Cellular and subcellular distribution of a cAMP-regulated prestalk protein and prespore protein in Dictyostelium discoideum: a study on the ontogeny of prestalk and prespore cells

We have analyzed a developmentally and spatially regulated prestalk-specific gene and a prespore-specific gene from Dictyostelium. The prestalk gene, pst-cathepsin, encodes a protein highly homologous to the lysosomal cysteine proteinases cathepsin H and cathepsin B. The prespore gene encodes a protein with some homology to the anti-bacterial toxin crambin and has been designated beejin. Using the lambda gtll system, we have made polyclonal antibodies directed against a portion of the protein encoded by pst-cathepsin and other antibodies directed against the beejin protein. Both antibodies stain single bands on Western blots. By immunofluorescence and Western blots, pst-cathepsin is not present in vegetative cells or developing cells during the first approximately 10 h of development. It then appears with a punctate distribution in a subset of developing cells. Beejin is detected only after approximately 15 h of development, also in a subset of cells. Pst-cathepsin is distributed in the anterior approximately 1/10 of migrating slugs and on the peripheral posterior surfaces of slugs. Beejin is distributed in the posterior region of slugs. Expression of both pst-cathepsin and beejin can be induced in subsets of isolated cultured cells by a combination of conditioned medium and extracellular cAMP in agreement with the regulation of the mRNAs encoding these proteins. We have used the antibodies as markers for cell type to examine the ontogeny and the spatial distribution of prestalk and prespore cells throughout multicellular development. Our findings suggest that prestalk cell differentiation is independent of position within the aggregate and that the spatial localization of prestalk cells within the multicellular aggregate arises from sorting of the prestalk cells after their induction. We have also found a class of cell in developing aggregates that contains neither the prestalk nor the prespore markers.     

Highly homologous filamin polypeptides have different distributions in avian slow and fast muscle fibers

The high molecular weight actin-binding protein filamin is located at the periphery of the Z disk in the fast adult chicken pectoral muscle (Gomer, R. H., and E. Lazarides, 1981, Cell, 23: 524-532). In contrast, we have found that in the slow anterior latissimus dorsi (ALD) muscle, filamin was additionally located throughout the l band as judged by immunofluorescence with affinity-purified antibodies on myofibrils and cryosections. The Z line proteins desmin and alpha-actinin, however, had the same distribution in ALD as they do in pectoral muscle. Quantitation of filamin and actin from the two muscle types showed that there was approximately 10 times as much filamin per actin in ALD myofibrils as in pectoral myofibrils. Filamin immunoprecipitated from ALD had an electrophoretic mobility in SDS polyacrylamide gels identical to that of pectoral myofibril filamin and slightly greater than that of chicken gizzard filamin. Two-dimensional peptide maps of filamin immunoprecipitated and labeled with 125I showed that ALD myofibril filamin was virtually identical to pectoral myofibril filamin and was distinct from chicken gizzard filamin.     

The synthesis and deployment of filamin in chicken skeletal muscle

During myogenesis in vitro the actin-binding protein filamin is present in myoblasts and early fused cells and is associated with α-actinin-containing filament bundles, as judged by double immunofluorescence using antibodies specific for these two proteins. Approximately one day after cell fusion, yet before the development of a-actinin-containing Z line striations, filamin disappears from the cells. Later in myogenesis, several days after the appearance of α-actinin-containing Z line striations, filamin reappears and accumulates in the cells. Double immunofluorescence with antibodies to filamin and vimentin (or desmin) reveals that the newly appearing filamin localizes now to the myofibril Z line and is visible there shortly before vimentin or desmin becomes associated with the Z line. Immunofluorescent localization of filamin in isolated chicken skeletal myofibrils and Z disc sheets indicates that filamin has the same distribution as desmin and vimentin; it surrounds each myofibril Z disc and forms honeycomb-like networks within each Z plane of the muscle fiber. Filamin may thus be involved in the transition of desmin and vimentin to the Z disc. Analysis of whole-cell extracts by SDS-polyacrylamide gel electrophoresis and by immunoautoradiography shows that filamin is present in myoblasts and in myotubes early after cell fusion. Concomitant with the absence of filamin fluorescence during the subsequent few days of myogenesis, the quantity of filamin is markedly reduced. During this time, metabolic pulse-labeling with ³⁵S-methionine reveals that the synthetic rate of filamin is also markedly reduced. As filamin fluorescence appears at the Z line, the quantity of filamin and its synthetic rate both increase. The removal of filamin from the cells suggests that filamin either may not be required, or may actually interfere with a necessary process, during the early stages of sarcomere morphogenesis. These results also indicate that the periphery of the Z disc is assembled in at least two distinct steps during myogenesis.     

