt cells. No statistical differences between the two cell types were observed in NEFA uptake or lipolysis. Small interference RNAs (siRNAs) against Tip47 were found to down-regulate protein levels for Tip47 by 85%. ADFP null cells treated with Tip47 siRNA retained the ability to form lipid droplets but to a lesser extent and shunted the utilization of exogenously added NEFA from triglycerides to phospholipids. These data support the hypothesis that Tip47 plays an important role in lipid metabolism. Tip47 and ADFP in peripheral tissues may play a critical role in regulating the formation and turnover, and hence metabolic consequences, of ectopic fat.

It is assumed that ERK2 in Dictyostelium is subject to adaptive regulation in response to constant extracellular ligand stimulation. We now show, to the contrary, that ERK2 remains active under continuous stimulation, differing from most ligand-activated pathways in chemotactically competent Dictyostelium and other cells. We show that the upstream phosphorylation pathway, responsible for ERK2 activation, transiently responds to receptor stimulation, whereas ERK2 dephosphorylation (deactivation) is inhibited by continuous stimulation. We argue that the net result of these two regulatory actions is a persistently active ERK2 pathway when the extracellular ligand (i.e., cAMP) concentration is held constant and that oscillatory production/destruction of secreted cAMP in chemotaxing cells accounts for the observed oscillatory activity of ERK2. We also show that pathways controlling seven-transmembrane receptor (7-TMR) ERK2 activation/deactivation function independently of G proteins and ligand-induced production of intracellular cAMP and the consequent activation of PKA. Finally, we propose that this regulation enables ERK2 to function both in an oscillatory manner, critical for chemotaxis, and in a persistent manner, necessary for gene expression, as secreted ligand concentration increases during later development. This work redefines mechanisms of ERK2 regulation by 7-TMR signaling in Dictyostelium and establishes new implications for control of signal relay during chemotaxis.

Adipose differentiation-related protein (ADRP) is localized to lipid droplets in most mammalian cells. ADRP, proposed to regulate fatty acid mobilization and lipid droplet formation, is linked to lipid accumulation in foam cells of human atherosclerotic lesions. In this report, we show that ADRP protein accumulates in Chinese hamster ovary fibroblastic cells cultured in the presence of oleic acid but is destabilized when fatty acid sources are removed from culture serum. The latter effect was blocked by the proteasome inhibitor MG132, whereas inhibitors of other proteolytic processes were ineffective. Pulse-chase experiments confirmed that ADRP degradation is inhibited by MG132. Conditions that stimulate ADRP degradation also promoted the covalent modification of ADRP by ubiquitin, whereas the addition of oleic acid to culture media, which promotes triacylglycerol deposition, blunted the appearance of ubiquitinated-ADRP. Treatment with MG132 increased the levels of ADRP associated with lipid droplets, as well as throughout the cytosol. Finally, we demonstrate that the disappearance of ADRP protein after the onset of perilipin expression during adipocyte differentiation is due to degradation by proteasomes Thus, proteolytic degradation of ADRP mediated through the ubiquitin/proteasome pathway appears to be a major mode for the post-translational regulation of ADRP.

Background: In the absence of Wnt stimulation, the transcriptional cofactor beta-catenin is destabilized via phosphorylation by protein kinase GSK3beta in complex with Axin family members. In the "canonical" Wnt signaling pathway, Disheveled (Dvl) is required to functionally inhibit the activity of the GSK3beta/Axin complex and thereby stabilize beta-catenin. Yet, the mechanisms that underlie Wnt regulation of GSK3 and stabilization of beta-catenin are still not fully appreciated., Results: Here, we examine time-dependent changes in protein-protein interactions that occur in response to Wnt treatment. We show that GSK3beta/Axin complexes are rapidly (t1/2 < 3 min) disrupted upon Wnt stimulation and that changes in GSK3beta/Axin association substantially precede both beta-catenin stabilization and Axin degradation. We further demonstrate that depletion of Galpha(o) or Galpha(q) will inhibit, respectively, the Wnt-induced disruption of GSK3beta/Axin2 and GSK3beta/Axin complexes and diminish Wnt stabilization of beta-catenin. We also show that direct activation of G proteins in vivo with GTPgammaS in the absence of exogenous Wnt will disrupt GSK3beta/Axin2 complexes and stabilize beta-catenin. Finally, we demonstrate an association of Galpha(o) with Fz that is also very rapidly (t1/2 < 1 min) perturbed upon Wnt-3a stimulation and that the Wnt-dependent effects on both GSK3beta/Axin2 and Galpha(o)/Fz are pertussis-toxin sensitive. Collectively, these data implicate a role for G proteins in the regulation of Wnt-mediated protein-protein interactions and signaling to beta-catenin., Conclusions: We conclude that rapid disruption of GSK3beta/Axin interactions in response to Wnt leads to the initial stabilization of beta-catenin and that Galpha(o) and Galpha(q) signaling contributes to Wnt-mediated GSK3beta/Axin disruption and the ultimate stabilization of beta-catenin.

