dictyNews Electronic Edition Volume 31, number 6 August 8, 2008 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. Upon publication of your paper, please send strains and plamids to the Dicty Stock Center. For more information see http://dictybase.org/StockCenter/Deposit.html. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ========= Abstracts ========= Correlated waves of actin filaments and PIP3 in Dictyostelium cells Yukako Asano, Akira Nagasaki, and Taro Q. P. Uyeda National Institute of Advanced Industrial Science and Technology (AIST) Cell Motility and the Cytoskeleton, in press Chemotaxis-deficient amiB-null mutant Dictyostelium cells show two distinct movements: 1) they extend protrusions randomly without net displacements; 2) they migrate persistently and unidirectionally in a keratocyte-like manner.   Here, we monitored the intracellular distribution of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to gain insight into roles PIP3 plays in those spontaneous motilities.  In keratocyte-like cells, PIP3 showed convex distribution over the basal membrane, with no anterior enrichment.   In stalled cells, as well as in wild type cells, PIP3 repeated wave-like changes, including emergence, expansion and disappearance, on the basal membrane.  The waves induced lamellipodia when they approached the cell edge, and the advancing speed of the waves was comparable to the migration speed of the keratocyte-like cells.  LY294002, an inhibitor of PI3 kinase, abolished PIP3 waves in stalled cells and stopped keratocyte-like cells.   These results together suggested that keratocyte-like cells are 'surfing' on the PIP3 waves by coupling steady lamellipodial protrusions to the PIP3 waves.  Simultaneous live observation of actin filaments and PIP3 in wild type or stalled amiB- cells indicated that the PIP3 waves were correlated with wave-like distributions of actin filaments.  Most notably, PIP3 waves often followed actin waves, suggesting that PIP3 induces local depolymerization of actin filaments.  Consistent with this idea, cortical accumulation of PIP3 was often correlated with local retraction of the periphery.  We propose that the waves of PIP3 and actin filaments are loosely coupled with each other and play important roles in generating spontaneous cell polarity. Submitted by: Taro Uyeda [t-uyeda@aist.go.jp] -------------------------------------------------------------------------------- Chemotaxis-mediated scission contributes to efficient cytokinesis in Dictyostelium. Akira Nagasaki, and Taro Q. P. Uyeda National Institute of Advanced Industrial Science and Technology (AIST) Cell Motility and the Cytoskeleton, in press Interphase amoeba of Entamoeba invadens are attracted to the furrowing region of a neighboring dividing cell to assist with the division.  A seemingly similar behavior has been observed in Dictyostelium discoideum, but in this case, it has not been shown whether the movements were truly directed toward the furrowing region or whether they have any relevance.   We thus used myosin II-null cells, which spend more time than wild type cells in cytokinesis, and successfully demonstrated that nearly half of the division events involve the attraction of a neighbor cell to the furrowing region.  Cells lacking the beta subunit of the trimeric G protein (Gbeta), which are incapable of chemotaxis, did not show such midwifery.  Culturing wild type cells flattened under agarose sheets also slowed the cytokinesis process, and this allowed us to demonstrate that phosphatidylinositol trisphosphate was enriched in the anterior region of midwifing cells, consistent with the view that midwifery in D. discoideum is also chemotaxis.   On substrates, while only 3.6% of wild type cells were multinucleate, 9% of Gbeta-null cells were multinucleate, and this was reduced to 3.4% when they were surrounded by wild type cells.  Conversely, multinucleated wild type cells increased to 6.8% when they were surrounded by Gbeta-null cells.   Thus, Gbeta-null cells frequently fail to divide because they cannot assist each other’s division and midwifery ensures successful cytokinesis in Dictyostelium discoideum. Submitted by: Taro Uyeda [t-uyeda@aist.go.jp] -------------------------------------------------------------------------------- Cell adhesion molecules regulate contractile ring-independent cytokinesis in Dictyostelium discoideum. Akira Nagasaki, Masamitsu Kanada, and Taro Q. P. Uyeda National Institute of Advanced Industrial Science and Technology (AIST) Cell Research, in press To investigate the roles of substrate adhesion in cytokinesis, we established  cell lines lacking paxillin (PAXB) or vinculin (VINA), and those expressing  the respective GFP fusion proteins in Dictyostelium discoideum.  As in  mammalian cells, GFP-PAXB and GFP-VINA formed focal adhesion-like  complexes on the cell bottom.  paxB- cells in suspension grew normally,  but on substrates, often failed to divide after regression of the furrow.  