[Show abstract][Hide abstract] ABSTRACT: 'Contact regions' are plasma membrane domains derived from areas of intercellular contact between aggregating Dictyostelium amebae (H.M. Ingalls et al. (1986). Proc. Nat. Acad. Sci. USA 83, 4779). Purified contact regions contain a prominent actin-binding protein with an M(r) of 34,000. Immunoblotting with monoclonal antibodies identifies this polypeptide as a 34,000 M(r) actin-bundling protein (known as 30 kDa protein), previously shown to be enriched in filopodia (M. Fechheimer (1987). J. Cell Biol. 104, 1539). About four times more 30 kDa protein by mass is associated with contact regions than is found in total plasma membranes isolated from aggregating cells. In agreement with these observations, immunostaining of the 30 kDa protein in aggregating cells reveals a prominent localization along the plasma membrane at sites of intercellular contact. By contrast, alpha-actinin does not appear to be significantly enriched at sites of cell to cell contact. Binding experiments using purified plasma membranes, actin and 30 kDa protein indicate that the 30 kDa protein is associated with the plasma membrane primarily through interactions with actin filaments. Calcium ions are known to decrease the interaction of actin with 30 kDa protein in solution. Surprisingly, membrane-associated complexes of actin and the 30 kDa protein are much less sensitive to dissociation by micromolar levels of free calcium ions than are complexes in solutions lacking membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
[Show abstract][Hide abstract] ABSTRACT: This chapter discusses the morphology and biochemistry of the membrane–cytoskeleton interface in the highly motile amebae of the cellular slime mold, Dictyostelium discoideum. This eukaryotic organism offers many advantages for the study of dynamic actin–membrane interactions. Amebae are phagocytes during log-phase growth, form multicellular aggregates during their well-characterized developmental cycle, and are chemotactic during both growth and development. The behavior, morphology, and biochemistry of D. discoideum amebae engaged in motile processes are strikingly like those of the best-characterized mammalian cell systems. D. discoideum is also amenable to genetic and molecular genetic analyses. Large quantities of amebae can be grown easily in axenic culture, and complementary DNA libraries, transformation systems, and mutants defective in motile functions are available. Much detailed information is available about the composition of the D. discoideum plasma membrane and about the composition and regulation of the cortical cytoskeleton. Actin polymerization is thought to occur at the cytoplasmic surfaces of plasma membranes during the persistent locomotion of polarized cells.
Advances in Molecular and Cell Biology 12/1990; 3:1-33. DOI:10.1016/S1569-2558(08)60442-7
[Show abstract][Hide abstract] ABSTRACT: Ponticulin is a 17,000-dalton transmembrane glycoprotein that is involved in the binding and nucleation of actin filaments by Dictyostelium discoideum plasma membranes. The major actin-binding protein isolated from these membranes by F-actin affinity chromatography, ponticulin also binds F-actin on blot overlays. The actin-binding activity of ponticulin in vitro is identical to that observed for purified plasma membranes: it resists extraction with 0.1 N NaOH, is sensitive to high salt concentrations, and is destroyed by heat, proteolysis, and thiol reduction and alkylation. A cytoplasmic domain of ponticulin mediates binding to actin because univalent antibody fragments directed against the cytoplasmic surface of this protein inhibit 96% of the actin-membrane binding in sedimenlation assays. Antibody specific for ponticulin emoves both ponticu-lin and the ability to reconstitute actin nucleation activity from detergent extracts of solubilized plasma membranes. Levels of plasma membrane ponticulin increase 2- to 3-fold during aggregation streaming, when cells adhere to each other and are highly motile. Although present throughout the plasma membrane, ponticulin is preferentially localized to some actin-rich membrane structures, including sites of cell-cell adhesion and arched regions of the plasma membrane reminiscent of the early stages of pseudopod formation. Ponticulin also is present but not obviously enriched at phagocytic cups of log-phase amebae. These results indicate that ponticulin may function in vivo to attach and nucleate actin filaments at the cytoplas-mic surface of the plasma membrane. A 17,000-dalton analogue of ponticulin has been identified in human polymorphonuclear leukocyte plasma membranes by immunoblotting and immunofluo-rescence microscopy. These findings suggest that the structure and function of ponticulin in motile cells has been evolutionarily conserved.
