Dicty News Electronic Edition Volume 23, number 10 September 24, 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 ============= GFP-golvesin constructs to study Golgi tubulation and post-Golgi vesicle dynamics in phagocytosis Guenther Gerisch , Aleksander Benjak 1a, Jana Koehler, Igor Weberb, and Natalie Schneiderc Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany a Present address: EMBL Heidelberg, Meyerhofstr. 1, D-69117 Heidelberg, Germany b Present address: Rudjer Boskovic Institute, Bijenicka cesta 54, 10000 Zagreb, Croatia c Present address: Kyoto University Biosimulation Center, Kyoto Research Park, Bldg. #4, 8F, Chudoji Awata-cho 93, Shimogyoku, Kyoto, Japan 600-8815 European Journal of Cell Biology, in press Dictyostelium cells are professional phagocytes that are optimally suited for the imaging of phagosome processing from particle uptake to exocytosis. In order to design fluorescent probes for monitoring membrane trafficking in the endocytic pathway, we have dissected a membrane protein, golvesin, and have linked fragments of its sequence to GFP. Endogenous golvesin is partitioned between the ER, the Golgi apparatus, endosomes, and the contractile vacuole complex. We have localized signals that are required for exit from the Golgi to post-Golgi compartments to the C-terminal region of the golvesin sequence. One GFP-tagged fragment turned out to be a highly specific Golgi marker and was used to demonstrate the interaction of Golgi tubules with phagosomes. Signals essential for the retrieval of golvesin at the end of phagosome processing were localized to the N-terminal region. A truncated golvesin construct escaping retrieval was employed in recording the delivery of a phagosomal protein to the plasma membrane. Applying this construct to a phagosome filled with multiple particles, we observed that the phagosome is segmented during exocytosis, meaning that sequential release of particles alternates with membrane fusion. Submitted by: Guenther Gerisch [gerisch@biochem.mpg.de] ----------------------------------------------------------------------------- Mobile actin clusters and traveling waves in cells recovering from actin depolymerization Guenther Gerisch 1, Till Bretschneider 1, Annette Mueller-Taubenberger 1, Evelyn Simmeth 1, Mary Ecke 1, Stefan Diez 2, and Kurt Anderson 2 1 Max-Planck-Institut fuer Biochemie, D-82152 Martinsried; 2 Max-Planck-Institut fuerÊmolekulare Zellbiologie und Genetik, Pfotenhauer Str. 108, D-01307 Dresden; Biophysical Journal, in press At the leading edge of a motile cell, actin polymerizes in close apposition to the plasma membrane. Here we ask how the machinery for force generation at a leading edge is established de-novo after the global depolymerization of actin. The depolymerization is accomplished by latrunculin A, and the re-organization of actin upon removal of the drug is visualized in Dictyostelium cells by total internal reflection fluorescence (TIRF) microscopy. The actin filament system is reorganized in three steps. First, F-actin assembles into globular complexes that move along the bottom surface of the cells at velocities up to 10 µm per minute. These clusters are transient structures that eventually disassemble, fuse or divide. In a second step, clusters merge into a contiguous zone at the cell border that spreads and gives rise to actin waves traveling on a planar membrane. Finally, normal cell shape and motility are resumed. These data show that the initiation of actin polymerization is separated in Dictyostelium from front protrusion, and that the coupling of polymerization to protrusion is a later step in the reconstitution of a leading edge. Submitted by: Guenther Gerisch [gerisch@biochem.mpg.de] ----------------------------------------------------------------------------- Chemotaxis of aggregating Dictyostelium cells Guenther Gerisch, and Mary Ecke Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany In: Key Experiments in Practical Developmental Biology (Eds. Manuel Mar’-Beffa and Jennifer Knight) Cambridge University Press, in press Objective of the Experiment In the course of Dictyostelium development, a multicellular organism is established by the aggregation of single cells. In the following experiment, the chemoattractant that guides cell movement in an aggregation field is replaced by treating cells with cyclic AMP diffusing out of a micropipette. By the use of a micropipette that is easily moved by a micromanipulator, the direction of diffusion gradients can be changed fast enough to study the response of the cells within the first few seconds of reorientation. Submitted by: Guenther Gerisch [gerisch@biochem.mpg.de] ----------------------------------------------------------------------------- GUANYLYL CYCLASES ACROSS THE TREE OF LIFE Pauline Schaap School