CSM News Electronic Edition Volume 7, number 14 November 23, 1996 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =========== Abstracts =========== Myosin light chain kinase gene disruption in Dictyostelium: A role for MLCK-A in cytokinesis and evidence for multiple MLCKs Janet L. Smith, Linda A. Silveira, and James A. Spudich Proc. Natl. Acad. Sci. USA 93:12321-6 We have created a strain of Dictyostelium that is deficient for the Ca2+/calmodulin-independent myosin light chain kinase MLCK-A. This strain undergoes cytokinesis less efficiently than wild type, which results in an increased frequency of multinucleate cells when grown in suspension. The MLCK-A- cells are able, however, to undergo development and to cap cross-linked surface receptors, processes that require myosin heavy chain. Phosphorylated regulatory light chain (RLC) is still present in MLCK-A- cells, indicating that Dictyostelium has one or more additional protein kinases capable of phosphorylating RLC. Concanavalin A treatment was found to induce phosphorylation of essentially all of the RLC in wild-type cells, but RLC phosphorylation levels in MLCK-A- cells are unaffected by concanavalin A. Thus MLCK-A is regulated separately from the otehr MLCK(s) in the cell. ------------------------------------------------------------------------ A marker of terminal stalk cell terminal differentiation in Dictyostelium Vicky Robinson*1 and Jeffrey Williams+2 1 Imperial Cancer Research Fund, Clare Hall Laboratory, S. Mimms, Herts EN6 3LD. 2 MRC Laboratory of Molecular Cell Biology and Department of Biology, University College London, Gower St, London WC1E 6BT * Present Address, The National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AR +To whom correspondence should be addressed Tel 0171 380 7254 Fax 0171 380 7254 Differentiation, in press. We describe the isolation of staB a Dictyostelium gene that is selectively expressed in stalk cells. If an aggregate enters culmination immediately staB is expressed in the bottom half of the stalk and in the basal disc but if a migratory slug is formed it is also expressed in the top of the stalk and in a disc of cells that surmounts the spore head. The latter two populations of cells derive from the rear part of the prestalk region, the pstO cells, which would therefore seem to change their transcriptional competency during slug migration The staB promoter contains a distal region that is required for expression within both the stalk and the basal disc and a proximal region that is only required for expression within the basal disc. This uncoupling of transcriptional specificities suggests that the signalling mechanisms that direct terminal differentiation in these two tissues differ in some way. --------------------------------------------------------------------- Clathrin Heavy Chain is Required for Spore Cell but not Stalk Cell Differentiation in Dictyostelium discoideum Maria L. Niswonger and Theresa J. O'Halloran Department of Cell Biology Duke University Medical Center Durham, NC 27710 Development, in press Previous studies of a clathrin-minus Dictyostelium cell line revealed important roles for clathrin heavy chain (clathrin) in endocytosis, secretion of lysosomal hydrolases, and osmoregulation. In this paper, we examine the contribution of clathrin-mediated membrane traffic to development in Dictyostelium discoideum. Clathrin-minus cells were delayed in early development. When exposed to starvation conditions, clathrin-minus cells streamed and aggregated more slowly than wild-type cells. Although clathrin-minus cells displayed only 40% the level of extracellular cyclic AMP binding normally found in wild-type cells, they responded chemotactically to extracellular cyclic AMP. Clathrin-minus cells down-regulated cyclic AMP receptors, but only to half the extent of wild-type cells. We found that the extent of development of clathrin-minus cells was variable and influenced by environmental conditions. Although the mutant cells always progressed beyond the tipped mound stage, the final structure varied from a finger-like projection to a short, irregular fruiting body. Microscopic examination of these terminal structures revealed the presence of intact stalks but a complete absence of spores. Clathrin-minus cells expressed prestalk (ecmA and ecmB) and prespore (psA and cotB) genes normally, but were blocked in expression of the sporulation gene spiA. Using clathrin-minus cells that had been transformed with various promoter-lacZ reporter constructs, we saw only partial sorting of clathrin-minus prestalk and prespore cells. Even when mixed with wild-type cells, clathrin-minus cells failed to sort correctly and never constructed functional