Dicty News Electronic Edition Volume 23, number 16 November 12, 2004 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Gamete fusion and cytokinesis preceding zygote establishment in the sexual process of Dictyostelium discoideum Kentaro Ishida, Toshihiro Hata, and Hideko Urushihara Develop.Growth & Differ. in press Cells of Dictyostelium discoideum become sexually mature under submerged and dark conditions, and fuse with opposite mating-type cells to form zygote giant cells, which gather surrounding cells and finally develop into dormant structures called macrocysts. In the present study, we found that the multinuclear fused cells formed during this process frequently underwent cytokinesis driven by random local movements. The split cells were capable of re-fusion, and repeated fission. These radical behaviors continued until the extensive cell aggregation started around the giant cells. Thus, gamete fusion and initiation of zygote development do not coincide in the mating of D. discoideum. Analyses by confocal microscopy and flow cytometry indicated that the cessation of the random movement followed pronuclear fusion, and that microtubule organizing centers (MTOCs), abundant in the fused cells at the beginning, gradually decreased and only one of them remained within the developed macrocyst. Some of the genes known to control cell movement, such as rasGEFB and rasS, increased shortly before the cessation of repeated fusion-fission and initiation of phagocytosis. These results suggest that the sequential molecular events are necessary in D. discoideum after gamete fusion to establish a new individuality of zygotes. Submitted by: Hideko Urushihara [hideko@biol.tsukuba.ac.jp] ----------------------------------------------------------------------------- Quantitative imaging of single live cells reveals spatiotemporal dynamics of multi-step signaling events of chemoattractant gradient sensing in Dictyostelium Xuehua Xu1, Martin Meier-Schellersheim2, Xuanmao Jiao1, Lauren E. Nelson1, and Tian Jin1* Laboratory of Immunogenetics1, Laboratory of Immunology2, National Institute of Allergy and Infectious Diseases, NIH Twinbrook II Facility, 12441 Parklawn Drive, Rockville, Maryland 20852, USA *Corresponding author Phone: 301-480-1430 Email: tjin@niaid.nih.gov Molecular biology of the cell, in press. Activation of G-protein-coupled chemoattractant receptors triggers dissociation of G alpha and G betagamma subunits. These subunits induce intracellular responses that can be highly polarized when a cell experiences a gradient of chemoattractant. Exactly how a cell achieves this amplified signal polarization is still not well understood. Here, we quantitatively measure temporal and spatial changes of receptor occupancy, G-protein activation by FRET imaging, and PIP3 levels by monitoring the dynamics of PHCrac-GFP translocation in single living cells in response to different chemoattractant fields. Our results provided the first direct evidence that G-proteins are activated to different extents on the cell surface in response to asymmetrical stimulations. A stronger, uniformly applied stimulation triggers not only a stronger G-protein activation but also a faster adaptation of downstream responses. When na•ve cells (which have not experienced chemoattractant) were abruptly exposed to stable cAMP gradients, G-proteins were persistently activated throughout the entire cell surface, whereas the response of PHCrac-GFP translocation surprisingly consisted of two phases, an initial transient and asymmetrical translocation around the cell membrane, followed by a second phase producing a highly polarized distribution of PHCrac-GFP. We propose a revised model of gradient sensing, suggesting an important role for locally controlled components that inhibit PI3Kinase activity. Submitted by: [Xuehua Xu