[Show abstract][Hide abstract] ABSTRACT: We have used a new combination of previously-described methods to obtain a 29-fold purification of plasma membranes from Dictyostelium discoideum. In this procedure, the pellet from a cell lysate is centrifuged through a high-pH sucrose gradient and then through a Renografin gradient. Electron microscopy shows that the resultant "Renografin membranes" are essentially homogeneous. As measured by enzymatic marker assays, contamination with mitochondria, lysosomes, and endoplasmic reticulum is minimal. As assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the protein composition of Renografin membranes is similar to that of highly purified membranes isolated using concanavalin A stabilization and detergent extraction. Using Renografin membranes, we have examined developmental changes in the membrane protein composition. In agreement with previous investigations, we observe major changes in lectin-binding glycoproteins and cell-surface-labeled proteins during the first 18 h of D. discoideum development. In contrast to most previous work, which may have employed plasma membranes of lesser purity, we also observe major changes in silver-stained membrane proteins. We conclude that many developmentally regulated proteins, previously thought to be minor membrane constituents, are a larger proportion of the plasma membrane than originally believed. The observed changes in membrane protein composition may correlate with changes in plasma membrane functions during development. For instance, ponticulin, the major salt-sensitive F-actin-binding protein in plasma membranes from vegetative cells, increases at least twofold in plasma membranes during early development when the cells are chemotaxing into large aggregates. The amount of plasma membrane ponticulin then decreases during the pseudoplasmodial stage.
[Show abstract][Hide abstract] ABSTRACT: Regions of plasma membrane involved in Dictyostelium discoideum intercellular adhesion resist solubilization with the nonionic detergent Triton X-100. Electron microscopy shows that these regions of the plasma membrane adhere to each other, forming many bi- and multilamellar structures. NaDodSO4/polyacrylamide gels of these regions contain major polypeptides at 225 kDa (residual myosin), 105 kDa, 88 kDa, 84 kDa, 47 kDa (residual actin), and 34 kDa. These membranes contain a subset of the total plasma membrane proteins, as analyzed by labeling of electrophoretically fractionated and blotted membrane proteins with radioiodinated Con A and by electrophoresis of membrane proteins from surface-labeled cells. Antibodies specific for gp80, a glycoprotein implicated in intercellular adhesion, intensely stain the 88-kDa and 84-kDa bands. Since these membrane regions resist Triton extraction, they appear to be stabilized by protein-protein interactions. Such stabilizing interactions may involve multivalent linkages with adjacent cells, or associations with intracellular actin and myosin, or both. Since these membranes appear to represent regions of intercellular contact, we call them "contact regions."
Proceedings of the National Academy of Sciences 08/1986; 83(13):4779-83. DOI:10.1073/pnas.83.13.4779 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In novel, low-speed sedimentation assays, highly purified, sonicated Dictyostelium discoideum plasma membrane fragments bind to F-actin beads (fluorescein-labeled F-actin on antifluorescein IgG-Sephacryl S-1000 beads). Binding was found to be (a) specific, since beads containing bound fluorescein-labeled ovalbumin or beads without bound fluorescein-labeled protein do not bind membranes, (b) saturable at approximately 0.6 microgram of membrane protein per microgram of bead-bound F-actin, (c) rapid with a t1/2 of 4-20 min, and (d) apparently of reasonable affinity since the off rate is too slow to be measured by present techniques. Using low-speed sedimentation assays, we found that sonicated plasma membrane fragments, after extraction with chaotropes, still bind F-actin beads. Heat-denatured membranes, proteolyzed membranes, and D. discoideum lipid vesicles did not bind F-actin beads. These results indicate that integral membrane proteins are responsible for the binding between sonicated membrane fragments and F-actin on beads. This finding agrees with the previous observation that integral proteins mediate interactions between D. discoideum plasma membranes and F-actin in solution (Luna, E.J., V. M. Fowler, J. Swanson, D. Branton, and D. L. Taylor, 1981, J. Cell Biol., 88:396-409). We conclude that low-speed sedimentation assays using F-actin beads are a reliable method for monitoring the associations between F-actin and membranes. Since these assays are relatively quantitative and require only micrograms of membranes and F-actin, they are a significant improvement over other existing techniques for exploring the biochemical details of F-actin-membrane interactions. Using F-actin beads as an affinity column for actin-binding proteins, we show that at least 12 integral polypeptides in D. discoideum plasma membranes bind to F-actin directly or indirectly. At least four of these polypeptides appear to span the membrane and are thus candidates for direct transmembrane links between the cytoskeleton and the cell surface.