Chloroquine resistance in Plasmodium falciparum malaria results from mutations in PfCRT, a member of a unique family of transporters present in apicomplexan parasites and Dictyostelium discoideum. Mechanisms that have been proposed to explain chloroquine resistance are difficult to evaluate within malaria parasites. Here we report on the targeted expression of wild-type and mutant forms of PfCRT to acidic vesicles in D. discoideum. We show that wild-type PfCRT has minimal effect on the accumulation of chloroquine by D. discoideum, whereas forms of PfCRT carrying a key charge-loss mutation of lysine 76 (e.g. K76T) enable D. discoideum to expel chloroquine. As in P. falciparum, the chloroquine resistance phenotype conferred on transformed D. discoideum can be reversed by the channel-blocking agent verapamil. Although intravesicular pH levels in D. discoideum show small acidic changes with the expression of different forms of PfCRT, these changes would tend to promote intravesicular trapping of chloroquine (a weak base) and do not account for reduced drug accumulation in transformed D. discoideum. Our results instead support outward-directed chloroquine efflux for the mechanism of chloroquine resistance by mutant PfCRT. This mechanism shows structural specificity as D. discoideum transformants that expel chloroquine do not expel piperaquine, a bisquinoline analog of chloroquine used frequently against chloroquine-resistant parasites in Southeast Asia. PfCRT, nevertheless, may have some ability to act on quinine and quinidine. Transformed D. discoideum will be useful for further studies of the chloroquine resistance mechanism and may assist in the development and evaluation of new antimalarial drugs.

We have identified two LIM domain proteins, LimF and ChLim, from Dictyostelium that interact with each other and with the small, Rab5-related, Rab21 GTPase to collectively regulate phagocytosis. To investigate in vivo functions, we generated cell lines that lack or overexpress LimF and ChLim and strains that express activating or inhibiting variants of Rab21. Overexpression of LimF, loss of ChLim, or expression of constitutively active Rab21 increases the rate of phagocytosis above that of wild type. Conversely, loss of LimF, overexpression of ChLim, or expression of a dominant-negative Rab21 inhibits phagocytosis. Our studies using cells carrying multiple mutations in these genes further indicate that ChLim antagonizes the activating function of Rab21-GTP during phagocytosis; in turn, LimF is required for Rab21-GTP function. Finally, we demonstrate that ChLim and LimF localize to the phagocytic cup and phago-lysosomal vesicles. We suggest that LimF, ChLim, and activated Rab21-GTP participate as a novel signaling complex that regulates phagocytic activity.

Dictyostelium discoideum has proven an exceptionally powerful system for studying numerous aspects of cellular and developmental functions. The relatively small ( approximately 34 Mb) chromosomal genome of Dictyostelium and high efficiency of targeted gene disruption have enabled researchers to characterize many specific gene functions. However, the number of selectable markers in Dictyostelium is restricted, as is the ability to perform effective genetic crosses between strains. Thus, it has been difficult to create multiple mutations within an individual cell to study epistatic relationships among genes or potential redundancies between various pathways. We now describe a robust system for the production of multiple gene mutations in Dictyostelium by recycling a single selectable marker, Blasticidin S resistance, using the Cre-loxP system. We confirm the effectiveness of the system by generating a single cell carrying four separate gene disruptions. Furthermore, the cells remain sensitive to transformation for additional targeted or random mutagenesis requiring Blasticidin selection and for functional expression studies of mutated or tagged proteins using other selectable markers.