spores. These results suggest three roles for clathrin during Dictyostelium development. First, clathrin increases the efficiency of early development. Second, clathrin enables proper and efficient patterning of prestalk and prespore cells during culmination. Third, clathrin is essential for differentiation of mature spore cells. --------------------------------------------------------------------- A HYDRODYNAMIC MODEL FOR DICTYOSTELIUM DISCOIDEUM MOUND FORMATION Bakhtier Vasiev, Florian Siegert and Cornelis J.Weijer J. Theor. Biol. in press Co-ordinated cell movement is the major mechanism controlling the multicellular morphogenesis of the slime mould Dictyostelium discoideum. Single cells aggregate chemotactically in response to propagating waves of cAMP to form a multicellular organism. Here we present a model to describe the formation of these multicellular aggregates. Cell movement is modelled as the flow of a compressible flui d controlled by cAMP induced chemotactic forces, frictional and adhesion forces and internal pressure. The model can simulate the whole early process of development from isolated single cells, format ion of bifurcating aggregation streams and formation of a three-dimensional aggregate with a single set of parameters. Direct comparison of simulations with experimental images of successive aggregat ion stages show a striking agreement. The model can also mimic alternative modes of morphogenesis frequently observed after disturbance of cAMP signalling or cell motility by chemicals or mutations. --------------------------------------------------------------------- Characterisation of functional myosin motor fragments S. E. Kurzawa*, D. J. Manstein, and M. A. Geeves* *Max-Planck-Institut fur Molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany; Max-Planck-Institut fur Medizinische Forschung, Jahnstr. 29, 69120 Heidelberg, Germany, and National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. Biochemistry, in press. The transient kinetic properties of the recombinant myosin head fragments M761 and M781, that both lack the light chain binding domain and correspond to the first 761 and 781 residues of Dictyostelium discoideum myosin II, were compared with those of the subfragment 1-like fragment M864 and a shorter catalytic domain fragment M754. The properties of M761, M781 and M864 are almost identical in regard to nucleotide binding, nucleotide hydrolysis, actin binding and the interactions between actin and nucleotide binding sites. Only the rate of the hydrolysis step was significantly faster for M761 and the affinity of M781 for actin significantly weaker than for M864. This indicates that the light chain binding domain plays no major role in the biochemical behaviour of the myosin head. In contrast loss of the peptide between 754 and 761 produced several major changes in the property of M754 as documented previously [Woodward, S.K.A., Geeves, M.A., & Manstein, D.J. Biochemistry 34, 16056-16064]. We further show that C-terminal extension of M761 with one or two a-actinin repeats has little effect on the behaviour the protein. --------------------------------------------------------------------- Analysis of cell movement and signalling during ring formation in an activated G1 mutant of Dictyostelium discoideum that is defective in prestalk zone formation. Rietdorf, J., Siegert, F., Dharmawardhane, S., Firtel,R.A. and Weijer, C.J. Developmental Biology, in press Mound formation in the cellular slime mould Dictyostelium results from the chemotactic aggregation of competent cells.Periodic cAMP signals propagate as multi-armed spiral waves and co-ordinate the movement of the cells. In the lateaggregate stage the cells differentiate into prespore and several prestalk cell types. Prestalk cells sort out chemotacticallyto form the tip, which then controls all further development. The tip organises cell movement via a scroll wave that converts to planar waves in the prespore zone leading to rotational cell movement in the tip and periodic forward movement in the prespore zone. Expression of a activated G1 protein under its own promoter leads to a severely altered morphogenesis from the mound stage onwards. Instead of forming a tipped mound, the cells form a ring-shaped structure without tip. Wave propagation pattern and dynamics during aggregation and mound formation in the mutant are indistinguishable from the parental strain AX3. However at the time of tip formation the spiral waves that organise the late aggregate do not evolve in a scroll organising centre in the tip but transform into a circularely closed (twisted) scroll ring wave. This leads to the formation of a doughnut shaped aggregate. During further