Dicty News Electronic Edition Volume 18, number 8 May 18, 2002 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ============= Abstracts ============= The Cdk5 homologue, Crp, regulates endocytosis and secretion in Dictyostelium and is necessary for optimum growth and differentiation Shiv K. Sharma1, Debra A. Brock2, Robin R. Ammann2 , Tiffany DeShazo3, Meenal Khosla1, Richard H. Gomer2,3 and Gerald Weeks1 1Department of Microbiology and Immunology, The University of British Columbia, Vancouver, BC V6T 1Z3 Canada 2Howard Hughes Medical Institute and 3Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main Street, Houston, TX 77005-1892 Developmental Biology, in press Summary Dictyostelium Crp is a member of the cyclin dependant kinase (Cdk) family of proteins. It is most related in sequence to mammalian Cdk5, which unlike other members of the family, has functions that are unrelated to the cell cycle. In order to better understand the function of Crp in Dictyostelium, we overexpressed a dominant negative form, Crp-D144N, under the control of the actin 15 promoter. Cells overexpressing Crp-D144N exhibit a reduced growth rate in suspension culture and reduced rates of fluid-phase endocytosis and phagocytosis. There is no reduction in Cdc2 kinase activity in extracts from cells overexpressing Crp-D144N, suggesting that the growth defect is not due to inhibition of Cdc2. In addition to the growth defect, the act15::crp-D144N transformants aggregate at a slower rate than wild-type cells and form large aggregation streams. These eventually break up to form small aggregates and most of these do not produce mature fruiting bodies. The aggregation defect is fully reversed in the presence of wild-type cells but terminal differentiation is only partially rescued. In act15::crp-D144N transformants, the countin component of the counting factor, a secreted protein complex that regulates the breakup of streams, mostly appears outside the cell as degradation products and the reduced level of the intact protein may at least partially account for the initial formation of the large aggregation streams. Our observations indicate that Crp is important for both endocytosis and efflux and that defects in these functions lead to reduced growth and aberrant development. ----------------------------------------------------------------------------- Clarke, M., Koehler, J., Arana, Q., Liu, T., Heuser, J., and Gerisch, G. Dynamics of the vacuolar H+-ATPase in the contractile vacuole complex and the endosomal pathway of Dictyostelium cells. J. Cell Sci., in press. ABSTRACT The vacuolar H+-ATPase (V-ATPase) is a multi-subunit enzyme that plays important roles in eukaryotic cells. In Dictyostelium, it is found primarily in membranes of the contractile vacuole complex, where it energizes fluid accumulation by this osmoregulatory organelle, and also in membranes of endo-lysosomes, where it serves to acidify the endosomal lumen. In the present study, a fusion was created between vatM, the gene encoding the 100-kDa transmembrane subunit of the V-ATPase, and the gene encoding Green Fluorescent Protein (GFP). When expressed in Dictyostelium cells, this fusion protein VatM-GFP was correctly targeted to contractile vacuole and endo-lysosomal membranes, and was competent to direct assembly of the V-ATPase enzyme complex. Protease treatment of isolated endosomes indicated that the GFP moiety, located on the C-terminus of VatM, was exposed to the cytoplasmic side of the endosomal membrane rather than to the lumenal side. VatM-GFP labeling of the contractile vacuole complex revealed clearly the dynamics of this pleiomorphic vesiculo-tubular organelle. VatM-GFP labeling of endosomes allowed direct visualization of the trafficking of vacuolar proton pumps in this pathway, which appeared to be entirely independent from the contractile vacuole membrane system. In cells whose endosomes were pre-labeled with TRITC-dextran and then fed yeast particles, VatM-GFP was delivered to newly formed yeast phagosomes with the same time course as TRITC-dextran, consistent with transfer via a direct fusion of endosomes with phagosomes. Several minutes were required before the intensity of the VatM-GFP labeling of new phagosomes reached the level observed in older phagosomes, suggesting that this fusion process was progressive and continuous. VatM-GFP was retrieved from the phagosome membrane prior to exocytosis of the indigestible remnants of the yeast particle. These data suggest that vacuolar proton pumps are recycled by fusion of advanced with newly-formed endosomes. ----------------------------------------------------------------------------- A Talin Fragment as an Actin Trap Visualizing Actin Flow in Chemotaxis, Endocytosis, and Cytokinesis Igor Weber, Jens Niewoehner, Aiping Du, Ursula Roehrig, and Guenther Gerisch Max-Planck-Institut fuer Biochemie, Martinsried, Germany Cell Motility and the Cytoskeleton, in press. ABSTRACT A C-terminal 63 kDa fragment of talin A from Dictyostelium discoideum forms a slowly dissociating complex with F-actin in vitro. This talin fragment (TalC63) has been tagged with GFP and used as a trap for actin filaments in chemotactic cell movement, endocytosis, and mitotic cell division. TalC63 efficiently sequesters actin filaments in vivo. Its translocation reflects the direction and efficiency of an actin flow. Along the body of a migrating Dictyostelium cell, this flow is directed from the front to the tail. If during chemotaxis one or two new fronts are induced, the flow is always directed away from these fronts. The flow thus reflects the re-programming of cell polarity in response to changing gradients of chemoattractant. In endocytosis, the fluorescent complexes are translocated to the base of a phagocytic or macropinocytic cup. During mitosis, the complexes of F-actin with TalC63 accumulate within the midzone of anaphase cells. If TalC63 is strongly expressed, the entire cleavage furrow is filled out by sequestered actin filaments, and cytokinesis is severely impaired. These cells are considered to mimic the phenotype of mutants deficient in the shredding of actin filaments that normally occurs in the mid-zone of a dividing cell. ----------------------------------------------------------------------------- Tubular-Vesicular Transformation in the Contractile Vacuole System of Dictyostelium. (Review) Guenther Gerisch*, John Heuser#, and Margaret Clarke+ * Max-Planck-Institut fuer Biochemie, Martinsried, Germany # Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, USA + Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA Cell Biology International, in press. ABSTRACT The contractile vacuole complex of Dictyostelium is the paradigm of a membrane system that undergoes tubular-vesicular transitions during its regular cycle of activities. This system acts as an osmoregulatory organelle in freshwater amoebae and protozoa. It collects fluid in a network of tubules and cisternae, and pumps it out of the cell through transient pores in the plasma membrane. Tubules and vacuoles are interconvertible. The tubular channels are associated with the cortical actin network and are capable of moving and fusing. The contractile vacuole complex is separate from vesicles of the endosomal pathway and preserves its identity in a dispersed state during cell division. We outline techniques to visualize the contractile vacuole system by electron and light microscopy. Emphasis is placed on GFP-fusion proteins that allow visualization of the dynamics of the contractile vacuole network in living cells. Proteins that control activities of this specialized organelle in Dictyostelium have been conserved during evolution and also regulate membrane trafficking in man. ----------------------------------------------------------------------------- [End Dicty News, volume 18, number 8]