Dicty News Electronic Edition Volume 14, number 10 April 29, 2000 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" ============= Abstracts ============= Rac1 GTPases Control Filopodia Formation, Cell Motility, Endocytosis, Cytokinesis, and Development in Dictyostelium Michel Dumontier, Petra Höcht, Ursula Mintert, and Jan Faix JCS, in press. SUMMARY The function of the highly homologous Rac1A, Rac1B, and Rac1C GTPases of the Dictyostelium Rac1 group was investigated. All three GTPases bound with an equal capacity to the IQGAP-related protein DGAP1, with a preference for the activated GTP-bound form. Strong overexpression of wild-type Rac1 GTPases N-terminally tagged with green fluorescent protein (GFP), predominantly induced the formation of numerous long filopodia. Remarkably, expression of the constitutively-activated GTPases resulted in dominant-negative phenotypes: these Rac1-V12 mutants completely lacked filopodia but formed numerous crown shaped structures resembling micropinosomes. Moreover, these mutants were severely impaired in cell motility, colony growth, phagocytosis, pinocytosis, cytokinesis, and development. Transformants expressing constitutively-inactivated Rac1-N17 proteins were similar to wild-type cells, but displayed abundant and short filopodia and exhibited a moderate defect in cytokinesis. Taken together, our results indicate that the three GTPases play an identical role in signaling pathways and are key regulators of cellular activities that depend on the re-organization of the actin cytoskeleton in Dictyostelium. ---------------------------------------------------------------------------- Two-step positioning of a cleavage furrow by cortexillin and myosin II Igor Weber, Ralph Neujahr, Aiping Du, Jana Koehler, Jan Faix and Guenther Gerisch Max-Planck-Institut fuer Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany Current Biology, in press. Background: The conventional myosin II is dispensable for mitotic division in Dictyostelium if the cells are attached to a substrate, but is required in suspension. Only a small fraction of attached myosin II-null cells fails to divide. Cortexillins are actin-bundling proteins important for the formation of a regular cleavage furrow, even in attached cells. Cortexillins translocate to the midzone of mitotic cells independent of their contact to a substrate. The cooperation of myosin II and cortexillin I in determining the position of a cleavage furrow is subject of the paper. Results: The fraction of attached myosin II-null cells that fail to divide is analysed using GFP-cortexillin I as a marker for priming of a cleavage furrow. We show that positioning of a cleavage furrow is split into two steps. In a myosin II- and substrate-independent process, cortexillin I delineates a zone around the equatorial region of the cell where a cleavage furrow can be formed. Myosin II reliably focusses the cleavage furrow to the middle of this cortexillin I zone. If asymmetric cleavage in the absence of myosin II partitions a cell into a binucleate and an anucleate portion, cell-surface ruffles are induced along the cleavage furrow, which lead to movement of the anucleate portion along the connecting strand towards the binucleate one. Conclusions: In myosin II-null cells, cleavage furrow positioning occurs in two steps: priming of the furrow region and actual cleavage, which may proceed in the middle or at one border of the cortexillin ring. A control mechanism acting at late cytokinesis prevents cell division into an anucleate and a binucleate portion, causing a displaced furrow to regress if it becomes aberrantly located on top of polar microtubule asters. ---------------------------------------------------------------------------- Discrete Interactions in Cell Adhesion Measured by Single-Molecule Force Spectroscopy Martin Benoit, Daniela Gabriel*, Guenther Gerisch* & Hermann E. Gaub LMU, Sektion Physik, Amalienstr 54, D-80799 Muenchen, Germany * MPI fuer Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany Nature Cell Biology, in press. Cell-to-cell adhesion mediated by specific cell-surface molecules is essential for multicellular development. We controlled the interaction between single cells and combined single-molecule force spectroscopy with genetic manipulation in order to measure de-adhesion forces at the resolution of individual cell-adhesion molecules. The measurements were focussed on contact site A as a prototype of cell adhesion proteins. The csA glycoprotein is specifically expressed in aggregating cells of Dictyostelium discoideum, which are engaged in the process of building up a multicellular organism. De-adhesion between two adjacent cell surfaces were found to be composed of discrete interactions characterized by an unbinding force of 23 ±8 pN measured at a rupture rate of 2.5 ±0.5 µm/sec. ---------------------------------------------------------------------------- [End Dicty News, volume 14, number 10]