Alternative splicing of Bim and Erk-mediated Bim(EL) phosphorylation are dispensable for hematopoietic homeostasis in vivo.

The pro-apoptotic BH3-only protein Bim has a major role in hematopoietic homeostasis, particularly in the lymphocyte compartment, where it strongly affects immune function. The three major Bim isoforms (Bim(EL), Bim(L) and Bim(S)) are generated by alternative splicing. Bim(EL), the most abundant isoform, contains a unique sequence that has been reported to be the target of phosphorylation by several MAP kinases. In particular, Erk1/2 has been shown to interact with Bim(EL) through the DEF2 domain of Bim(EL) and specifically phosphorylate this isoform, thereby targeting it for ubiquitination and proteasomal degradation. To examine the physiological importance of this mechanism of regulation and of the alternative splicing of Bim, we have generated several Bim knock-in mouse strains and analyzed their hematopoietic system. Although mutation in the DEF2 domain reduces Bim(EL) degradation in some circumstances, this mutation did not significantly increase Bim's pro-apoptotic activity in vivo nor impact on the homeostasis of the hematopoietic system. We also show that Bim(EL) and Bim(L) are interchangeable, and that Bim(S) is dispensable for the function of Bim. Hence, we conclude that physiological regulation of Bim relies on mechanisms independent of its alternative splicing or the Erk-dependent phosphorylation of Bim(EL).

Stimulation of innate immune responses by malarial glycosylphosphatidylinositol via pattern recognition receptors.

The glycosylphosphatidylinositol (GPI) anchor of Plasmodium falciparum is thought to function as a critical toxin that contributes to severe malarial pathogenesis by eliciting the production of proinflammatory responses by the innate immune system of mammalian hosts. Analysis of the fine structure of P. falciparum GPI suggests a requirement for the presence of both core glycan and lipid moieties in the recognition and signalling of parasite glycolipids by host immune cells. It has been demonstrated that GPI anchors of various parasitic protozoa can mediate cellular immune responses via members of the Toll-like family of pattern recognition receptors (TLRs). Recent studies indicate that GPI anchors of P. falciparum and other protozoa are preferentially recognized by TLR-2, involving the MyD88-dependent activation of specific signalling pathways that mediate the production of proinflammatory cytokines and nitric oxide from host macrophages in vitro. However, the contribution of malaria GPI toxin to severe disease syndromes and the role of specific TLRs or other pattern recognition receptors in innate immunity in vivo is only just beginning to be characterized. A better understanding of the molecular mechanisms underlying severe malarial pathogenesis may yet lead to substantial new insights with important implications for the development of novel therapeutics for malaria treatment.

Mutant Rac1B expression in Dictyostelium: effects on morphology, growth, endocytosis, development, and the actin cytoskeleton.

Rac1 is a small G-protein in the Ras superfamily that has been implicated in the control of cell growth, adhesion, and the actin-based cytoskeleton. To investigate the role of Rac1 during motile processes, we have established Dictyostelium cell lines that conditionally overexpress epitope-tagged Dictyostelium discoideum wild-type Rac1B (DdRac1B) or a mutant DdRac1B protein. Expression of endogenous levels of myc- or GFP-tagged wild-type DdRac1B had minimal effect on cellular morphologies and behaviors. By contrast, expression of a constitutively active mutant (G12-->V or Q61-->L) or a dominant negative mutant (T17-->N) generated amoebae with characteristic cellular defects. The morphological appearance of actin-containing structures, intracellular levels of F-actin, and cellular responses to chemoattractant closely paralleled the amount of active DdRac1B, indicating a role in upregulating actin cytoskeletal activities. Expression of any of the three mutants inhibited cell growth and cytokinesis, and delayed multicellular development, suggesting that DdRac1B plays important regulatory role(s) during these processes. No significant effects were observed on binding or internalization of latex beads in suspension or on intracellular membrane trafficking. Cells expressing DdRac1B-G12V exhibited defects in fluid-phase endocytosis and the longest developmental delays; DdRac1B-Q61L produced the strongest cytokinesis defect; and DdRac1B-T17N generated intermediate phenotypes. These conditionally expressed DdRac1B proteins should facilitate the identification and characterization of the Rac1 signaling pathway in an organism that is amenable to both biochemical and molecular genetic manipulations.

Membrane cytoskeleton: PIP(2) pulls the strings.

