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.

Switching of filamin polypeptides during myogenesis in vitro.

During chicken skeletal myogenesis in vitro, the actin-binding protein filamin is present at first in association with actin filament bundles both in myoblasts and in myotubes early after fusion. Later in mature myotubes it is found in association with myofibril Z disks. These two associations of filamin are separated by a period of several days, during which the protein is absent from the cytoplasm of differentiating myotubes (Gomer, R., and E. Lazarides, 1981, Cell, 23:524-532). To characterize the two classes of filamin polypeptides we have compared, by two-dimensional peptide mapping, 125I-labeled filamin immunoprecipitated from myoblasts and fibroblasts to filamin immunoprecipitated from mature myotubes and adult skeletal myofibrils. Myoblast filamin is highly homologous to fibroblast and purified chicken gizzard filamins. Mature myotube and adult myofibril filamins are highly homologous but exhibit extensive peptide differences with respect to the other three classes of filamin. Comparison of peptide maps from immunoprecipitated 35S-methionine-labeled filamins also shows that fibroblast and myoblast filamins are highly homologous but show substantial peptide differences with respect to mature myotube filamin. Filamins from both mature myotubes and skeletal myofibrils exhibit a slightly higher electrophoretic mobility than gizzard, fibroblast, and myoblast filamins. Short pulse-labeling studies show that mature myotube filamin is synthesized as a lower molecular weight variant and is not derived from a higher molecular weight precursor. These results suggest that myoblast and mature myotube filamins are distinct gene products and that during skeletal myogenesis in vitro one class of filamin polypeptides is replaced by a new class of filamin polypeptides, and that the latter is maintained into adulthood.

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.

The cell density factor CMF regulates the chemoattractant receptor cAR1 in Dictyostelium.

Starving Dictyostelium cells aggregate by chemotaxis to cAMP when a secreted protein called conditioned medium factor (CMF) reaches a threshold concentration. Cells expressing CMF antisense mRNA fail to aggregate and do not transduce signals from the cAMP receptor. Signal transduction and aggregation are restored by adding recombinant CMF. We show here that two other cAMP-induced events, the formation of a slow dissociating form of the cAMP receptor and the loss of ligand binding, which is the first step of ligand-induced receptor sequestration, also require CMF. Vegetative cells have very few CMF and cAMP receptors, while starved cells possess approximately 40,000 receptors for CMF and cAMP. Transformants overexpressing the cAMP receptor gene cAR1 show a 10-fold increase of [3H]cAMP binding and a similar increase of [125I]CMF binding; disruption of the cAR1 gene abolishes both cAMP and CMF binding. In wild-type cells, downregulation of cAR1 with high levels of cAMP also downregulates CMF binding, and CMF similarly downregulates cAMP and CMF binding. This suggests that the cAMP binding and CMF binding are closely linked. Binding of approximately 200 molecules of CMF to starved cells affects the affinity of the majority of the cAR1 cAMP receptors within 2 min, indicating that an amplifying mechanism allows one activated CMF receptor to regulate many cARs. In cells lacking the G-protein beta subunit, cAMP induces a loss of cAMP binding, but not CMF binding, while CMF induces a reduction of CMF binding without affecting cAMP binding, suggesting that the linkage of the cell density-sensing CMF receptor and the chemoattractant cAMP receptor is through a G-protein.

A density-sensing factor regulates signal transduction in Dictyostelium.

Dictyostelium discoideum initiates development when cells overgrow their bacterial food source and starve. To coordinate development, the cells monitor the extracellular level of a protein, conditioned medium factor (CMF), secreted by starved cells. When a majority of the cells in a given area have starved, as signaled by CMF secretion, the extracellular level of CMF rises above a threshold value and permits aggregation of the starved cells. The cells aggregate using relayed pulses of cAMP as the chemoattractant. Cells in which CMF accumulation has been blocked by antisense do not aggregate except in the presence of exogenous CMF. We find that these cells are viable but do not chemotax towards cAMP. Videomicroscopy indicates that the inability of CMF antisense cells to chemotax is not due to a gross defect in motility, although both video and scanning electron microscopy indicate that CMF increases the frequency of pseudopod formation. The activations of Ca2+ influx, adenylyl cyclase, and guanylyl cyclase in response to a pulse of cAMP are strongly inhibited in cells lacking CMF, but are rescued by as little as 10 s exposure of cells to CMF. The activation of phospholipase C by cAMP is not affected by CMF. Northern blots indicate normal levels of the cAMP receptor mRNA in CMF antisense cells during development, while cAMP binding assays and Scatchard plots indicate that CMF antisense cells contain normal levels of the cAMP receptor. In Dictyostelium, both adenylyl and guanylyl cyclases are activated via G proteins. We find that the interaction of the cAMP receptor with G proteins in vitro is not measurably affected by CMF, whereas the activation of adenylyl cyclase by G proteins requires cells to have been exposed to CMF. CMF thus appears to regulate aggregation by regulating an early step of cAMP signal transduction.