Dictyostelium discoideum grow unicellularly, but develop as multicellular organisms. At two stages of development, their underlying symmetrical pattern of cellular organization becomes disrupted. During the formation of the multicellular aggregate, individual non-polarized cells re-organize their cytoskeletal structures to sequester specific intracellular signaling elements for activation by and directed movement within chemoattractant gradients. Subsequently, response to secreted morphogens directs undifferentiated populations to adopt different cell fates. Using a combination of cellular, biochemical and molecular approaches, workers have now begun to understand the mechanisms that permit Dictyostelium (and other chemotactic cells) to move directionally in shallow chemoattractant gradients and the transcriptional regulatory pathways that polarize cell-fate choice and initiate pattern formation.

The perilipin/ADRP/TIP47 (PAT) proteins localize to the surface of intracellular neutral lipid droplets. Perilipin is essential for lipid storage and hormone regulated lipolysis in adipocytes, and perilipin null mice exhibit a dramatic reduction in adipocyte lipid stores. A significant fraction of the approximately 200 amino acid N-terminal region of the PAT proteins consists of 11-mer helical repeats that are also found in apolipoproteins and other lipid-associated proteins. The C-terminal 60% of TIP47, a representative PAT protein, comprises a monomeric and independently folded unit. The crystal structure of the C-terminal portion of TIP47 was determined and refined at 2.8 A resolution. The structure consists of an alpha/beta domain of novel topology and a four-helix bundle resembling the LDL receptor binding domain of apolipoprotein E. The structure suggests an analogy between PAT proteins and apolipoproteins in which helical repeats interact with lipid while the ordered C-terminal region is involved in protein:protein interactions.

Chemotactic cells, including neutrophils and Dictyostelium discoideum, orient and move directionally in very shallow chemical gradients. As cells polarize, distinct structural and signaling components become spatially constrained to the leading edge or rear of the cell. It has been suggested that complex feedback loops that function downstream of receptor signaling integrate activating and inhibiting pathways to establish cell polarity within such gradients. Much effort has focused on defining activating pathways, whereas inhibitory networks have remained largely unexplored. We have identified a novel signaling function in Dictyostelium involving a Galpha subunit (Galpha9) that antagonizes broad chemotactic response. Mechanistically, Galpha9 functions rapidly following receptor stimulation to negatively regulate PI3K/PTEN, adenylyl cyclase, and guanylyl cyclase pathways. The coordinated activation of these pathways is required to establish the asymmetric mobilization of actin and myosin that typifies polarity and ultimately directs chemotaxis. Most dramatically, cells lacking Galpha9 have extended PI(3,4,5)P(3), cAMP, and cGMP responses and are hyperpolarized. In contrast, cells expressing constitutively activated Galpha9 exhibit a reciprocal phenotype. Their second message pathways are attenuated, and they have lost the ability to suppress lateral pseudopod formation. Potentially, functionally similar Galpha-mediated inhibitory signaling may exist in other eukaryotic cells to regulate chemoattractant response.

Dictyostelium discoideum is a powerful and genetically tractable model system used for the study of numerous cellular molecular mechanisms including chemotaxis, phagocytosis and signal transduction. The past 2 years have seen a significant expansion in the scope and accessibility of online resources for Dictyostelium. Recent advances have focused on the development of a new comprehensive online resource called dictyBase (http://dictybase.org). This database not only provides access to genomic data including functional annotation of genes, gene products and chromosomal mapping, but also to extensive biological information such as mutant phenotypes and corresponding reference material. In conjunction with additional sites (http://genome. imb-jena.de/dictyostelium/, http://dictyensembl. bioch.bcm.tmc.edu and http://www.sanger.ac.uk/Projects/D&#95;discoideum/) from the genome sequencing and assembly centers, these improvements have expanded the scope of the Dictyostelium databases making them accessible and useful to any researcher interested in comparative and functional genomics in metazoan organisms.

Lipolysis in adipocytes governs the release of fatty acids for the supply of energy to various tissues of the body. This reaction is mediated by hormone-sensitive lipase (HSL), a cytosolic enzyme, and perilipin, which coats the lipid droplet surface in adipocytes. Both HSL and perilipin are substrates for polyphosphorylation by protein kinase A (PKA), and phosphorylation of perilipin is required to induce HSL to translocate from the cytosol to the surface of the lipid droplet, a critical step in the lipolytic reaction (Sztalryd C., Xu, G., Dorward, H., Tansey, J. T., Contreras, J.A, Kimmel, A. R., and Londos, C. (2003) J. Cell Biol. 161, 1093-1103). In the present paper we demonstrate that phosphorylation at one of the two more recently discovered PKA sites within HSL, serines 659 and 660, is also required to effect the translocation reaction. Translocation does not occur when these serines residues are mutated simultaneously to alanines. Also, mutation of the catalytic Ser-423 eliminates HSL translocation, showing that the inactive enzyme does not migrate to the lipid droplet upon PKA activation. Thus, HSL translocation requires the phosphorylation of both HSL and perilipin.