development, the doughnut increases in diameter and the twisted scroll wave converts into a train of planar waves, resulting in periodic rotational cell movement. Although biochemical consequences resulting from this mutation are still unclear, it must affect prestalk cell differentiation. The mutant produces the normal proportion of prespore cells but is unable to form functional prestalk cells, i.e. prestalk cells with an ability to sort out from the prespore cells and form a prestalk zone. Failure of sorting leads to an altered signal geometry that then directs ring formation. This mutant demonstrates the importance of prestalk cell sorting for the stabilisation of the scroll wave that organizes the centre in the tip. PLEASE NOTE: The full paper can be downloaded from our homepage, http://www.zi.biologie.uni-muenchen.de/zoologie/dicty/dicty.html --------------------------------------------------------------------- A mutation in the cAMP signaling pathway affects sexual development of Dictyostelium discoideum Hajime Shimizu, Takahiro Morio, Hideki D. Shimizu and Hideko Urushihara Institute of Biological Sciences, University of Tsukuba Tsukuba, Ibaraki 305 Japan Development, Growth & Differentiation. in press. Amoebae of cellular slime molds have two developmental modes, asexual fruiting body formation and sexual macrocyst formation. How developmental choice is made is an interesting subject of wide impor-tance. Light exposure and dry conditions are favorable for asexual de-velopment, while conditions of darkness and high humidity are so for sexual development. In Dictyostelium discoideum, the latter conditions enhance zygote formation which determines the fate of surrounding cells for sexual development. Here we describe a mutant (TMC1) defective in the post-fusion aggregation of cells during sexual development. This mutant is also aggregationless in asexual development, and the level of cAMP receptor is reduced. Correspondingly, a series of existing mutants with defects in cAMP signaling pathways showed the same sexual phenotype as TMC1. These results suggest that molecular mechanisms of development are shared by the two alternative developmental modes. --------------------------------------------------------------------- Editor's Note: The following abstract appeared in the last issue of the CSM-News, but some recipients have reported that the abstract was truncated. It is reproduced here for those who did not receive the entire abstract. Stimulation by DIF causes an increase of intracellular Ca2+in Dictyostelium discoideum M. Azhar1, P.K. Kennady2, G. Pande2 and Vidyanand Nanjundiah1* 1Developmental Biology and Genetics Laboratory, Indian Institute of Science, Bangalore-560012, India. 2Centre for Cellular and Molecular Biology, Hyderabad 5000 07, India. Exper. Cell Res. in press. Using fluorescence-activated cell sorting (FACS), we have studied the effect of the differentiation-inducing factor DIF on cellular Ca2+ in Dictyostelium discoideum. We have shown previously that freshly starved or post-aggregation amoebae are heterogenous with respect to the amounts of cellular Ca2+ that they contain; the L or 'low Ca2+' class exhibits a prespore tendency and the H or 'high Ca2+' class exhibits a prestalk tendency [Azhar et al., Exp Cell Res. 227, 344-351, 1996] Upon adding DIF there is an approximately two-fold increase in the relative fraction of amoebae falling in the H class within two minutes. A major part of the increase is caused by Ca2+ influx from the extracellular medium. Therefore a rise in the level of cellular Ca2+ is an early step in the signal transduction pathway following stimulation by DIF. Also, in parallel with cellular heterogeneity in respect of Ca2+ content, there is also a heterogeneity in the response to DIF. The main result of this study is that the two classes persist after stimulation with 10nM DIF-1 but the proportions of cells belonging to them change significantly. With t1.5 amoebae, the H ('high Ca2+') class increases in relative size from an average of 18.47% to 34.36% within 2 minutes. In the case of t9 amoebae, about 12.44% fall in the H class and DIF-1 application raises this to 23.07% (on average) after 2 minutes. The nearly two-fold elevation in the H:L ratio caused by DIF-1 remains stable until 15 minutes in all experiments (long-term monitoring was not attempted). Functional heterogeneity, indicating a future prestalk or prespore tendency, is a pervasive feature of the life cycle of D. discoideum. The response to DIF-1 likewise mirrors a qualitative pre-aggregative difference between the two classes of amoebae. --------------------------------------------------------------------- [End CSM News, volume 7, number 14]