Dicty News Electronic Edition Volume 14, number 12 June 12, 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" =================================== Postdoctoral Position Available =================================== Funding is available to support a Postdoctoral Fellow interested in an interdisciplinary approach to study the control of actin cytoskeleton in motile cells. Traditional biochemical and molecular genetic approaches will be combined with novel laser-optical equipment and real-time fluorescence microscopy. My laboratory is interested in how motile cells control actin cytoskeleton to exert forces. We have developed novel laser-optical instrumentation that can monitor subcellular mechanical properties in real-time. In addition to proof-of-principle studies with animal cell lines and reconstituted actin gels, we have new data measuring actin mechanics during phagocytosis by Dictyostelium discoideum. Forces peak during uptake and are very brief (~10s). Simultaneous molecular localization of GFP-tagged species with the mechanical forces should help resolve the temporal order of events. The Fellow will focus on the biological aspects of the study, but will also help develop and adapt existing techniques and technologies. Candidates should have a PhD in an area of Biochemistry, Cell or Molecular Biology and enthusiasm to explore new areas in a multidisciplinary approach. Starting salary will be determined based on experience and potential. Applicants should indicate their qualifications and interest in the project, a CV and contact information for individuals willing to support the application (names, addresses, email and phone). The position will be open later this Fall, and applicants should indicate their availability with a start date. Dr. Scot C. Kuo Dept. of Biomedical Engineering The Johns Hopkins University 720 Rutland Avenue, Ross 724 Baltimore, MD 21205 email: sckuo@bme.jhu.edu web: http://www.bme.jhu.edu/~skuo/ ============= Abstracts ============= Crossing the finish line of development: regulated secretion of Dictyostelium proteins Supriya Srinivasan, Hannah Alexander and Stephen Alexander Division of Biological Sciences, 303 Tucker Hall, University of Missouri, Columbia, MO 65211-7400, USA. Trends in Cell Biology 10: 215-219 (June 2000) SUMMARY The genesis of the spore coat of Dictyostelium represents an exquisite example of developmentally regulated protein secretion. The proteins that are destined to be assembled into the spore coat extracellular matrix are stored in unique prespore vesicles that are triggered to secrete their contents at terminal differentiation. The regulation of this process is unfolding with the identification of the individual proteins in these vesicles. ---------------------------------------------------------------------------- A coccoid bacterial parasite of Naegleria sp. ( Schizopyrenida, Vahlkampfiidae) inhibits cyst formation of its host but not transformation to the flagellate stage. Rolf Michel1*, Karl-Dieter Müller2, Bärbel Hauröder1, Lothar Zöller1 1Central Institute of the Federal Armed Forces Medical Services, D-56065 Koblenz/ Germany 2Instituf für Medizinische Mikrobiologie, Universität Essen, D-45147 Essen/ Germany ACTA PROTOZOOLOGICA, in press. Summary A non- thermophilic Naegleria species isolated from an aquarium for ornamental fishes harboured coccoid Gram-negative bacteria of 1µm in diameter as endocytobionts multiplying by binary fission. These intracytoplasmic bacteria, called "Knic" inhibited cyst formation of their host, but not its transformation to the flagellate stage. Consequently the resulting flagellates contained these intracellular parasites as could be verified by electron microscopy. The endocytic bacteria could not be grown on eight different bacteriological media under various atmospheric conditions. The ribbon-like roughly hexagonal surface of the bacteria as shown by SEM is responsible for the spiny appearence as seen by TEM. The successful axenisation of the infected host amoebae made it possible to harvest pure bacterial suspensions for cocultivation assays with different free living amoebae and Dictyostelum discoideum. As a result, an extremely wide host spectrum of the endocytobionts could be determined including other Naegleria spp., Acanthamoebae, members of the genera Hartmannella, Vahlkampfia, and Balamuthia. Successful infection of four additional Naegleria strains with the coccoid endocytobionts resulted also in impairment of the capabilty to form cysts but did not interfere with transformation to the flagellate stage. Dictyostelium discoideum as a member of the Acrasiales was also highly susceptible to infection with the Gram-negative bacteria inducing a strong disturbance of their normal aggregation pattern. ---------------------------------------------------------------------------- Microtubule Organization and the Effects of GFP-tubulin Expression in Dictyostelium discoideum. M. Kimble, C. Kuzmiak, K. N. McGovern, E.L. de Hostos. Department of Biology, University of South Florida, Tampa, FL 33620-5150. Department of Pathology, University of California, San Francisco, VAMC 113B, 4150 Clement St., San Francisco, CA 94121 Cell Motility & the Cytoskeleton, in press. Summary: We have labeled microtubules in living Dictyostelium amoebae by incorporation of a GFP-a-tubulin fusion protein. The GFP-a-tubulin incorporates into microtubules and, as reported by others (Neujahr et al., 1998), the labeled microtubules are highly motile. Electron microscopy (EM) analysis of the distribution and organization of microtubules in the amoebae shows that some cytoplasmic microtubules form close associations. These associations could allow motor proteins attached to one microtubule to walk along an adjacent microtubule and thus generate some of the observed motility. Protein blot analysis indicates that the GFP-a-tubulin incorporates into microtubules at a lower efficiency than does the endogenous a-tubulin. EM and immunofluorescence (IF) analyses suggest that the GFP-a-tubulin interferes with microtubule nucleation. We have also observed an increased sensitivity of the GFP- a-tubulin expressing cells to blue light, as compared to wild-type cells. These results suggest that although GFP-a-tubulin can be used as a marker for microtubules in living cells, the use of this marker is not recommended for certain types of studies such as assembly dynamics. ---------------------------------------------------------------------------- The internal phosphodiesterase RegA is essential for the suppression of lateral pseudopods during Dictyostelium chemotaxis Deborah Wessels, Hui Zhang, Joshua Reynolds, Karla Daniels, Paul Heid, Sijie Lu, Adam Kuspa, Gad Shaulsky, WIlliam F. Loomis and David R. Soll Molecular Biology of the Cell, in press Dictyostelium strains in which the gene encoding the cytoplasmic cAMP phosphodiesterase RegA is inactivated form small aggregates. This defect was corrected by introducing copies of the wild type regA gene, indicating that the defect was solely the consequence of the loss of the phosphodiesterase. Using a computer assisted motion analysis system, regA- mutant cells were found to show little sense of direction during aggregation. When labeled wild type cells were followed in a field of aggregating regA- cells, they also failed to move in an orderly direction, indicating that signaling was impaired in mutant cell cultures. However, when labeled regA- cells were followed in a field of aggregating wild type cells, they again failed to move in an orderly fashion, primarily in the deduced fronts of waves, indicating that the chemotactic response was also impaired. Since wild type cells must assess both the increasing spatial gradient and the increasing temporal gradient of cAMP in the front of a natural wave, the behavior of regA- cells was motion-analyzed first in simulated temporal waves in the absence of spatial gradients and then in spatial gradients in the absence of temporal waves. Our results demonstrate that RegA is neither involved in assessing the direction of a spatial gradient of cAMP nor in distinguishing between increasing and decreasing temporal gradients of cAMP. However, RegA is essential for specifically suppressing lateral pseudopod formation during the response to an increasing temporal gradient of cAMP, a necessary component of natural chemotaxis. We discuss the possibility that RegA functions in a network that regulates myosin phosphorylation by controlling internal cAMP levels, and in support of that hypothesis demonstrate that myosin II does not localize in a normal fashion to the cortex of regA- cells in an increasing temporal gradient of cAMP. ---------------------------------------------------------------------------- [End Dicty News, volume 14, number 12]