Akey step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase (HSL) from the cytosol to the surface of the lipid storage droplet. Adipocytes from perilipin-null animals have an elevated basal rate of lipolysis compared with adipocytes from wild-type mice, but fail to respond maximally to lipolytic stimuli. This defect is downstream of the beta-adrenergic receptor-adenylyl cyclase complex. Now, we show that HSL is basally associated with lipid droplet surfaces at a low level in perilipin nulls, but that stimulated translocation from the cytosol to lipid droplets is absent in adipocytes derived from embryonic fibroblasts of perilipin-null mice. We have also reconstructed the HSL translocation reaction in the nonadipocyte Chinese hamster ovary cell line by introduction of GFP-tagged HSL with and without perilipin A. On activation of protein kinase A, HSL-GFP translocates to lipid droplets only in cells that express fully phosphorylatable perilipin A, confirming that perilipin is required to elicit the HSL translocation reaction. Moreover, in Chinese hamster ovary cells that express both HSL and perilipin A, these two proteins cooperate to produce a more rapidly accelerated lipolysis than do cells that express either of these proteins alone, indicating that lipolysis is a concerted reaction mediated by both protein kinase A-phosphorylated HSL and perilipin A.

Cells migrating directionally toward a chemoattractant source display a highly polarized cytoskeletal organization, with F-actin localized predominantly at the anterior and myosin II at the lateral and posterior regions. Dictyostelium discoideum has proven a useful system for elucidating signaling pathways that regulate this chemotactic response. During development, extracellular adenosine 3', 5' monophosphate (cAMP) functions as a primary signal to activate cell surface cAMP receptors (cARs). These receptors transduce different signals depending on whether or not they are coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins) (see the STKE Connections Maps). Multiple G protein-stimulated pathways interact to establish polarity in chemotaxing D. discoideum cells by localizing F-actin at their leading edge and by regulating the phosphorylation state and assembly of myosin II. Many of the molecular interactions described are fundamental to the regulation of chemotaxis in other eukaryotic cells.

Perilipin A coats the lipid storage droplets in adipocytes and is polyphosphorylated by protein kinase A (PKA); the fact that PKA activates lipolysis in adipocytes suggests a role for perilipins in this process. To assess whether perilipins participate directly in PKA-mediated lipolysis, we have expressed constructs coding for native and mutated forms of the two major splice variants of the perilipin gene, perilipins A and B, in Chinese hamster ovary fibroblasts. Perilipins localize to lipid droplet surfaces and displace the adipose differentiation-related protein that normally coats the droplets in these cells. Perilipin A inhibits triacylglycerol hydrolysis by 87% when PKA is quiescent, but activation of PKA and phosphorylation of perilipin A engenders a 7-fold lipolytic activation. Mutation of PKA sites within the N-terminal region of perilipin abrogates the PKA-mediated lipolytic response. In contrast, perilipin B exerts only minimal protection against lipolysis and is unresponsive to PKA activation. Since Chinese hamster ovary cells contain no PKA-activated lipase, we conclude that the expression of perilipin A alone is sufficient to confer PKA-mediated lipolysis in these cells. Moreover, the data indicate that the unique C-terminal portion of perilipin A is responsible for its protection against lipolysis and that phosphorylation at the N-terminal PKA sites attenuates this protective effect.

Asymmetric body axis formation is central to metazoan development. Dictyostelium establishes an anterior/posterior axis utilizing seven-transmembrane cAMP morphogen receptors (CARs) and GSK3-mediated signal transductions that has a parallel with metazoan Wnt/Frizzled-GSK3 pathways. In Dictyostelium, GSK3 promotes posterior cell patterning but inhibits anterior cell differentiation. Tyrosine kinase ZAK1 mediates GSK3 activation. We now show that CAR4 regulates a tyrosine phosphatase that inhibits GSK3 activity. We have also identified essential phosphotyrosines in GSK3, confirmed their role in activated/deactivated regulation and cell fate decisions, and relate them to the predicted 3D structure of GSK3beta. CARs differentially regulate GSK3 activity by selectively activating a tyrosine phosphatase or kinase for pattern formation. The findings may provide a comparative understanding of CAR-GSK3 and Wnt/Frizzled-GSK3 pathways.

