CSM News Electronic Edition Volume 3, number 4 July 23, 1994 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web through www.nwu.edu. =========== Abstracts =========== Anne L. Hitt, Tze Hong Lu and Elizabeth J. Luna. Ponticulin Is an Atypical Membrane Protein. J. Cell Biol., in press. We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single-copy gene encodes a developmentally-regulated message that is high during growth and early development, but drops precipitously during cell streaming at ~8 hours of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha-helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically-oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (A. L. Hitt, J. H. Hartwig, and E. J. Luna, accompanying manuscript). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s). ----------------------------------------------------------------------- Anne L. Hitt, John H. Hartwig, and Elizabeth J. Luna. Ponticulin Is the Major High-affinity Link Between the Plasma Membrane and the Cortical Actin Network in Dictyostelium. J. Cell Biol., in press. Interactions between the plasma membrane and underlying actin-based cortex have been implicated in membrane organization and stability, the control of cell shape, and various motile processes. To ascertain the function of high-affinity actin-membrane associations, we have disrupted by homologous recombination the gene encoding ponticulin, the major high-affinity actin-membrane link in Dictyostelium discoideum amoebae. Cells lacking detectable amounts of ponticulin message and protein also are deficient in high-affinity actin-membran e binding by several criteria. First, only 10-13% as much endogenous actin co-sediments through sucrose with crude plasma membranes from ponticulin-minus cells, as compared with membranes from the parental strain. Second, purified plasma membranes exhibit little or no binding or nucleation of exogenous actin in vitro. Finally, only 10-30% as much endogenous actin partitions with plasma membranes from ponticulin-minus cells after these cells are mechanically unroofed with polylysine-coated coverslips. The loss of the cell=D5s major actin-binding membrane protein appears to be surprisingly benign under laboratory conditions. Ponticulin-minus cells grow normally in axenic culture and pinocytose FITC-dextran at the same rate as do parental cells. The rate of phagocytosis of particles by ponticulin-minus cells in growth media also is unaffected. By contrast, after initiation of development, cells lacking ponticulin aggregate faster than the parental cells. Subsequent morphogenesis proceeds asynchronously, but viable spores can form. These results indicate that ponticulin is not required for cellular translocation, but apparently plays a role in cell patterning during development. ---------------------------------------------------------------------- [End CSM-News, volume 3, number 4]