The Journal of Cell Biology 08/1984; 99(1 Pt 1):58-70. · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The isozymes patterns of ovarian malate dehydrogenase (MDH) from various anuran amphibian series were analyzed by isoelectric focusing (IEF). Extensive variability was observed in both the soluble (sMDH) and mitochondrial (mMDH) patterns with as few as two and as many nine bands being visualized in different species. The mean pIs for sMDH ranged from 4.5 to 8.3 and those for mMDH fell between 5.1 and 8.2. The sMDHs are considerably more heat labile in Rana species living in northern latitudes compared to those from southern states. Inhibition with p-chlormercuribenzoate (PCMB) revealed the importance of sulfhydryl groups for the activity of sMDHs, while the functional requirement for these groups in mMDHs appears to be of lesser importance. Observations from these studies lend support to the accumulating evidence that Rana pipiens from such southern locations as New Mexico may have undergone speciation.
[Show abstract][Hide abstract] ABSTRACT: Innovel,low-speed sedimentationassays,highlypurified,sonicatedDictyostelium discoideum plasma membrane fragmentsbind toF-actinbeads (fluorescein-labeledF-actinon antifluoresceinIgG-SephacrylS-1000 beads).Bindingwas found tobe (a)specific,sincebeads containingbound fluorescein-labeledovalbumin or beads without bound fluorescein-labeled proteindo not bind membranes, (b)saturableat-0.6ug of membrane proteinpermicrogram ofbead-bound F-actin,(c)rapidwitha b,,of4-20 min,and (d)apparentlyofreasonableaffinity since the offrate istoo slow to be measured by present techniques. Using low-speed sedimentationassays,we found thatsonicatedplasma membrane fragments,afterextraction with chaotropes, stillbind F-actinbeads. Heat-denatured membranes, proteolyzed mem- branes,and D.discoideumlipidvesiclesdidnot bind F-actinbeads.These resultsindicatethat integralmembrane proteinsare responsibleforthe binding between sonicated membrane fragmentsand F-actinon beads.Thisfindingagreeswiththe previousobservationthatintegral proteinsmediate interactionsbetween D.discoideum plasma membranes and F-actinin solution(Luna,E.J.,V.M.Fowler,J.Swanson, D.Branton,and D.L.Taylor,1981,J.CellBiol., 88:396-409).We conclude thatlow-speed sedimentation assaysusingF-actinbeads are a reliablemethod formonitoringthe associationsbetween F-actinand membranes .Sincethese assaysarerelatively quantitativeand requireonlymicrograms ofmembranes and F-actin,they are a significantimprovement over other existingtechniques forexploringthe biochemical detailsofF-actin-membrane interactions . Using F-actinbeads asan affinitycolumn foractin-bindingproteins,we show thatatleast 12 integralpolypeptides in D.discoideumplasma membranes bind to F-actindirectlyor indirectly .At leastfourof these polypeptidesappear to span the membrane and are thus candidatesfordirecttransmembrane linksbetween the cytoskeletonand the cellsurface.