CSM News Electronic Edition Volume 1, number 15 September 18, 1993 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [129.105.233.50]. ============ Abstracts ============ The Formation of Liquid Crystals From Actin Filaments. Ruth Furukawa, Robin Kundra, and Marcus Fechheimer, Dept. of Zoology , University of Georgia Athens, GA 30602 Bichemistry, in press. ABSTRACT Actin is crosslinked by actin-binding proteins in the cytoplasm to form either isotropic or highly oriented anisotropic structures. The inherent orientation among actin filaments could influence whether an isotropic or highly oriented anisotropic structure is formed. A highly oriented state can arise spontaneously through the formation of liquid crystals as predicted by polymer theory. In this study, the ability of filamentous actin to form liquid crystalline domains was detected using the anisotropic component of scattered light and by observation of birefringence. As liquid crystalline domains formed, the intensity of the anisotropic component of scattered light increased, and birefringent macroscopic oriented domains were directly observed. The formation of liquid crystalline domains was dependent on the concentration of actin filaments, and on the average filament length controlled by varying the ratio of gelsolin to actin monomers. The concentration of actin filaments required to form liquid crystalline domains increased moderately as the average length was decreased. At a fixed actin concentration, orientation among the filaments attained a maximum value at a ratio of actin to gelsolin in the range from 1500-2000, and decreased as the ratio was increased or decreased from this range. The results are not well explained by theoretical treatments for liquid crystal formation by either monodisperse, charged rods or wormlike chains. Differences from the theoretical predictions for formation of liquid crystals are most likely due to the polydisperse filament length distribution of actin. This phenomenon may have important effects on the structural and rheological properties of the cytoplasm in living cells. --------------------------------------------------------------- Analysis of cellular interactions involved in differential control of prestalk genes in Dictyostelium discoideum. Yohko Yamada and Koji Okamoto Department of Botany, Faculty of Science, Kyoto University, Kyoto 606-01, Japan Developmental Biology, in press. Abstract Prestalk specific genes, ecmA and ecmB in Dictyostelium are both induced by cAMP and DIF-1. In an attempt to understand the control mechanism of the differential expression of the two genes in multicellular aggregates, we examined the requirement for additional cellular interaction using low cell density cultures. We show that the whole process of inducing expression of these genes depends on high cell density. However, cells which have become responsive to DIF-1 expressed both genes at a low cell density in the presence of DIF-1 when incubated in the medium previously conditioned by developing cells. 8-Br-cAMP, which is believed to penetrate cell membrane and activate protein kinase A, induced ecmB, but not ecmA, in the absence of the conditioned medium. These results suggest that there may be a specific inducer of ecmB in the conditioned medium which acts via activation of protein kinase A. Previously the two genes were shown to respond differently to cAMP in late development at high cell densities, where there were cellular interactions. However cAMP given to low density plated-cells inhibited the conditioned medium-dependent induction of both genes to the same extent, suggesting that cAMP itself does not directly show the different effects on the two genes. --------------------------------------------------------------- Cytoplasmic dynein plays a role in mammalian mitotic spindle formation. Vaisberg, E.A., Koonce, M.P., & McIntosh, J.R. J. Cell Biology, in press Abstract The formation and functioning of a mitotic spindle depends not only on the assembly/disassembly of microtubules but also on the action of motor enzymes. Cytoplasmic dynein has been localized to spindles but whether or how it functions in mitotic processes is not yet known. We have cloned and expressed DNA fragments that encode the putative ATP-hydrolytic sites of the cytoplasmic dynein heavy chains from Dictyostelium and HeLa cells. Monospecific antibodies raised to the resulting polypeptides inhibit dynein motor activity in vitro and their injection into mitotic mammalian cells blocks the formation of spindles in prophase or during recovery from nocodazole treatment at later stages of mitosis. Cells arrest with unseparated centrosomes and form monopolar spindles. The injected antibodies have no detectable effect on chromosome attachment to a bipolar spindle or on motions during anaphase. These data suggest that cytoplasmic dynein plays a unique and important role in the initial events of bipolar spindle formation, while any later roles it may play