Anne L. Hitt

Direct binding of F-actin to ponticulin, an integral plasma membrane glycoprotein.

We have developed an 125I-labeled F-actin blot overlay assay for the identification of F-actin-binding proteins after transfer to nitrocellulose from SDS-polyacrylamide gels. Two major F-actin-binding proteins from Dictyostelium discoideum, a cytoplasmic 30 kDa protein and a 17 kDa integral membrane protein, and two minor membrane polypeptides of 19 kDa and 15 kDa were detected by this method. Using F-actin affinity and immunoaffinity chromatography, the 17 kDa polypeptide was identified as ponticulin, a previously described actin-binding glycoprotein from D. discoideum plasma membranes (Wuestehube, L.J., and Luna, E.J., [1987]: J. Cell Biol. 105:1741-1751). The binding of F-actin to ponticulin on blots is specific because unlabeled F-actin competes with 125I-labeled F-actin and because G-actin does not bind. Nitrocellulose-bound ponticulin displays binding characteristics similar to those of purified plasma membranes in solution, e.g., F-actin binding is sensitive to high salt and to elevated temperatures. Under optimal conditions, 125-I-labeled F-actin blot overlays are at least as sensitive as are immunoblots with an antibody specific for ponticulin. When blotted onto nitrocellulose after 2-D gel electrophoresis, all isoforms of ponticulin and of the 19 kDa and 15 kDa polypeptides appear to bind F-actin in proportion to their abundance. Thus the actin-binding activies of these proteins do not appear to be regulated by modifications that affect isoelectric point. However, the actin-binding activity of nitrocellulose-bound ponticulin is diminished when the protein is exposed to reducing agents, suggesting an involvement of disulfide bond(s) in ponticulin function. The 125I-labeled F-actin blot overlay assay also may enable us to identify F-actin-binding proteins in other cell types and should provide a convenient method for monitoring the purification of these proteins.

Development of a fluorescent F-actin blot overlay assay for detection of F-actin binding proteins.

Interactions between cellular proteins and filamentous (F) actin are key to many cellular functions, e.g., cell motility, endocytosis, cell:cell adhesion, and cell:substrate adhesion. Previously, a functional assay using 125I-labeled F-actin to detect a subset of F-actin binding proteins by blot overlay was developed. We have modified this assay to use the fluorescent label, Alexa 488, in place of 125Iodine. The detection limit for Alexa 488-labeled actin using a Molecular Dynamics STORM 860 Fluorescence/PhosphorImager was as little as 100pg of labeled actin. The Alexa 488 F-actin assay detects the same proteins from Dictyostelium discoideum and with approximately the same sensitivity (approximately 10 microg/ml F-actin final concentration) as the analogous 125I-labeled F-actin blot overlay. The use of Alexa 488 F-actin for blot overlay assays requires no radioactive materials and generates no hazardous waste. Assays can be performed on the laboratory bench top and the blots imaged directly with a blue laser scanner, either wet or dry. In addition, the Alexa 488 fluorophore is highly resistant to photobleaching, does not decay, and may be stored frozen or lyophilized. Alexa 488 F-actin is a stable, cost-effective, nonhazardous probe used for rapid identification of a subset of F-actin binding proteins.

Identification of a second member of the ponticulin gene family and its differential expression pattern.

We have identified a homologue (ponB) of the ponticulin gene (ponA), an F-actin binding protein, in the expressed sequence tag library generated to mRNA isolated from fusion-competent cells of Dictyostelium discoideum. PonB is predicted to have many of the same characteristics as ponticulin. Both proteins are predicted to possess a cleaved signal peptide, a glycosyl anchor, an amphipathic beta-strand structure and six conserved cysteines. Because of the sequence similarity and predicted conserved structures, this gene constitutes the second member of a ponticulin gene family. Unlike ponticulin, ponB is not expressed in axenically grown cells or during the asexual reproductive phase of D. discoideum. PonB is expressed by cells grown on bacterial lawns and by cells induced to be fusion-competent, i.e., gametes. The expression of ponB correlates with the appearance of a new F-actin binding activity in cell lysates of bacterially grown ponA(-) cells. By immunofluorescence microscopy, ponB appears to be localized to vesicles and to the plasma membrane of bacterially grown cells. Because ponticulin is the major high-affinity link between the plasma membrane and the cytoskeleton, the ponticulin gene family is likely to be part of the redundant system of proteins involved in connecting the cytoskeleton to the plasma membrane.

Role of Ponticulin in Pseudopod Dynamics, Cell-Cell Adhesion, and Mechanical Stability of an Amoeboid Membrane Skeleton

ELIZABETH J. LUNA’, ANNE L. HITT’, DAMON SHUTT*, DEBORAH WESSELS*, DAVID SOLL*, PAT JAY3, CHRIS HUG3, ELLIOT L. ELSON3, ALEX VESLEY4, GREGORY P. DOWNEY4, MICHAEL WANG’, STEVEN M. BLOCK’, WADE SIGURDSON6, AND FREDERICK SACHS6 ’ University of Massachusetts Medical Center, Worcester Foundation Campus, 222 Maple Ave, Shrewsbury, Massachusetts 01545; * Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242; 3 Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63 110; 4 Department of Medicine, University of Toronto, Ontario, ON M5S lA8, Canada; 5 Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544; and 6 Department of Biophysics, SUNY at Buffalo, Buffalo, New York 14214 Ponticulin is a transmembrane protein that constitutes the major high-affinity link between the actin cytoskele- ton and the plasma membrane of the soil amoeba Dictyo- stelium discoideum. As one of the few membrane proteins known to contain both a cytoplasmic domain and a glyco- syl anchor, ponticulin accounts for about 90% of the actin- binding and actin-nucleating activities of isolated plasma membranes. The function of ponticulin in vivo is being deduced by analyzing mutant amoebae in which the single-copy ponticulin gene has been disrupted by homologous recom- bination. These cells are deficient in high-affinity actin- membrane binding, as determined by co-sedimentation of actin and membrane vesicles from freshly broken cells, electron microscopic analysis, and in vitro assays of actin- membrane binding and membrane-mediated actin nucle- ation. Thus ponticulin’s role as a major link between the actin cytoskeleton and the plasma membrane has been confirmed both in vivo and in vitro. Because ponticulin