Teaching a biology laboratory course using Dictyostelium.

The Dictyostelium discoideum model system is a powerful tool for undergraduate cell biology teaching laboratories. The cells are biologically safe, grow at room temperature and it is easy to experimentally induce, observe, and perturb a breadth of cellular processes making the system amenable to many teaching lab situations and goals. Here we outline the advantages of Dictyostelium, discuss laboratory courses we teach in three very different educational settings, and provide tips for both the novice and experienced Dictyostelium researcher. With this article and the extensive sets of protocols and tools referenced here, implementing these labs, or parts of them, will be relatively straightforward for any instructor.

Elastic modulus of Dictyostelium is affected by mechanotransduction.

The stiffness of adherent mammalian cells is regulated by the elasticity of substrates due to mechanotransduction via integrin-based focal adhesions. Dictyostelium discoideum is an ameboid protozoan model organism that does not carry genes for classical integrin and can adhere to substrates without forming focal adhesions. It also has a life cycle that naturally includes both single-cellular and multicellular life forms. In this article, we report the measurements of the elastic modulus of single cells on varied substrate stiffnesses and the elastic modulus of the multicellular "slug" using atomic force microscopy (AFM) as a microindenter/force transducer. The results show that the elastic modulus of the Dictyostelium cell is regulated by the stiffness of the substrate and its surrounding cells, which is similar to the mechanotransduction behavior of mammalian cells.

The PCH family member proline-serine-threonine phosphatase-interacting protein 1 targets to the leukocyte uropod and regulates directed cell migration.

Pombe Cdc15 homology (PCH) family members have emerged as important regulators of membrane-cytoskeletal interactions. Here we show that PSTPIP1, a PCH family member expressed in hematopoietic cells, regulates the motility of neutrophil-like cells and is a novel component of the leukocyte uropod where it colocalizes with other uropod components, such as type I PIPKIgamma. Furthermore, we show that PSTPIP1 association with the regulator of endocytosis, dynamin 2, and PSTPIP1 expression impairs transferrin uptake and endocytosis. We also show that PSTPIP1 localizes at the rear of neutrophils with a subpopulation of F-actin that is specifically detected by the binding of an F-actin probe that detects a more stable population of actin. Finally, we show that actin polymerization, but not the microtubule network, is necessary for the polarized distribution of PSTPIP1 toward the rear of the cell. Together, our findings demonstrate that PSTPIP1 is a novel component of the leukocyte uropod that regulates endocytosis and cell migration.

Neutrophil chemotaxis in a patient with neonatal-onset multisystem inflammatory disease and Muckle-Wells syndrome.

BACKGROUND: Neonatal-onset multisystem inflammatory disease (NOMID)/chronic infantile neurologic, cutaneous, and articular syndrome is an autoinflammatory disease characterized by urticarial rash, arthropathy, and central nervous system inflammation. OBJECTIVE: To describe a 13-year-old girl with overlapping symptoms of NOMID and Muckle-Wells syndrome who has a mutation in cryopyrin (NALP3). METHODS: We examined neutrophil migration using transwell assay and time-lapse videomicroscopy. We also examined p38 mitogen-activated protein kinase (MAPK) activation in patient and control neutrophils using Western blot analysis. RESULTS: Neutrophil defects in chemotactic migration were found to a variety of chemoattractants, including interleukin 8, N-formyl-methionyl-leucyl-phenylalanine, complement C5a, and leukotriene B4. Her neutrophils exhibited elevated basal and stimulated p38 MAPK activation in response to interleukin 8, N-formyl-methionyl-leucyl-phenylalanine, complement C5a, and leukotriene B4. CONCLUSIONS: This study is the first, to our knowledge, to demonstrate defects in neutrophil chemotaxis and p38 MAPK signaling in a patient with NOMID and Muckle-Wells syndrome and a cryopyrin mutation.

Secretory IgA induces antigen-independent eosinophil survival and cytokine production without inducing effector functions.

BACKGROUND: Eosinophils in human beings reside in tissues, especially the mucosal tissues of the gastrointestinal tract and inflamed airways. Secretory IgA (S-IgA) is the predominant antibody secreted by these tissues and likely plays a role in the innate immune response. OBJECTIVE: Because eosinophils and S-IgA are often colocalized in mucosal tissues, we examined the potential regulatory effects of S-IgA without antigens on survival, gene expression, and effector functions of human eosinophils. METHODS: Eosinophils were incubated with S-IgA in solution without antigens (soluble S-IgA) or with S-IgA immobilized to mimic multivalent antigen cross-linking. Eosinophil activation was monitored by superoxide anion generation and degranulation. Survival was assessed between 24 and 96 hours. Gene and protein expression were examined by microarray and ELISA. Eosinophil lysates were examined by immunoblot for extracellular signal-regulated kinase (ERK) phosphorylation. RESULTS: Immobilized S-IgA stimulated eosinophil superoxide production and degranulation; soluble S-IgA did not. Although immobilized S-IgA inhibited eosinophil survival in vitro, soluble S-IgA enhanced survival; this involved autocrine production of GM-CSF. Soluble S-IgA without antigens induced increases in mRNA levels of various cytokines, chemokines, signal transduction molecules, antiapoptotic factors, and cell surface markers. By using ELISA, we confirmed protein expression of selected mediators. Eosinophil interaction with soluble S-IgA likely involves FcalphaRI (CD89) and ERK pathway activation. CONCLUSION: Secretory IgA without multivalent antigens may regulate survival and gene expression of eosinophils. Eosinophils in mucosal tissues can be either primed for action (cytokine production and survival) or fully activated (degranulation and superoxide release) by different forms of S-IgA.