of Life Sciences, University of Dundee, UK Frontiers in Bioscience, in press TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Animals 3.1. The cyclase catalytic domain 3.2. Vertebrate cGMP signaling, a brief survey 3.3. Invertebrate guanylyl cyclases 4. Fungi 5. Dictyostelids 6. Plants 6.1. Flowering plants 6.2. Chlorophyte green algae 7. Alveolates 8. Discicristates 9. Prokaryotes 10. Summary and perspective 11. Acknowledgement 12. References 1. Abstract This review explores the origins, diversity and functions of guanylyl cyclases in cellular organisms. In eukaryotes both cGMP and cAMP are produced by the conserved class III cyclase domains, while prokaryotes use five more unrelated catalysts for cyclic nucleotide synthesis. The class III domain is found embedded in proteins with a large variety of membrane topologies and other functional domains, but the vertebrate guanylyl cyclases take only two forms, the receptor guanylyl cyclases with single transmembrane domain and the soluble enzymes with heme binding domain. The invertebrates additionally show a soluble guanylyl cyclase that cannot bind heme, while the more basal metazoans may lack the heme binding enzymes altogether. Fungi, the closest relatives of the metazoans, completely lack guanylyl cyclases, but they appear again in the Dictyostelids, the next relative in line. Remarkably, the two Dictyostelid guanylyl cyclases have little in common with the vertebrate enzymes. There is a soluble guanylyl cyclase, which shows greatest sequence and structural similarity to the vertebrate soluble adenylyl cyclase, and a membrane-bound form with the same configuration as the dodecahelical adenylyl cyclases of vertebrates. There is a difference, the pseudosymmetric C1 and C2 catalytic domains have swapped position in the Dictyostelium enzyme. Unlike the vertebrate guanylyl cyclases, the Dictyostelium enzymes are activated by heterotrimeric G-proteins. Swapped C1 and C2 domains are also found in the structurally similar guanylyl cyclases of ciliates and apicomplexans, but these enzymes additionally harbour an amino-terminal ATPase module with ten transmembrane domains. G-protein regulation could not be demonstrated for these enzymes. Higher plants lack class III cyclase domains, but an unexplored wealth of guanylyl cyclases is present in the green alga Chlamydomonas. Progenitors of all structural variants of the eukaryote guanylyl cyclases are found among the prokaryote adenylyl cyclases. This and the close similarity of many guanylyl cyclases to adenylyl cyclases suggests a paraphyletic origin for the eukaryote enzymes with multiple events of conversion of substrate specificity. Submitted by: Pauline Schaap [p.schaap@dundee.ac.uk] ----------------------------------------------------------------------------- A Rapid and Efficient Method to Generate Multiple Gene Disruptions in Dictyostelium Using a Single Selectable Marker and the Cre-loxP System Jan Faix ^ ", Lisa Kreppel * ` ", Gad Shaulsky +, Michael Schleicher ^, and Alan R. Kimmel * * Laboratory of Cellular and Developmental Biology, NIDDK National Institutes of Health, Bethesda, MD 20892-8028, USA ^ A. Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-UniversitŠt 80336 MŸnchen, Germany + Department of Molecular and Human Genetics Baylor College of Medicine, Houston, TX 77030, USA Nucelic Acids Research, in press Dictyostelium has proven an exceptionally powerful system for studying numerous aspects of cellular and developmental functions. The relatively small (~34 Mb) chromosomal genome of Dictyostelium and high efficiency of targeted gene disruption have enabled researchers to characterize many specific gene functions. However, the number of selectable markers in Dictyostelium is restricted, as is the ability to perform effective genetic crosses between strains. Thus, it has been difficult to create multiple mutations within an individual cell to study epistatic relationships among genes or potential redundancies between various pathways. We now describe a robust system for the production of multiple gene mutations in Dictyostelium by recycling a single selectable marker, Blasticidin S-resistance, using the Cre-loxP system. We confirm the effectiveness of the system by generating a single cell carrying 4 separate gene disruptions. Furthermore, the cells remain sensitive to transformation for additional targeted or random mutagenesis requiring Blasticidin selection and for functional expression studies of mutated or tagged proteins using other selectable markers. Submitted by: Alan Kimmel [ark1@helix.nih.gov] Please Note: all vectors essential for the procedure will be made available thru the Dicty Stock Center - Alan ============================================================================== [End Dicty News, volume 23, number 10]