Wayne M. Grant

Cofilin Produces Newly Polymerized Actin Filaments that Are Preferred for Dendritic Nucleation by the Arp2/3 Complex

One of the earliest events in the process of cell motility is the massive generation of free actin barbed ends, which elongate to form filaments adjacent to the plasma membrane at the tip of the leading edge. Both cofilin and Arp2/3 complex have been proposed to contribute to barbed end formation during cell motility. Attempts to assess the functions of cofilin and Arp 2/3 complex in vivo indicate that both cofilin and Arp2/3 complex contribute to actin polymerization: cofilin by severing and Arp2/3 by nucleating and branching. In order to determine if the activities of cofilin and Arp2/3 complex interact, we employed a light microscope-based assay to visualize actin polymerization directly in the presence of both proteins. The results indicate that cofilin generates barbed ends to increase the mass of freshly polymerized F-actin but does not directly affect the activity of Arp2/3 complex. However, while ADP, ADP-Pi, and newly polymerized ATP-filaments are all capable of supporting Arp2/3-mediated branching, newly polymerized F-actin supports most of the Arp2/3-induced branch formation. The results suggest that, in vivo, cofilin contributes to barbed end formation by inducing the initial increase in the number of barbed ends leading to increased ATP-F-actin, which in turn supports higher levels of dendritic nucleation by active Arp2/3 complex.

Nucleoplasmic Calcium Is Required for Cell Proliferation

Ca2+ signals regulate cell proliferation, but the spatial and temporal specificity of these signals is unknown. Here we use selective buffers of nucleoplasmic or cytoplasmic Ca2+ to determine that cell proliferation depends upon Ca2+ signals within the nucleus rather than in the cytoplasm. Nuclear Ca2+ signals stimulate cell growth rather than inhibit apoptosis and specifically permit cells to advance through early prophase. Selective buffering of nuclear but not cytoplasmic Ca2+ signals also impairs growth of tumors in vivo. These findings reveal a major physiological and potential pathophysiological role for nucleoplasmic Ca2+ signals and suggest that this information can be used to design novel therapeutic strategies to regulate conditions of abnormal cell growth.

Characterisation of a fatty acid and retinol binding protein orthologue from the hookworm Ancylostoma ceylanicum.

Hookworms, bloodfeeding intestinal nematodes, infect nearly one billion people in resource limited countries and are a leading cause of anaemia and malnutrition. Like other nematodes, hookworms lack the capacity to synthesise essential fatty acids de novo and therefore must acquire those from exogenous sources. The cDNA corresponding to a putative Ancylostoma ceylanicum fatty acid and retinol binding protein-1 (AceFAR-1) was amplified from adult hookworm mRNA. Studies using quantitative reverse transcriptase real-time PCR demonstrate that AceFAR-1 transcripts are most abundant in the earliest developmental stages of the parasite, and greater in females than males. Using in vitro assays, the recombinant AceFAR-1 (rAceFAR-1) was shown to bind individual fatty acids with equilibrium dissociation constants in the low micromolar range. The pattern of fatty acid uptake by live adult worms cultured ex vivo was similar to the in vitro binding profile of rAceFAR-1, raising the possibility that the native protein may be involved in acquisition of fatty acids by A. ceylanicum. Animals vaccinated orally with rAceFAR-1 and the mucosal adjuvant cholera toxin exhibited a statistically significant (40-47%) reduction in intestinal worm burden compared with controls immunized with antigen or adjuvant alone. Together, these data suggest a potential role for AceFAR-1 in hookworm biology, making it a potentially valuable target for drug and vaccine development.

Functional Coupling of Chromogranin with the Inositol 1,4,5-Trisphosphate Receptor Shapes Calcium Signaling

Chromogranins A and B are high capacity, low affinity calcium (Ca2+) storage proteins that bind to the inositol 1,4,5-trisphosphate-gated receptor (InsP3 R). Although most commonly associated with secretory granules of neuroendocrine cells, chromogranins have also been found in the lumen of the endoplasmic reticulum (ER) of many cell types. To investigate the functional consequences of the interaction between the InsP3 R and the chromogranins, we disrupted the interaction between the two proteins by adding a chromogranin fragment, which competed with chromogranin for its binding site on the InsP3R. Responses were monitored at the single channel level and in intact cells. When using InsP3 R type I incorporated into planar lipid bilayers and activated by cytoplasmic InsP3 and luminal chromogranin, the addition of the fragment reversed the enhancing effect of chromogranin. Moreover, the expression of the fragment in the ER of neuronally differentiated PC12 cells attenuated agonist-induced intracellular Ca2+ signaling. These results show that the InsP3R/chromogranin interaction amplifies Ca2+ release from the ER and that chromogranin is an essential component of this intracellular channel complex.

CAERULEIN-INDUCED INTRACELLULAR PANCREATIC ZYMOGEN ACTIVATION IS DEPENDENT UPON CALCINEURIN

Aberrant cytosolic Ca(2+) flux in pancreatic acinar cells is critical to the pathological pancreatic zymogen activation observed in acute pancreatitis, but the downstream effectors are not known. In this study, we examined the role of Ca(2+)-activated protein phosphatase 2B (or calcineurin) in zymogen activation. Isolated pancreatic acinar cells were stimulated with supraphysiological caerulein (100 nM) with or without the calcineurin inhibitors FK506 or cell-permeable calcineurin inhibitory peptide (CiP). Chymotrypsin activity was measured as a marker of zymogen activation, and the percent amylase secretion was used as a measure of enzyme secretion. Cytosolic Ca(2+) changes were recorded in acinar cells loaded with the intermediate Ca(2+)-affinity dye fluo-5F using a scanning confocal microscope. A 50% reduction in chymotrypsin activity was observed after pretreatment with 1 microM FK506 or 10 microM CiP. These pretreatments did not affect amylase secretion or the rise in cytosolic Ca(2+) after caerulein stimulation. These findings suggest that calcineurin mediates caerulein-induced intra-acinar zymogen activation but not enzyme secretion or the initial caerulein-induced cytosolic Ca(2+) signal.