dictyNews Electronic Edition Volume 28, number 14 May 25, 2007 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 dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ========= Abstracts ========= Note: The Xu et al. abstract from last week's dictyNews had an error in the journal. The corrected version is below. Locally controlled inhibitory mechanisms are involved in eukaryotic GPCR-mediated chemosensing Xuehua Xu1, Martin Meier-Schellersheim2, Jianshe Yan1 and Tian Jin1* J. Cell Biology, in press GPCR signaling mediates a balance of excitatory and inhibitory activities that regulate Dictyostelium chemosensing to cAMP. The molecular nature and kinetics of these inhibitors are unknown. We report that transient cAMP stimulations induce PIP3 responses without a refractory period, suggesting that GPCR-mediated inhibition accumulates and decays slowly. Moreover, exposure to cAMP gradients leads to asymmetric distribution of the inhibitory components. The gradients induce a stable accumulation of the PIP3 reporter PHCrac-GFP in the front of cells near the cAMP source. Rapid withdrawal of the gradient led to the re-association of G-protein subunits, and the return of the PIP3 phosphatase PTEN and PHCrac-GFP to their pre-stimulus distribution. Reapplication of cAMP stimulation produces a clear PHCrac-GFP translocation to the back but not to the front, indicating that a stronger inhibition is maintained in the front of a polarized cell. Our study demonstrates a novel spatiotemporal feature of currently unknown inhibitory mechanisms acting locally on the PI3K activation pathway. Submitted by: Tian Jin [TJIN@niaid.nih.gov] -------------------------------------------------------------------------------- The Real Factor for Polypeptide Elongation in Dictyostelium Cells is EF-2B, not EF-2A Tomoko Yoshino, Yasuo Maeda and Aiko Amagai Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan BBRC, in press Polypeptide elongation factor 2 (EF-2) plays an essential role in protein synthesis and is believed to be indispensable for cell proliferation. Recently, it has been demonstrated that there are two kinds of EF-2 (EF-2A and EF-2B with 76.6% of sequence identity at the amino acid level) in Dictyostelium discoideum. Although the knockout of EF-2A slightly impaired cytokinesis, EF-2A null cells exhibited almost normal protein synthesis and cell growth, suggesting that there is another molecule capable of compensating for EF-2 function. Since EF-2B is the most likely candidate, we examined its function using ef-2b knockdown cells prepared by the RNAi method. Our results strongly suggest that EF-2B is required for protein synthesis and cell proliferation, functioning as the real EF-2. Interestingly, the expressions of ef-2a and ef-2b mRNAs during development are reversely regulated, and the ef-2b expression is greatly augmented in ef-2a null cells. Submitted by: Aiko Amagai [aiamagai@mail.tains.tohoku.ac.jp] -------------------------------------------------------------------------------- PTEN Plays a Role in the Suppression of Lateral Pseudopod FormationÊ During Dictyostelium Motility and Chemotaxis Deborah Wessels, Daniel F. Lusche, Spencer Kuhl, Paul Heid andÊ David R. Soll Journal of Cell Science, in press It has been suggested that the PIP3 phosphatase and tensin homolog, PTEN, plays a fundamental role in Dictyostelium discoideum chemotaxis. To identify that role, the behavior of a pten- mutant was quantitatively analyzed using 2D and 3D computer-assisted methods. pten- cells were capable of polarizing and translocating in the absence of attractant, and sensing and responding to spatial gradients, temporal gradients and natural waves of attractant. However, all of these responses were compromised (i.e., less efficient) due to the fundamental incapacity of pten- cells to suppress lateral pseudopod formation and turning. This defect was equally manifested in the absence as well as presence of attractant. PTEN, which is constitutively localized in the cortex of polarized cells, was found essential for the attractant-stimulated increase in cortical myosin II and F-actin that is responsible for the increased suppression of pseudopods during chemotaxis. PTEN, therefore, plays a fundamental role in the suppression of lateral pseudopod formation, a process essential for the efficiency of locomotion and chemotaxis, but not in directional sensing. Submitted by: Deb Wessels [deborah-wessels@uiowa.edu] -------------------------------------------------------------------------------- A vesicle surface tyrosine kinase regulates phagosome maturation Jun Fangà, Joseph A. Brzostowskià, Stephen Ou, Nilgun Isik, Vinod Nair,^ and Tian Jin* Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, NIH, Twinbrook II Facility, 12441 Parklawn Drive, Rockville, Maryland 20852, USA ^ Research Technologies Section/RTB Rocky Mountain Laboratories/NIAID/NIH 903 South 4th Street, Hamilton, MT 59840 J. Cell Biology, in press Phagocytosis is crucial for host defense against microbial pathogens and for obtaining nutrients in Dictyostelium discoideum. Phagocytosed particles are delivered via a complex route from phagosomes to lysosomes for degradation, but the molecular mechanisms involved in the phagosome maturation process are not well understood. Here, we identify a novel vesicle associated receptor tyrosine kinase-like protein, VSK3, in D. discoideum. We demonstrate how VSK3 is involved in phagosome maturation. VSK3 resides on the membrane of late endosomes/lysosomes with its C-terminal kinase domain facing the cytoplasm. Inactivation of VSK3 by gene disruption reduces the rate of phagocytosis in cells, which is rescued by re-expression of VSK3. We found that the in vivo function of VSK3 depends on the presence of the kinase domain and vesicle localization. Furthermore, VSK3 is not essential for engulfment, but instead, is required for the fusion of phagosomes with late endosomes/lysosomes. Our findings suggest that localized tyrosine kinase signaling on the surface of endosome/lysosomes represents a control mechanism for phagosome maturation. Submitted by: Tian Jin [TJIN@niaid.nih.gov] ============================================================== [End dictyNews, volume 28, number 14]