Intracellular neutral lipid storage droplets are essential organelles of eukaryotic cells, yet little is known about the proteins at their surfaces or about the amino acid sequences that target proteins to these storage droplets. The mammalian proteins Perilipin, ADRP, and TIP47 share extensive amino acid sequence similarity, suggesting a common function. However, while Perilipin and ADRP localize exclusively to neutral lipid storage droplets, an association of TIP47 with intracellular lipid droplets has been controversial. We now show that GFP-tagged TIP47 co-localizes with isolated intracellular lipid droplets. We have also detected a close juxtaposition of TIP47 with the surfaces of lipid storage droplets using antibodies that specifically recognize TIP47, further indicating that TIP47 associates with intracellular lipid storage droplets. Finally, we show that related proteins from species as diverse as Drosophila and Dictyostelium can also target mammalian or Drosophila lipid droplet surfaces in vivo. Thus, sequence and/or structural elements within this evolutionarily ancient protein family are necessary and sufficient to direct association to heterologous intracellular lipid droplet surfaces, strongly indicating that they have a common function for lipid deposition and/or mobilization.

Background: Seven-transmembrane receptor (7-TMR)-G protein networks are molecular sensors of extracellular signals in all eukarya. These pathways cycle through activated (sensitized) and inhibited (desensitized) states, and, while many of the molecular components for signal activation have been described, inhibitory mechanisms are not well characterized. In Dictyostelium, 7-TM cAMP receptors direct chemotaxis and development but also regulate the periodic synthesis of their own ligand, the chemoattractant/morphogen cAMP. We now demonstrate through loss-of-function/gain-of-function studies that the novel heterotrimeric Galpha9 protein subunit regulates an inhibitory pathway during early Dictyostelium development for the cAMP signal response., Results: galpha9 null cells form more cAMP signaling centers, are more resistant to compounds that inhibit cAMP signaling, and complete aggregation sooner and at lower cell densities than wild-type cells. These phentoypes are consistent with the loss of an inhibitory signaling pathway during development of galpha9 null cells. Cells expressing constitutively activated Galpha9 are defective in cAMP signaling center formation and development at low cell density and display an increased sensitivity to cAMP signal inhibition that is characteristic of enhanced suppression of the cAMP signal response. Finally, we demonstrate that galpha9 null cells, which have been codeveloped with a majority of wild-type cells, primarily establish cAMP signaling centers and are able to non-autonomously direct wild-type cells to adopt a galpha9 null-like phenotype., Conclusions: We suggest that Galpha9 functions in an inhibitory-feedback pathway that regulates cAMP signaling center formation and propagation. Galpha9 may be part of the mechanism that regulates lateral signal inhibition or that modulates receptor desensitization.

Dictyostelium is an attractive model system for the study of mechanisms basic to cellular function or complex multicellular developmental processes. Recent advances in Dictyostelium genomics have generated a wide spectrum of resources. However, much of the current genomic sequence information is still not currently available through GenBank or related databases. Thus, many investigators are unaware that extensive sequence data from Dictyostelium has been compiled, or of its availability and access. Here, we discuss progress in Dictyostelium genomics and gene annotation, and highlight the primary portals for sequence access, manipulation and analysis (http://genome.imb-jena.de/dictyostelium/; http://dictygenome.bcm.tmc.edu/; http://www.sanger. ac.uk/Projects/D&#95;discoideum/; http://www.csm.biol. tsukuba.ac.jp/cDNAproject.html).

Perilipin coats the lipid droplets of adipocytes and is thought to have a role in regulating triacylglycerol hydrolysis. To study the role of perilipin in vivo, we have created a perilipin knockout mouse. Perilipin null (peri(-/-)) and wild-type (peri(+/+)) mice consume equal amounts of food, but the adipose tissue mass in the null animals is reduced to approximately 30% of that in wild-type animals. Isolated adipocytes of perilipin null mice exhibit elevated basal lipolysis because of the loss of the protective function of perilipin. They also exhibit dramatically attenuated stimulated lipolytic activity, indicating that perilipin is required for maximal lipolytic activity. Plasma leptin concentrations in null animals were greater than expected for the reduced adipose mass. The peri(-/-) animals have a greater lean body mass and increased metabolic rate but they also show an increased tendency to develop glucose intolerance and peripheral insulin resistance. When fed a high-fat diet, the perilipin null animals are resistant to diet-induced obesity but not to glucose intolerance. The data reveal a major role for perilipin in adipose lipid metabolism and suggest perilipin as a potential target for attacking problems associated with obesity.