Trypsin Potentiates Human Fibrocyte Differentiation

Trypsin-containing topical treatments can be used to speed wound healing, although the mechanism of action is unknown. To help form granulation tissue and heal wounds, monocytes leave the circulation, enter the wound tissue, and differentiate into fibroblast-like cells called fibrocytes. We find that 20 to 200 ng/ml trypsin (concentrations similar to those used in wound dressings) potentiates the differentiation of human monocytes to fibrocytes in cell culture. Adding trypsin inhibitors increases the amount of trypsin needed to potentiate fibrocyte differentiation, suggesting that the potentiating effect is dependent on trypsin proteolytic activity. Proteases with other site specificities such as pepsin, endoprotease GluC, and chymotrypsin do not potentiate fibrocyte differentiation. This potentiation requires the presence of albumin in the culture medium, and tryptic fragments of human or bovine albumin also potentiate fibrocyte differentiation. These results suggest that topical trypsin speeds wound healing by generating tryptic fragments of albumin, which in turn potentiate fibrocyte differentiation.     

A chemorepellent inhibits local Ras activation to inhibit pseudopod formation to bias cell movement away from the chemorepellent

The ability of cells to sense chemical gradients is essential during development, morphogenesis, and immune responses. Although much is known about chemoattraction, chemorepulsion remains poorly understood. Proliferating Dictyostelium cells secrete a chemorepellent protein called AprA. AprA prevents pseudopod formation at the region of the cell closest to the source of AprA, causing the random movement of cells to be biased away from the AprA. Activation of Ras proteins in a localized sector of a cell cortex helps to induce pseudopod formation, and Ras proteins are needed for AprA chemorepulsion. Here we show that AprA locally inhibits Ras cortical activation through the G protein–coupled receptor GrlH, the G protein subunits Gβ and Gα8, Ras protein RasG, protein kinase B, the p21-activated kinase PakD, and the extracellular signal–regulated kinase Erk1. Diffusion calculations and experiments indicate that in a colony of cells, high extracellular concentrations of AprA in the center can globally inhibit Ras activation, while a gradient of AprA that naturally forms at the edge of the colony allows cells to activate Ras at sectors of the cell other than the sector of the cell closest to the center of the colony, effectively inducing both repulsion from the colony and cell differentiation. Together, these results suggest that a pathway that inhibits local Ras activation can mediate chemorepulsion.     

A secreted cell number counting factor represses intracellular glucose levels to regulate group size in dictyostelium

Developing Dictyostelium cells form evenly sized groups of approximately 2 x 10(4) cells. A secreted 450-kDa protein complex called counting factor (CF) regulates group size by repressing cell-cell adhesion and myosin polymerization and by increasing cAMP-stimulated cAMP production, actin polymerization, and cell motility. We find that CF regulates group size in part by repressing internal glucose levels. Transformants lacking bioactive CF and wild-type cells with extracellular CF depleted by antibodies have high glucose levels, whereas transformants oversecreting CF have low glucose levels. A component of CF, countin, affects group size in a manner similar to CF, and a 1-min exposure of cells to countin decreases glucose levels. Adding 1 mm exogenous glucose negates the effect of high levels of extracellular CF on group size and mimics the effect of depleting CF on glucose levels, cell-cell adhesion, cAMP pulse size, actin polymerization, myosin assembly, and motility. These results suggest that glucose is a downstream component in part of the CF signaling pathway and may be relevant to the observed role of the insulin pathway in tissue size regulation in higher eukaryotes.     