Cell-autonomous determination of cell-type choice in Dictyostelium development by cell-cycle phase.

The developmental fate of individual cells has been examined in a system that allows Dictyostelium discoideum cells to differentiate in the absence of aggregation. The results show that the propensity of single amoebae to differentiate into either prespore or prestalk cells occurs by a cell-autonomous mechanism dependent on the cell's position in the cell cycle at the initiation of development. Cells that divide between approximately 1 1/2 hours before and approximately 40 minutes after the differentiation-inducing starvation become prestalk, whereas cells dividing at other times become prespore cells. These results suggest mechanisms by which an initial proportioning of the two cell types within the aggregate is achieved.

Intercellular signalling. Knowing that you're among friends.

Factors that are simultaneously secreted and sensed are used by cells to monitor local cell density; a recently discovered factor of this type controls the transformation-competence of Bacillus subtilis.

Cell-density sensing: come on inside and tell us about it.

The developmental pathway chosen by a Bacillus subtilis cell is influenced by the local cell density. To sense cell density, the cell monitors at least three different secreted signal peptides, two of which are detected by a new type of transduction mechanism involving their specific transport into the cell.

Spatial and temporal regulation of a foreign gene by a prestalk-specific promoter in transformed Dictyostelium discoideum.

We analyzed a developmentally regulated prestalk-specific gene from Dictyostelium discoideum encoding a cathepsin-like protease. A hybrid gene was constructed by fusing 2.5 kilobases of 5' flanking sequences and part of the coding region of the gene in-frame to the Escherichia coli beta-glucuronidase gene and was transformed into D. discoideum cells. In cells transformed with this vector, the gene fusion showed the same temporal regulation as the endogenous gene during multicellular development and, like endogenous prestalk genes, was highly inducible by cyclic AMP in in vitro cell cultures. Moreover, immunofluorescence studies showed that the fusion protein had the same spatial distribution within the migrating pseudoplasmodium as the endogenous gene. The results indicate that the regions of the D. discoideum prestalk-specific cathepsin gene contain all the necessary information for proper temporal, spatial, and cyclic AMP regulation of a prestalk cell-type gene in D. discoideum transformants and leads the way for experiments to identify the cell-type-specific regulatory elements.

Cell cycle phase, cellular Ca2+ and development in Dictyostelium discoideum.

In Dictyostelium discoideum, the initial differentiation of cells is regulated by the phase of the cell cycle at starvation. Cells in S and early G2 (or with a low DNA content) have relatively high levels of cellular Ca2+ and display a prestalk tendency after starvation, whereas cells in mid to late G2 (or with a high DNA content) have relatively low levels of Ca2+ and display a prespore tendency. We found that there is a correlation between cytosolic Ca2+ and cell cycle phase, with high Ca2+ levels being restricted to cells in the S and early G2 phases. As expected on the basis of this correlation, cell cycle inhibitors influence the proportions of amoebae containing high or low Ca2+. However, it has been reported that in the rtoA mutant, which upon differentiation gives rise to many more stalk cells than spores (compared to the wild type), initial cell-type choice is independent of cell cycle phase at starvation. In contrast to the wild type, a disproportionately large fraction of rtoA amoebae fall into the high Ca2+ class, possibly due to an altered ability of this mutant to transport Ca2+.

A cell-density sensing factor regulates the lifetime of a chemoattractant-induced G alpha-GTP conformation.

Starving Dictyostelium discoideum cells monitor the local density of other starving cells by simultaneously secreting and sensing CMF. CMF regulates signal transduction through the chemoattractant cAMP receptor, cAR1. cAR1 activates a heterotrimeric G protein by stimulating G alpha 2 to release GDP and bind GTP. We show here that the rate of cAMP-stimulated GTP hydrolysis in membranes from cells exposed to CMF is roughly 4 times slower than in membranes from untreated cells, even though the rate of GTP binding is the same. This hydrolysis is abolished in cells lacking G alpha 2. Our data thus suggest that CMF regulates cAMP signal transduction in part by prolonging the lifetime of the G alpha 2-GTP complex.

Paralog chromatography.

As a mixed mode ligand, a small peptide can mimic an antibody's paratope (antigen recognition site). This report describes the construction of a representative set of paratope analogs, or "paralogs," which can be conjugated to a chromatographic sorbent to combine desirable characteristics of traditional high-performance liquid chromatography columns with the specificity of a moderate affinity antibody. The broad utility of this novel set of protein separatory reagents is illustrated on the complex mixture of proteins in a yeast lysate.

Complexity of the Neurospora crassa circadian clock system: multiple loops and oscillators.

Organisms from bacteria to humans use a circadian clock to control daily biochemical, physiological, and behavioral rhythms. We review evidence from Neurospora crassa that suggests that the circadian clock is organized as a network of genes and proteins that form coupled evening- and morning-specific oscillatory loops that can function autonomously, respond differently to environmental inputs, and regulate phase-specific outputs. There is also evidence for coupled morning and evening oscillator loops in plants, insects, and mammals, suggesting conservation of clock organization. From a systems perspective, fungi provide a powerful model organism for investigating oscillator complexity, communication between oscillators, and addressing reasons why the system has evolved to be so complex.