are redundant with other motor proteins. Possible mechanisms of dynein's involvement in mitosis are discussed. ---------------------------------------------------------------------- [[ The following abstracts are decoded copies of the abstracts that Peter's lab distributed via the mail server a week or so ago. ]] PHOSPHOLIPASE C IN DICTYOSTELIUM DISCOIDEUM. cAMP surface-receptor and G-protein regulated activity in vitro. Anthony A. Bominaar, Fanja Kesbeke and Peter J.M. Van Haastert. ABSTRACT The cellular slime mould Dictyostelium discoideum shows several responses following stimulation with the chemoattractant cAMP, including a transient rise in cAMP, cGMP and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. In this paper the regulation of phospholipase C in vitro is described. Under our experimental conditions commercial phosphatidylinositol 4,5-bisphosphate [PtdInsP2] can not be used to analyze phospholipase C activity in Dictyostelium lysates, because it is hydrolysed mainly to glycerophosphoinositol instead of Ins(1,4,5)P3. Enzyme activity was determined using endogenous, unlabelled PtdInsP2 as a substrate. The product was measured by isotope-dilution-assay and identified as authentic Ins(1,4,5)P3. Since phospholipase C is strictly Ca2+ dependent with an optimal concentration range from 10-6 to 10-4 M, cell lysates were prepared in EGTA and the enzyme reaction was started by adding 10-5 M free Ca2+. Phospholipase C activity increased 2 fold during Dictyostelium development up to 8 hours of starvation, after which the activity declined to less than 10 % of the vegetative level. Enzyme activity in vitro increased up to 2-fold following stimulation of cells with the agonist cAMP in vivo. Addition of 10-5 M GTPþS during lysis activated the enzyme to the same extent, and this effect was antagonized by GDPáS. These results strongly suggest that surface cAMP receptors and G-proteins regulate phospholipase C during Dictyostelium development. ------------------------------------------------------------------- PHOSPHOLIPASE C IN DICTYOSTELIUM DISCOIDEUM. Identification of stimulatory and inhibitory surface receptors and G-proteins. Anthony A. Bominaar and Peter J.M. Van Haastert ABSTRACT A combined biochemical and genetic approach was used to show that phospholipase C in the cellular slime mould Dictyostelium is under dual regulation by the chemoattractant cAMP. This dual regulation involves stimulatory and inhibitory surface receptors and G-proteins. In wild type cells both cAMP and GTPþS stimulated phospholipase C. In contrast, mutant fgd A, lacking the G-protein à-subunit Gà2, showed no stimulation by either cAMP or GTPþS, indicating that Gà2 is the stimulatory G-protein. In mutant fgd C cAMP did not stimulate phospholipase C, but stimulation by GTPþS was normal, suggesting that the defect in this mutant is upstream of the stimulatory Gà2. Inhibition of phospholipase C was achieved in wild type cells by the partial antagonist 3'NH-cAMP. This inhibition was no longer observed in transformed cell-lines lacking either the surface cAMP receptor cAR1 or the G-protein à- subunit Gà1; in these cells the agonist cAMP still activated phospholipase C. These results indicate that Dictyostelium phospholipase C is regulated via a stimulatory and an inhibitory pathway. The inhibitory pathway is composed of the surface receptor cAR1 and the G-protein G1. The stimulatory pathway consists of an unknown cAMP receptor (possibly the fgd C gene product) and the G-protein G2. --------------------------------------------------------------------- Redundant Regulatory Elements Account For The Developmental Control Of A Ribosomal Protein Gene Of Dictyostelium discoideum Ruey Ken and Charles K. Singleton Department of Molecular Biology Box 1820, Station B Vanderbilt University Nashville, TN 37235 ABSTRACT In Dictyostelium discoideum , ribosomal protein genes along with other growth specific genes appear to be coordinately regulated, primarily in response to differences in the translational capacity of developing versus growing cells. In particular, expression of the members of this large class of genes is rapidly and dramatically deactivated when the developmental program is initiated and growth and division cease. In order to understand the mechanisms behind the deactivation event and how it is coupled to the transition from growth to development, we have analyzed the promoter of the V18 gene, a ribosomal protein gene characteristic of this class of growth specific genes. We have delineated three discrete regions involved in the transcription and regulation of the V18 gene. An initiator region which appears to function in a TATA-independent manner was required for transcription and establishing start site utilization. Two regions upstream of this were defined, both of which were found to independently confer proper developmental regulation. ------------------------------------------------------------------- [[End CSM-News.v1.#15]]