For a number of reasons including ease of cell culture, genetic manipulation and experimental design the social amoeba Dictyostelium discoideum has long been a model system for investigating the morphological and molecular events of chemotaxis and development. Starvation of Dictyostelium initiates a 24-h developmental process which begins with the pulsed secretion of cAMP by a fraction of the amoebae towards which neighboring amoebae chemotax. Interaction of the secreted cAMP with the G-protein-coupled cAMP receptor (cAR1) on the plasma membranes of neighboring cells initiates a series of molecular and morphological events including enhanced expression of cAR1 and adenylyl cyclase A (ACA), cell elongation and polarization and chemotaxis. Release of G from the heterotrimeric G-protein coupled to cAR1 activates myosin II. G also activates two synergistic and partially redundant RasC- and RasG-signaling pathways. One pathway activates target of rapomycin complex 2 (TORC2) and protein kinase B (PKB) initiating polymerization of actin at the front of the cell, which, together with contraction of actomyosin II at the rear, supports chemotaxis towards the aggregation centers. A second Ras pathway activates phosphatidylinositol 3-kinase (PI3K) at the cells leading edge, which catalyzes the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3) to which cytoplasmic regulator of adenylyl cyclase (CRAC) binds and activates membrane-associated ACA. PIP3 also contributes to the TORC2 pathway which induces actin polymerization. TORC2 contributes to activation of ACA and, independently of G, binding of cAMP to cAR1 leads to phosphorylation and activation of extracellular signal regulated kinase 2 (ERK2) which increases cAMP concentration by inhibiting its hydrolysis by a phosphodiesterase. ACA-containing vesicles translocate to the rear of chemotaxing cells where secretion of cAMP creates a cell-to-cell cAMP signal relay resulting in head-to-tail streams of cells that aggregate into tight mounds of 100,000 or more cells in about 12 h. Over the next 12 h, the multi-cellular mounds differentiate through several morphological stages developing into mature fruiting bodies comprising a spore head supported by a stalk. In an appropriate nutritional environment, spores germinate into amoebae and the life-cycle begins anew. Recently, we have been studying the role of actin and actin-binding proteins in chemotaxis and cell devleopment in the Dictyostelium model system. Last year we reported (Shu et al., 2012) that actin cross-linking proteins cortexillin I and II have essential roles in Dictyostelium chemotaxis and thier development to mature fruiting bodies. The single knockout of either ctxI or ctxII impairs, and their double knockout completely inhibits, cell streaming and development by decreasing the secretion of cAMP. The Dictyostelium genome contains another gene with deduced amino acid sequence similar to ctxI and ctxII, particularly in the actin-binding C-terminal half, but less similar than ctxI and ctxII are to each other. The protein has been referred to as cortexillin III (ctxIII). We have now found that ctxIII co-localizes at the cell cortex with F-actin and ctxI and ctxII. However, in contrast to ctxI and ctxII, ctxIII acts as a negative regulator of endocytosis and cells growth. Whereas ctxI and II-null cells grow more slowly than wild-type cells, ctxIII- knockout cells grow faster than wild-type cells both in suspension and on agar plates. Over-expression of ctxIII inhibits cytokinesis and produces large multi-nucleated cells. Also, whereas ctxI and ctxII are required for normal chemotaxis, ctxIII-null cells chemotax and stream normally although they form small and broken mounds and, therefore, smaller fruiting bodies. CtxIII also acts as a negative regulator of phagocytosis, pinocytosis and exocytosis. The possible involvement of ctx I and II in these processes has not been determined. As we showed last year, the double knockout of ctxI and II alters the actin cytoskeleton inducing thick actin bundles in the cell cortex. We are now studying whether the actin cytoskeleton is disturbed in ctxIII-null cells and whether purified ctxIII crosslinks actin filaments into parallel bundles as do ctxI and ctxII. We will also determine if ctxIII has a PIP2-binding site, which ctxI has but not ctxII has not.
|Liu, Xiong; Shu, Shi; Yu, Shuhua et al. (2014) Biochemical and biological properties of cortexillin III, a component of Dictyostelium DGAP1-cortexillin complexes. Mol Biol Cell 25:2026-38|
|Shu, Shi; Liu, Xiong; Kriebel, Paul W et al. (2012) Actin cross-linking proteins cortexillin I and II are required for cAMP signaling during Dictyostelium chemotaxis and development. Mol Biol Cell 23:390-400|
|Liu, Xiong; Shu, Shi; Hong, Myoung-Soon S et al. (2010) Mutation of actin Tyr-53 alters the conformations of the DNase I-binding loop and the nucleotide-binding cleft. J Biol Chem 285:9729-39|
|Shu, Shi; Liu, Xiong; Kriebel, Paul W et al. (2010) Expression of Y53A-actin in Dictyostelium disrupts the cytoskeleton and inhibits intracellular and intercellular chemotactic signaling. J Biol Chem 285:27713-25|
|Brzeska, Hanna; Hwang, Kae-Jung; Korn, Edward D (2008) Acanthamoeba myosin IC colocalizes with phosphatidylinositol-4,5-bisphosphate at the plasma membrane due to the high concentration of negative charge. J Biol Chem :|