Gene identification by shotgun antisense.

Shotgun antisense is a technique to make a random set of mutant cells or organisms in such a way that one can select an interesting mutant and then sequence part of the mutated gene within a day. In addition to the fantastic rapidity with which one can identify the mutated gene, there are more advantages of this technique over other mutagenesis techniques: (1) one can identify genes that when completely repressed are lethal; (2) one can select which sets of genes will be mutated; and (3) genes that are expressed from multiple copies can be repressed and thus identified.

Not being the wrong size.

Size regulation is a never-ending problem. Many of us worry that parts of ourselves are too big whereas other parts are too small. How organisms--and their tissues--are programmed to be a specific size, how this size is maintained, and what might cause something to become the wrong size, are key problems in developmental biology. But what are the mechanisms that regulate the size of multicellular structures?

Skelemin, a cytoskeletal M-disc periphery protein, contains motifs of adhesion/recognition and intermediate filament proteins.

In striated muscle, myofibrils are anchored to an interconnecting cytoskeleton of desmin intermediate filaments. Skelemin (195 kDa) may be a link between myofibrils and the intermediate filament cytoskeleton. Skelemin partitions with desmin to the insoluble cytoskeleton, and increases the thickness of reconstituted intermediate filaments. Concentrated at the M-disc periphery, skelemin may also contact myosin filaments. We used immunoscreening to isolate a mouse muscle cDNA which encodes a protein with a calculated molecular mass of 185 kDa. Anti-skelemin antibodies bound to the protein products of each of three nonoverlapping regions of the open reading frame. Antibodies directed against the protein products of each one-third of the cDNA react with a 195-kDa muscle protein and stain the M-disc indistinguishably from the original anti-skelemin antibodies, suggesting that the cDNA encodes skelemin. A single skelemin mRNA is detected in muscle but not non-muscle tissues, consistent with immunostaining results. Skelemin is a member of a family of myosin-associated proteins containing fibronectin type III and immunoglobulin superfamily C2 motifs. Skelemin is unique in this family in having intermediate filament core-like motifs, one near each terminus. We hypothesize that skelemin could interact with myosin or myosin-associated proteins through its fibronectin and/or immunoglobulin motifs, and with intermediate filaments through intermediate filament-like motifs.

PSF and CMF, autocrine factors that regulate gene expression during growth and early development of Dictyostelium.

Throughout growth and development, Dictyostelium cells secrete autocrine factors that accumulate in proportion to cell density. At sufficient concentration, these factors cause changes in gene expression. Vegetative Dictyostelium cells continuously secrete prestarvation factor (PSF). The bacteria upon which the cells feed inhibit their response to PSF, allowing the cells to monitor their own density in relation to that of their food supply. At high PSF/bacteria ratios, which occur during late exponential growth, PSF induces the expression of several genes whose products are needed for cell aggregation. When the food supply has been depleted, PSF production declines, and a second density-sensing pathway is activated. Starving cells secrete conditioned medium factor (CMF), a glycoprotein of Mr 80 kDa that is essential for the development of differentiated cell types. Antisense mutagenesis has shown that cells lacking CMF cannot aggregate, and preliminary data suggest that CMF regulates cAMP signal transduction. Calculations indicate that a mechanism of simultaneously secreting and recognizing a signal molecule, as used by Dictyostelium to monitor cell density, could also be used to determine the total number of cells in a tissue.

A developmentally regulated cell surface receptor for a density-sensing factor in Dictyostelium.

Conditioned medium factor (CMF) is an 80-kDa glycoprotein which is the signal in a cell density-sensing system used by developing Dictyostelium cells. CMF is slowly secreted by cells when they starve, and the extracellular level of CMF then becomes an indicator of the density of starving cells. To examine how CMF is sensed, we have made bacterially synthesized recombinant CMF and found that it has as much activity as native CMF, indicating that glycosylation is not part of the active site of CMF. Expression of recombinant fragments of CMF indicates that the active site lies within an 88-amino acid region near the N terminus. To determine whether CMF is sensed by cell surface receptors, we examined binding of iodinated recombinant CMF to live cells. We found saturable binding to 6-h starved cells at 3.9 x 10(4) molecules/cell with a KD of 2.1 nM. The binding saturates in 30 min, and a Scatchard plot indicates that there is only one class of receptor. The binding is competed off by the addition of either the native or recombinant CMF, or the 88-amino acid active fragment region; no binding competition is seen from the nonactive regions or other proteins. Very little binding to vegetative cells is seen, with maximal binding seen in cells starved for 6-8 h. The amount of cell surface CMF binding then decreases during later development. Normal levels of CMF binding are seen to CMF- cells, indicating that CMF is not required for the accumulation of its own receptor.