The perilipins are the most abundant proteins at the surfaces of lipid droplets in adipocytes and are also found in steroidogenic cells. To investigate perilipin function, perilipin A, the predominant isoform, was ectopically expressed in fibroblastic 3T3-L1 pre-adipocytes that normally lack the perilipins. In control cells, fluorescent staining of neutral lipids with Bodipy 493/503 showed a few minute and widely dispersed lipid droplets, while in cells stably expressing perilipin A, the lipid droplets were more numerous and tightly clustered in one or two regions of the cytoplasm. Immunofluorescence microscopy revealed that the ectopic perilipin A localized to the surfaces of the tiny clustered lipid droplets; subcellular fractionation of the cells using sucrose gradients confirmed that the perilipin A localized exclusively to lipid droplets. Cells expressing perilipin A stored 6-30-fold more triacylglycerol than control cells due to reduced lipolysis of triacylglycerol stores. The lipolysis of stored triacylglycerol was 5 times slower in lipid-loaded cells expressing perilipin A than in lipid-loaded control cells, when triacylglycerol synthesis was blocked with 6 microm triacsin C. This stabilization of triacylglycerol was not due to the suppression of triacylglycerol lipase activity by the expression of perilipin A. We conclude that perilipin A increases the triacylglycerol content of cells by forming a barrier that reduces the access of soluble lipases to stored lipids, thus inhibiting triacylglycerol hydrolysis. These studies suggest that perilipin A plays a major role in the regulation of triacylglycerol storage and lipolysis in adipocytes.

Until recently, protein kinase GSK3 (glycogen synthase kinase 3), an essential component for cell-fate specification, had been considered a constitutively activated enzyme subject to developmentally regulated inhibition through hierarchical, linear signaling paths. Data from various systems now indicate more complex scenarios involving activating as well as inhibiting circuits, and the differential formation of multi-protein complexes that antagonistically affect GSK3 function.

Inhibition of GSK3 by 7-TM Wnt/wg receptor signaling is critical for specifying embryonic cell fate patterns. In Dictyostelium, the 7-TM cAMP receptors regulate GSK3 by parallel, antagonistic pathways to establish a developmental body plan. We describe here a novel tyrosine kinase, ZAK1, downstream of 7-TM cAMP receptor signaling that is required for GSK3 activation during development. zak1-nulls have reduced GSK3 activity and are defective in GSK3-regulated developmental pathways. Moreover, recombinant ZAK1 phosphorylates and activates GSK3 in vitro. We propose that ZAK1 is a positive regulator of GSK3 activity required for cell pattern formation in Dictyostelium and speculate that similar mechanisms exist to antagonize Wnt/wg signaling for metazoan cell fate specification.

Previously it was demonstrated using a model precursor that processing at the N terminus of the HIV-1 protease (PR) precedes processing at its C terminus. We now show the expression, purification, and kinetics of the autoprocessing reaction of a PR precursor linked to 53 amino acids of the native flanking transframe region (DeltaTFP-p6(pol)) of Gag-Pol and containing its two native cleavage sites. The PR contains the two cysteine residues exchanged to alanines, mutations that do not alter the kinetics or the structural stability of the mature PR. DeltaTFP-p6(pol)-PR, which encompasses the known PR inhibitor sequence Glu-Asp-Leu within DeltaTFP, undergoes cleavage at the DeltaTFP/p6(pol) and p6(pol)/PR sites in two consecutive steps to produce the mature PR. Both DeltaTFP-p6(pol)-PR and p6(pol)-PR exhibit low intrinsic enzymatic activity. The appearance of the mature PR is accompanied by a large increase in catalytic activity. It follows first-order kinetics in protein concentration with a rate constant of 0.13 +/- 0.01 min(-1) in 0.1 M acetate at pH 4.8. The pH-rate profile for the observed first-order rate constant is bell-shaped with two ionizable groups of pK(a) 4.9 and 5.1. The rate constant also exhibits approximately 7-fold higher sensitivity to urea denaturation as compared with that of the mature PR, suggesting that the cleavage at the N terminus of the PR domain from the precursor leads to the stabilization of the dimeric structure.