The  efficient cytokinesis of paxB- cells in suspension is not due to shear  forces to assist abscission, since they divided normally in static suspension  culture as well.  Double knockout strains lacking mhcA, which codes for  myosin II, and paxB or vinA displayed cytokinetic defects more severe than  each single knockout strain.  In mitotic wild-type cells, GFP-PAXB was  diffusely distributed on the basal membrane, but was strikingly condensed  along the polar edges in mitotic mhcA- cells.  These results are consistent  with our idea that Dictyostelium expresses two forms of cytokinesis, one  that is contractile ring-dependent and adhesion-independent, and the other  that is contractile ring-independent and adhesion-dependent, and that the  latter requires PAXB and VINA.  Furthermore, that paxB- cells fail to divide  more frequently in the presence of substrate adhesion suggests additional  signaling roles of this adhesion molecule.  Submitted by: Taro Uyeda [t-uyeda@aist.go.jp] -------------------------------------------------------------------------------- PI3-kinase signaling contributes to orientation in shallow gradients and enhances speed in steep chemoattractant gradients   Leonard Bosgraaf, Ineke Keizer-Gunnink and Peter J.M. Van Haastert J. Cell Sci, in  press Dictyostelium cells that chemotax toward cAMP produce phosphatidylinositol (3,4,5) trisphosphate (PIP3) at the leading edge, which has been implicated in actin reorganization and pseudopod extension. However, in the absence of PIP3 signaling cells will chemotax via alternative pathways. Here we examined the potential contribution of PIP3 to chemotaxis of wild type cells. The results show that steep cAMP gradients (larger than 10% concentration difference across the cell) induce strong PIP3 patches at the leading edge, which has little effect on the orientation but strongly enhances the speed of the cell. Using a new sensitive method for PIP3 detection that corrects for the volume of cytosol in pixels at the boundary of the cell, we show that in shallow cAMP gradient (less than 5% concentration difference across the cell) PIP3 is still somewhat enriched at the leading edge. Cells lacking PI3-kinase activity exhibit poor chemotaxis in these shallow gradients. Due to the reduced speed and orientation in steep and shallow gradients, respectively, cells lacking PIP3 signaling require 2 to 6 fold longer times to reach a point source of chemoattractant compared to wild type cells. These results show that although PI3K signaling is dispensable for chemotaxis, it gives the wild type an advantage over mutant cells. Submitted by: Peter Van Haastert [P.J.M.van.Haastert@rug.nl] -------------------------------------------------------------------------------- Sorting of the v-SNARE VAMP7 in Dictyostelium discoideum: a role for more than one Adaptor Protein (AP) complex. Nelly Bennett a*, François Letourneur b, Michel Ragno a and Mathilde Louwagie c. a Commissariat a l’Energie Atomique, iRTSV, Laboratoire de Biochimie et Biophysique des Systemes Integres, CNRS UMR5092, Universite Joseph Fourier, 38054 Grenoble, France. b Institut de Biologie et Chimie des Proteines, UMR5086-CNRS/Universite Lyon I, IFR 128 BioSciences Lyon-Gerland, 69367 Lyon, France. c Commissariat a l’Energie Atomique, iRTSV, Laboratoire d’Etude de la Dynamique des Proteomes, INSERM U880, Universite Joseph Fourier, 38054 Grenoble, France. * Corresponding author: nelly.bennett@cea.fr Experimental Cell Research, in press Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptors (SNAREs) participate in the specificity of membrane fusions in the cell. The mechanisms of specific SNARE sorting are still however poorly documented. We investigated the possible role of Adaptor Protein (AP) complexes in sorting of the Dictyostelium discoideum v-SNARE VAMP7. In live cells, GFP-VAMP7 is observed in the membrane of endocytic compartments. It is also observed in the plasma membrane of a small proportion of the cells. Mutation of a potential dileucine motif dramatically increases the proportion of cells with GFP-VAMP7 in their plasma membrane, strongly supporting the participation of an AP complex in VAMP7 sorting to the endocytic pathway. A partial increase occurs in knockout cells for the medium subunits of AP-2 and AP-3 complexes, indicating a role for both AP-2 and AP-3. VAMP7, as well as its t-SNAREs partners syntaxin 8 and Vti1, are co-immunoprecipitated with each of the medium subunits of the AP-1, AP-2, AP-3 and AP-4 complexes. This result supports the conclusion that VAMP7 directly interacts with both AP-2 and AP-3. It also raises the hypothesis of an interaction with AP-1 and AP-4. GFP-VAMP7 is retrieved from the endocytic pathway at and/or before the late post-lysosomal stage through an AP-independent mechanism. Submitted by: Nelly Bennett [nelly.bennett@cea.fr] ============================================================== [End dictyNews, volume 31, number 6]