Hyperosmotic stress-induced reorganization of actin bundles in Dictyostelium cells over-expressing cofilin.

BACKGROUND: Cofilin is a low-molecular weight actin-modulating protein, which binds to, severs, and depolymerizes actin filaments in vitro. Aip1, an actin-interacting protein, was recently identified as a product of a gene on a multicopy plasmid which suppresses the temperature-sensitive phenotype of a cofilin mutant in Saccharomyces cerevisiae. Actin cytoskeleton plays an essential role in resistance to hyperosmotic stress in Dictyostelium discoideum. The roles of cofilin and Aip1 in this resistance are not known. RESULTS: In response to hyperosmotic stress, D. discoideum cells round up. This stress-induced morphological change involves the redistribution of cofilin, together with actin filaments, into cortical contractile portions of the cells, followed by their contraction. Over-expression of cofilin increases and thickens cortical actin bundles in cells. The bundles become tight and are reorganized into a ring-shaped structure in response to hyperosmotic stress. The ring structure of actin bundles had two characteristic bands across them; bright and dark bands, heavily stained and not stained with phalloidin. In the bundles, straight filaments with a diameter of 5.3-nm were aligned parallel by cross-bridge structures. In cells lacking the myosin-II heavy chain, the bundles, which were induced by an over-expression of cofilin, shortened and became straight following hyperosmotic stress, forming a polygonal structure. D. discoideum Aip1/Wrp2 enhanced the severing of actin filaments by cofilin in vitro and colocalized with cofilin in cells, including those that were over-expressing cofilin before and after exposure to hyperosmotic stress. CONCLUSIONS: Cofilin plays a pivotal role in concert with Aip1/Wrp2 in the reorganization of actin architectures into bundles that contract in a myosin-II-independent manner, in response to hyperosmotic stress.

Monomer arrangement in HSP90 dimer as determined by decoration with N and C-terminal region specific antibodies.

Electron microscopy using the low-angle rotary shadowing replica method showed that the HSP90 dimer consists of four globular domains aligning in a tandem fashion. When decorated with two monoclonal antibodies against epitopes mapped on the N-terminal region of HSP90, these antibodies bound to both ends of the HSP90 dimer. A C-terminal region specific antibody was shown to bind to the side of HSP90. These results support a model for HSP90 dimer whereby two HSP90 monomers are arranged in an antiparallel fashion and dimerize through the C-terminal domain. Treatment of HSP90 at elevated temperatures or with ATP at room temperature, though not with ADP, induces molecular transformation of the linear HSP90 dimer into an O-ring-shaped structure.

A novel 66-kDa stress protein, p66, associated with the process of cyst formation of Physarum polycephalum is a Physarum homologue of a yeast actin-interacting protein, AIP1.

When exposed to various stresses including heat shock, myxoamoebae, growing haploid cells of Physarum polycephalum, show marked morphological changes and consequently become disk-shaped microcysts. We have found that p66 is induced exclusively in the course of microcyst formation and has an actin-binding activity. In this study, we purified p66 to homogeneity and isolated a p66 cDNA. The deduced protein sequence contained 601 amino acids and showed 31% identity to a yeast actin-interacting protein, AIP1. Northern blot analysis revealed that the amount of p66 mRNA was significantly increased by heat shock in myxoamoebae but not in plasmodia. Thus, p66 seems to be a developmentally-expressed stress protein which regulates the rearrangement of actin organization during microcyst formation in P. polycephalum.

Activation of a Ca(2+)-dependent protein kinase in response in heat shock in the myxoamoebae of a true slime mold, Physarum polycephalum.

Protein kinase activities in myxoamoebae of a true slime mold, Physarum polycephalum, were investigated in response to heat shock. In-gel assay detected an apparent activation of a Ca(2+)-dependent, 53-kDa protein kinase that phosphorylated casein but not histone H1. This enzyme needed co-presence of Mg2+ ion with Ca2+ for activity. Treatment with calf intestinal alkaline phosphatase did not affect the heat-inducible 53-kDa protein kinase activity at all. The effects of protein kinase inhibitors were examined, and staurosporine suppressed the activity of this enzyme completely. H-7 decreased the activity to about 20% and HA-1004 to 65%. These results suggest that this protein kinase that may phosphorylate tyrosine and serine/threonine residues of target proteins is activated by heat shock in Physarum cells, and the activation is not regulated via phosphorylation by putative protein kinase(s) that may act at an upstream position in the signaling cascade(s).

Creatine kinase MB measured by fluorometric enzyme immunoassay and immunochemiluminescence.

Creatine kinase MB (CK-MB) was measured in serum by a fluorometric enzyme immunoassay on the Stratus analyzer and by an immunochemiluminometric assay using the Ciba Corning Magic Lite System. Both methods were standardized against purified CK-MB, with Stratus underestimating by 20 percent and Magic Lite overestimating by 28 percent. The assays proved sensitive and linear; however, at a CK-MB concentration of 7.0 micrograms per L, Stratus gave unacceptable inter-assay precision. No cross-reactivity was observed with CK-MM or CK-BB and elevated triglycerides, bilirubin, and hemoglobin did not interfere. Correlations with an immunoradiometric assay (Embria), using 522 samples, gave: Stratus = 0.999 (Embria) -3.3; r = 0.969, and Magic Lite = 1.225 (Embria) -3.03; r = 0.971. When using Magic Lite, results from 40 acute myocardial infarction (AMI) patients gave a mean CK-MB value of 93.8 micrograms per L (range: 9.2 to 428 micrograms per L) at the peak of enzyme release and a mean value of 69.6 micrograms per L (range: 6.7 to 319 micrograms per L) when using Stratus. Both methods proved to be highly sensitive and specific in the diagnosis of AMI; however, the need for standardization of CK-MB assays is stressed.