A single cell density-sensing factor stimulates distinct signal transduction pathways through two different receptors

In Dictyostelium discoideum, cell density is monitored by levels of a secreted protein, conditioned medium factor (CMF). CMFR1 is a putative CMF receptor necessary for CMF-induced G protein-independent accumulation of the SP70 prespore protein but not for CMF-induced G protein-dependent inositol 1,4,5-trisphosphate production. Using recombinant fragments of CMF, we find that stimulation of the inositol 1,4,5-trisphosphate pathway requires amino acids 170-180, whereas SP70 accumulation does not, corroborating a two-receptor model. Cells lacking CMFR1 do not aggregate, due to the lack of expression of several important early developmentally regulated genes, including gp80. Although many aspects of early developmental cAMP-stimulated signal transduction are mediated by CMF, CMFR1 is not essential for cAMP-stimulated cAMP and cGMP production or Ca(2+) uptake, suggesting the involvement of a second CMF receptor. Exogenous application of antibodies against either the region between a first and second or a second and third possible transmembrane domain of CMFR1 induces SP70 accumulation. Antibody- and CMF-induced gene expression can be inhibited by recombinant CMFR1 corresponding to the region between the first and third potential transmembrane domains, indicating that this region is extracellular and probably contains the CMF binding site. These observations support a model where a one- or two-transmembrane CMFR1 regulates gene expression and a G protein-coupled CMF receptor mediates cAR1 signal transduction.     

A precise group size in Dictyostelium is generated by a cell-counting factor modulating cell-cell adhesion

A remarkable aspect of Dictyostelium development is that cells form evenly sized groups of approximately 2 x 10(4) cells. A secreted 450 kDa protein complex called counting factor (CF) regulates the number of cells per group. We find that CF regulates group size by repressing cell-cell adhesion. In both experiments and computer simulations, high levels of CF (and thus low adhesion) result in aggregation streams breaking up into small groups, while no CF (and thus high adhesion) results in no stream breakup and large groups. These results suggest that in Dictyostelium and possibly other systems a secreted factor regulating cell-cell adhesion can regulate the size of a group of cells.     

Genetic diversity of Dinaric brown bears (Ursus arctos) in Croatia with implications for bear conservation in Europe

Brown bears have lost most of their range on the European continent. The remaining western populations are small, isolated and highly endangered. The Dinaric-Pindos brown bear population is the western-most stable population and the fourth largest in Europe. It has been recognized as a potential source for recolonization of populations whose survival is at risk. Indeed, several translocations of Dinaric bears to Italy, Austria and France have recently been made. Despite the importance of the Dinaric bear population, its genetic status remains poorly understood. Using tissue samples from 156 hunted or accidentally killed Dinaric bears in Croatia, this study analysed genetic diversity at 12 microsatellite loci, as well as population structure and past reductions in size. In addition, a subset of 59 samples was used to assess diversity of the mitochondrial DNA control region. The results indicate that Dinaric bears have high nuclear genetic diversity, as compared to other extant brown bear populations, despite genetic evidence of a bottleneck caused by past persecutions. However, haplotype diversity was low, probably as a result of male-biased dispersal and female philopatry. Not surprisingly, no evidence of population sub-structure was found using nuclear markers, as the bear habitat has remained continuous and the highway network has been built only recently. Management should focus on maintaining habitat connectivity and keeping the effective population size as large as possible. In addition, when removing individuals, care should be taken not to further deplete the population of rare haplotypes. A coordinated transboundary management of the entire Dinaric-Pindos brown bear population should be a priority for its long-term conservation.