- American Cancer Society Senior Fellowship, University of California, San Diego, 1979-1981
- Visiting Scientist, German Cancer Research Center, 1979
- American Cancer Society Fellowship, University of California, San Diego, 1977-1979
- Ph.D., University of Rochester, 1977
Our laboratory group studies signaling cascades essential for eukaryotic growth and development, using molecular, genetic, cellular, and biochemical techniques, and the model eukaryote Dictyostelium, which grow as individual phagocytic cells in enriched media, but develop multicellularly upon nutrient depletion. We have defined cell autonomous and non-autonomous signal transduction pathways that drive transitional decisions to promote growth and/or regulate development. We probe a variety of signaling pathways which are shared in complex systems to better understand mechanisms and circuits for focus on human diseases. In certain instances, we extend studies to mammalian models.
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. Using Dictyostelium as a model, we 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, we were able to demonstrate directed migration of Dictyostelium 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. 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. 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.
Chemotaxis and cell migration also play pivotal roles in normal physiological processes such as embryogenesis, inflammation, and wound healing, as well as in pathological processes including chronic inflammatory disease and cancer metastasis. Novel chemotaxis/migration inhibitors are, thus, desirable for developing effective therapeutics and probing molecular mechanisms. We describe a fluorescence-based phenotypic assay in a 1536-well plate format for high-throughput screening of novel inhibitors of chemotaxis/migration within complex libraries of thousands of compounds. Although the assay utilizes the unique cellular response properties of Dictyostelium, the compounds identified are able to inhibit chemotaxis of mammalian cells. In addition, a parallel cell cytotoxicity counter-screen with an ATP content assay is described that eliminates cytotoxic compounds from the screen. This novel compound screening approach enables rapid identification of novel lead compounds that inhibit chemotaxis in human and other cells for drug development and research tools.
This laboratory also investigates molecular processes required for establishing a terminally differentiated organism from a homogeneous population of totipotent cells and is defining signal transduction pathways that specify developmental cell fates and pattern formation. By characterizing receptor-mediated cascades and nuclear targets, our research serves to identify mechanisms that are basic to multicellular differentiation. Organizing the embryonic body plan is a major requisite governing early metazoan development, with morphogen signaling central to establishing and specifying cell fates. 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. We suggest that these data may similarly mirror defects in CHARGE syndrome, an extremely severe, multi-system congenital disorder caused by loss-of-functions mutations in the human CHD type III variant CHD7, a potential ortholog of Dictyostelium ChdC.
We also maintain active collaborations to understand molecular mechanisms required for lipogenic and lipolytic action in mammalian cells. Excessive cellular lipid storage can be a risk factor for metabolic disorders, including insulin resistance, cardiovascular disease, and hepatic steatosis. Intracellular lipid droplets are unique organelles that store metabolic precursors of cellular energy, membrane biosynthesis, steroid hormone synthesis, and signaling. We first discovered the Perilipins (PLINs) as a 5 member multi-protein family that targets lipid droplet surfaces and regulates lipid storage and hydrolysis in mammalian cells. Each Plin member has unique lipid association properties, cell expression patterns and cell-specific functions.
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 transcription factors that stimulate expression of genes important for FA oxidation, whereas genes involved in glucose uptake and oxidation are suppressed. 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.
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.
Myocardial triglycerides stored in lipid droplets are important in regulating the intracellular delivery of fatty acids for energy generation in mitochondria, for membrane biosynthesis, and as agonists for intracellular signaling. Previously, we showed that deficiency in the lipid droplet protein perilipin 5 (Plin5) markedly reduces triglyceride storage in cardiomyocytes and increases the flux of fatty acids into phospholipids. Here, we investigated whether Plin5 deficiency in cardiomyocytes alters mitochondrial function. We found that Plin5 deficiency reduced mitochondrial oxidative capacity. Furthermore, in mitochondria from Plin5-/- hearts, the fatty acyl composition of phospholipids in mitochondrial membranes was altered and mitochondrial membrane depolarization was markedly compromised. These findings suggest that mitochondria isolated from hearts deficient in Plin5, have specific functional defects.
- Phosphorylation of chemoattractant receptors regulates chemotaxis, actin reorganization and signal relay.
- Brzostowski JA, Sawai S, Rozov O, Liao XH, Imoto D, Parent CA, Kimmel AR.
- J Cell Sci (2013 Oct 15) 126:4614-26. Abstract/Full Text
- Regulation of nucleosome positioning by a CHD Type III chromatin remodeler and its relationship to developmental gene expression in Dictyostelium.
- Platt JL, Kent NA, Kimmel AR, Harwood AJ.
- Genome Res (2017 Apr) 27:591-600. Abstract/Full Text
Research in Plain Language
We are interested in processes that control how cells sense nutrients to stimulate or limit cell growth and establish stem-cell fate decisions for differentiation into the many tissue types. We study cell-cell communication, but also intracellular signaling circuits that regulate patterns of gene expression in cell-specific manners.
A major step during early development is the organization of the embryonic body plan. Morphogens are secreted chemical messengers that convey signals that establish embryonic axis organization by helping to specify different cell fates. We study the receptors that recognize morphogen signals, and the cellular pathways that they activate. When stimulated, various types of receptors promote or inhibit specific differentiation patterns for axis formation. We also have examined signals and mechanisms that direct cell movement and sense cell density. Collectively, our studies may reveal new mechanisms for cell fate specification, but also for tumor suppression.
Obesity has wide systemic impact beyond an expanded lipid storage in adipocytes and increased circulating free fatty acids and triacylglycerides. We collaborate to understand the molecular mechanisms that are required for regulated lipid storage and mobilization and to limit excessive cellular lipid storage and the accompanying risks for metabolic syndrome, including insulin resistance, cardiovascular disease, and hepatic steatosis.