Dicty News Electronic Edition Volume 16, number 5 March 3, 2001 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dictybase.org" ============== Abstracts ============== Dictyostelium Cells' Cytoplasm as Active Viscoplastic Body Wolfgang Feneberg*, Monika Westphal§ and Erich Sackmann* * Technische Universitaet Muenchen, Department of Physics E22, James-Franck-Strasse, D-85748 Garching, Germany § Max-Planck-Institut fuer Biochemie, Research Group of Cell Dynamics, D-82152 Martinsried, Germany European Biophysics Journal, in press. Abstract: We applied a recently developed microrheology technique based on colloidal magnetic tweezers to measure local viscoelastic moduli and active forces in cells of dictyostelium discoideum. The active transport of nonmagnetic beads taken up by phagocytosis was analyzed by single particle tracking which allowed to measure the length of straight steps and the corresponding velocities of the movements. The motion consists of a superposition of nearly straight long-range steps (step length in the micrometer range) and local random walks (step widths about 0.1 micro m). The velocities for the former type of motion range from 1 to 3 micro m/s. They decrease with increasing bead size and are attributed to rapid active transport along microtubuli. The short-range local motions exhibit velocities of less than 0.5mm/s and reflect the internal dynamics of the cytoplasm. Viscoelastic response curves were measured by application of force pulses with amplitudes varying between 50pN and 400pN. Analysis of the response curves in terms of mechanical equivalent circuits yielded cytoplasmic viscosities varying between 10 and 350PaŚs. Simultaneous analysis of the response curves and of the bead trajectories showed that the motion of the beads is determined by the local yield stress within the cytoplasmic scaffold and cisternae, which varies between sigma = 30PaŚm and 250PaŚm. The motion of intra-cellular particles is interpreted in terms of visco-plastic behaviour and the apparent viscosity is a measure for the reciprocal rate of bond-breakage within the cytoplasmatic network. The viscoelastic moduli are interpreted as dynamic quantities which depend sensitively on the amplitude of the forces and the rate of bond-breakage is determined by the Arrhenius-Kramers law with the activation energy being reduced by the work performed by the applied force. In agreement with previous work we provide evidence that the myosin II deficient cells exhibit higher yield stresses suggesting that the function of myosin II as cross-linker is taken over by the other (non-active) cross-linkers. ----------------------------------------------------------------------------- Tyrosine phosphorylation-independent nuclear translocation of a Dictyostelium STAT in response to DIF signalling *Masashi Fukuzawa, *Tsuyoshi Araki, Iris Adrian and Jeffrey G. Williams+ School of Life Sciences, University of Dundee, MSI/WTB Complex, Dow Street, Dundee DD1 5EH UK + Corresponding author (j.g.williams@dundee.ac.uk) *These two authors contributed equally to this work Molecular Cell, in press SUMMARY We describe a Dictyostelium STAT, Dd-STATc, that regulates the speed of early development and the timing of terminal differentiation. Dd-STATc also functions as a repressor, that directs graded expression of the ecmA gene in different prestalk cell populations. Developing Dictyostelium cells produce a chlorinated hexaphenone, DIF, that directs prestalk cell differentiation. Dd-STATc is tyrosine phosphorylated, dimerises and translocates to the nucleus when cells are exposed to DIF. Surprisingly however, SH2 domain-phosphotyrosine interaction is not necessary for the DIF-induced nuclear translocation of Dd-STATc. In this respect Dd-STATc activation resembles several recently described, non-canonical mammalian STAT signalling processes. We show instead that DIF mediates nuclear translocation via sequences located in the divergent, N-terminal half of the Dd-STATc molecule. ----------------------------------------------------------------------------- Receptor Mediated Activation of Heterotrimeric G-proteins in Living Cells Chris Janetopoulos*, Tian Jin*, and Peter Devreotes*+ Department of Biological Chemistry Johns Hopkins Medical Institutions Baltimore, MD 21205 *Present address: Department of Cell Biology and Anatomy, Johns Hopkins Medical Institutions, Baltimore, MD 21205 Science, In Press Abstract Receptor mediated activation of heterotrimeric guanine nucleotide binding proteins (G-proteins) was visualized in living D. discoideum cells by monitoring fluorescence resonance energy transfer (FRET) between a- and b- subunits fused to cyan and yellow fluorescent proteins. The G-protein heterotrimer rapidly dissociated and reassociated upon addition and removal of chemoattractant. During continuous stimulation G-protein activation reached a dose-dependent steady state level. Even though physiological responses subsided, the activation did not decline. Thus, adaptation occurs at another point in the signaling pathway and occupied receptors, whether or not phosphorylated, catalyze the G-protein cycle. Construction of similar energy transfer pairs of mammalian G-proteins should enable direct in situ mechanistic studies and applications such as drug screening and searching for ligands for new G-protein coupled receptors. ----------------------------------------------------------------------------- [End Dicty News, volume 16, number 5]