A recent application of optical tweezers has shown that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels control adhesion of the membrane bilayer to the underlying cytoskeleton, by regulated direct binding of PIP(2) to cytoskeletal proteins and/or indirect effects on cytoskeleton structure.

A serpentine receptor-dependent, Gbeta- and Ca(2+) influx-independent pathway regulates mitogen-activated protein kinase ERK2 in Dictyostelium.

Ca(2+) influx and mitogen-activated protein (MAP) kinase activation are important phenomena in signal transduction, which are often interconnected. We investigated whether serpentine receptor-dependent, Gbeta-independent activation of MAP kinase ERK2 by chemoattractant cyclic AMP (cAMP) is mediated by Ca(2+) influx in the social amoeba Dictyostelium discoideum. We generated a D. discoideum double mutant, which harbours a temperature-sensitive Gbeta subunit and expresses the apoaequorin protein. Utilizing this mutant, we demonstrate that cAMP induced Ca(2+) influx into intact D. discoideum cells can be blocked completely at both the permissive and the restrictive temperature, by using either gadolinium ions or Ruthenium Red. Under the same experimental conditions, these substances do not abolish cAMP stimulation of ERK2 at either temperature. We conclude that there is a Gbeta- and Ca(2+) influx-independent pathway for the receptor-dependent activation of MAP kinase ERK2 in D. discoideum.

Intracellular Ca2+ signals in Dictyostelium chemotaxis are mediated exclusively by Ca2+ influx.

We measured folate- and cAMP-induced changes in cytoplasmic free calcium concentration ([Ca2+]i) using recombinant aequorin reconstituted in living Dictyostelium cells with coelenterazine-h. The resulting semi-synthetic protein displayed increased sensitivity to Ca2+ allowing accurate measurement of chemoattractant-induced transients at low resting levels. Both folate- and cAMP-induced Ca2+ responses were developmentally regulated, exhibited remarkably similar kinetics and were dependent on the relative rather than the absolute magnitude of increases in attractant concentration. They began after a short delay of 5-10 seconds, leading to a maximum increase in cytosolic calcium concentration after approximately 25 seconds and a return to basal level within approximately 60 seconds after stimulation. Responses elicited by the two chemoattractants were dose-dependent and saturated between 4 and 20 microM. They depended on the presence of free extracellular calcium ions and were inhibited in a concentration-dependent manner between 10(-4) and 10(-5) M. In accordance with 45Ca2+-uptake measurements by Milne and Coukell (J. Cell Biol. (1991) 112, 103-110), both responses were also completely inhibited by 15 microM Ruthenium Red, 15 microM carbonylcyanide m-chlorophenyl-hydrazone (CCCP) and 500 microM gadolinium ions. Under conditions that prohibited influx of Ca2+ from the extracellular medium there were no detectable changes in [Ca2+]i that could be related to a separate release of the ion from intracellular stores. Together, these results show that the Ca2+ signals involved in chemotaxis correlate temporally with actin depolymerization (not polymerization) and are mediated by Ca2+ influx, not IP3-mediated intracellular release.

A slow sustained increase in cytosolic Ca2+ levels mediates stalk gene induction by differentiation inducing factor in Dictyostelium.

During Dictyostelium stalk cell differentiation, cells vacuolate, synthesize a cellulose cell wall and die. This process of programmed cell death is accompanied by expression of the prestalk gene ecmB and induced by the differentiation inducing factor DIF. Using cell lines expressing the recombinant Ca2+-sensitive photoprotein apoaequorin, we found that 100 nM DIF increases cytosolic Ca2+ ([Ca2+]i) levels from approximately 50 to 150 nM over a period of 8 h. The Ca2+-ATPase inhibitor 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ) induced a similar increase in [Ca2+]i levels and induced expression of the prestalk gene ecmB to the same level as DIF. The [Ca2+]i increases induced by DIF and BHQ showed similar kinetics and preceded ecmB gene expression by approximately 1-2 h. The Ca2+ chelator 1,2-bis(o-aminophenoxy)-ethane-N,N,N'N'-tetra-acetic acid (BAPTA) efficiently inhibited the BHQ-induced [Ca2+]i increase and blocked DIF-induced expression of the ecmB gene. These data indicate that the effects of DIF on stalk gene expression are mediated by a sustained increase in [Ca2-]i. Sustained [Ca2+]i elevation mediates many forms of programmed cell death in vertebrates. The Dictyostelium system may be the earliest example of how this mechanism developed during early eukaryote evolution.