SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications, trials & patents

Abstract

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. Dictyostelium is a unique model for studying the complex and interactive cyclic nucleotide signaling pathways that regulate multicellular development. Dictyostelium grow as individual single-cells, but in the absence of nutrients, they initiate a multicellular developmental program. Central to this is secreted cAMP, a primary GPCR-response signal. Activated cAMP receptors at the cell surface direct a number of downstream signaling pathways, including synthesis of the intracellular second messengers cAMP and cGMP. These, in turn, activate a series of downstream targets that direct chemotaxis within extracellular cAMP gradients, multicellular aggregation, and, ultimately, cell-specific gene expression, morphogenesis, and cytodifferentiation. Extracellular cAMP and intracellular cAMP and cGMP exhibit rapid fluctuations in concentrations and are, thus, subject to exquisite regulation by both synthesis and degradation. The Dictyostelium genome encodes seven phosphodiesterases (PDEs) that degrade cyclic nucleotides to nucleotide 5'-monophosphates. Each PDE has a distinct structure, substrate specificity, regulatory input, cellular localization, and developmentally regulated expression pattern. The intra- or extra-cellular localizations and enzymatic specificities for cAMP or cGMP are essential for degradative precision at different developmental stages. The diverse PDEs, the nucleotide cyclases, and the target proteins for cAMP and cGMP in Dictyostelium. We link the major molecular, cellular, and developmental events regulated by cyclic nucleotide signaling, with emphasis to the input of each PDE and consequence of loss-of-function mutations. We have also related the structures and functions of the Dictyostelium PDEs with those of humans and in context to potential therapeutic understandings. The recognition and killing of infectious bacteria by macrophage and other phagocytic cells is a paramount defense strategy within the innate immune response. Multiple compounds are released by bacteria, and different phagocytic cells express specific chemoattractant receptors with distinct binding specificities; activation of these receptors directs a complex series of intracellular signaling pathways that guide directed cell migration toward a bacterial source. However, many bacterial chemoattractants are structurally related to molecules that are synthesized by and, thus, intrinsic to phagocytic cells. If these molecules were released from cells and also recognized by a chemoattractant receptor, they could create a confounding signal that would interfere with directional sensing of the bona fide bacterially-released chemoattractant gradient; this would be distinct from an auto-immune response. We investigated approaches that could suppress such interfering signals, without disrupting bacterial sensing and targeting of infectious bacteria. As are macrophage, Dictyostelium are professional phagocytes with highly sensitive chemoattraction toward gram positive and gram negative bacteria. Dictyostelium recognize several bacterially-secreted compounds. The Dictyostelium chemoattractant G protein coupled receptor Far1 is activated by bacterially-secreted folate. Folate is part of the family of pterin/pteridine molecules and is essential for Dictyostelium (and mammalian) cell growth. Dictyostelium, in addition, synthesize several structurally related pteridine molecules. We present evidence for different mechanisms that allow Dictyostelium to distinguish bacterial signals for chemotaxis from endogenous compounds. Through structure/function studies, we show that Far1 activation affinity is highly sensitive to very minor structural modifications relative to folate and shows strong preference to secreted bacterial products than to structurally similar but distinct endogenous molecules. Dictyostelium secrete a deaminase that will modify compounds to render them inactive. Cells can very rapidly inactivate environmental signals in close proximity, but are still able to recognize an active incoming signal, before it becomes deaminated and inactivated; this mechanism would also reinforce the strength of the exogenous chemoattractant gradient for enhanced directional sensing. Surprisingly, anti-folates (e.g. methotrexate) are resistant to this inactivation. In addition, we show that the secretion of endogenous compounds is minimized during times of active chemotaxis to bacteria. Finally, Far1 in Dictyostelium had been suggested to directly bind and respond to lipopolysaccharides (LPS) through their core region saccharide motifs for gram negative bacterial recognition. We show that highly purified preparations of LPS containing core region saccharides and distal O polysaccharides can synergize modestly with a non-saturating dose of folate to promote an enhanced signal response. In context to PAMPS, Pathogen Associated Molecular Patterns, Far1 is potential pathogen (pattern) recognition receptor (PRR) for bacterial 2-amino pteridine compounds. Post-transcriptional processes mediated by mRNA binding proteins represent important control points in the regulation of gene expression. TTP members are RNA binding and destabilizing proteins, characterized by a central specific tandem zinc finger and C-terminal, CNOT1 binding domain. We had characterized TtpA, the single TTP member of the family in Dictyostelium discoideum, and, using null, frame-shift, cysteine point mutants within the TZF domain, which prevents mRNA binding, and a carboxyl-terminal mutation, which removes the CNOT1 binding domain, explored the role TTP in Dictoystelium. All the mutants exhibit reproducibly increased expression of six specific transcripts, suggesting relief from TTP post-transcriptional repression. All six target mRNAs contain an identical A/U-rich instability motif in their 3’UTRs; the motif element is able to confer TtpA regulation to a heterologous mRNA and regulation is lost upon mutation of the motif. Using co-IP and MS/MS peptide mapping, in conjunction with yeast 2-hybrid screens, we identified several proteins that interact with TtpA. These include CNOT1, 14-3-3, as well as unique proteins with metabolic functions. Interaction regions have been modelled in AlphaFold and are being tested directly. Further data show differences related to the presence or absence of active TtpA in nutrient-rich or -depleted media. We have mapped regions in TtpA that may modulate TtpA activity and are following connection to mRNA- and protein-binding interactions.