(and and and illustrates the large number of fertilization tubules observed upon examination of these samples in the fluorescence microscope. filamentous actin, the method yielded 2C3 106 fertilization tubules/g protein, representing up to a 360-fold enrichment of these organelles. Examination by negative stain electron microscopy demonstrated that the purified fertilization tubules were morphologically indistinguishable from fertilization tubules on intact, activated mt+ gametes, retaining both the extracellular fringe and the internal array of actin filaments. Several proteins, including actin as well as two surface proteins identified by biotinylation studies, copurified with the fertilization tubules. Most importantly, the isolated mt+ fertilization tubules bound to the apical Ampicillin Trihydrate ends of activated mt? gametes between the two flagella, the site of the mt? mating structure; CTSD a single fertilization tubule bound per cell, binding was specific for gametes, Ampicillin Trihydrate and fertilization tubules isolated from trypsin-treated, activated mt+ gametes did not bind to activated mt? gametes. The defining moment of fertilization is the adhesion and fusion of the plasma membranes of two interacting gametes. Although gametes are nonfusogenic when they first encounter their partners, their initial adhesive interactions generate a signal transduction cascade that renders the gametes capable of cell fusion (Snell and White, 1996). In multicellular organisms, adhesion-induced signaling prepares sperm for adhesion and fusion of the plasma membranes by inducing the acrosome reaction, an exocytic event that exposes previously cryptic membrane domains that contain adhesion/fusion molecules (Primakoff et al., 1980; Ward and Kopf, 1993). Moreover, hydrolytic enzymes released from the sperm during the acrosome reaction digest the extracellular matrix surrounding the egg, thereby exposing the adhesion/fusion molecules on the egg plasma membrane (Primakoff et al., 1980). Although the molecules that mediate adhesion and fusion vary widely among species (Glabe, 1985; Hong and Vacquier, 1986; Blobel et al., 1992; Snell and White, 1996), evolution appears to have favored a common cellular structure, an actin-filled cell protrusion, that provides the scaffolding for presentation of adhesion/fusion proteins (for example see Monroy, 1985; Yanagimachi, 1988). Microscopic analysis of spermCegg interactions in mouse and many other vertebrate and invertebrate species has shown that sperm selectively bind to the microvillus-rich portion of the egg plasma membrane (Tegner and Epel, 1976; Hylander and Summers, 1977; Yanagimachi, 1988; Foltz and Lennarz, 1992; Hart et al., 1992; Ohlendieck et al., 1994). In a manner analogous to eggs, the adhesion/fusion proteins on sperm also appear to be restricted to specialized regions of the plasma membrane. Although eutherian sperm bind and fuse via their apparently microvillus-free equatorial region, the male gametes in most other organisms studied form an apically localized, actin-filled cell protrusion or fusion organelle similar to a microvillus (Jessen et al., 1973; Tilney et al., 1973; Tilney, Ampicillin Trihydrate 1975; Yanagimachi, 1988). The nearly universal use of actin-filled fusion organelles in fertilization suggests that the regulated fusion of cell membranes requires a complex interplay of adhesion/fusion molecules and other, as yet unidentified features of these microvillus-like structures. Therefore, and by analogy to studies of fusion of intracellular membranes (Rothman, 1994), a complete understanding of the cellular and molecular events that underlie gamete fusion will require both the development of methods for isolating these domains of the cell surface in a structurally and functionally intact form as well as assays for in vitro reconstitution of adhesion and fusion. To this end, we have begun to study a fusion organelle, the Ampicillin Trihydrate mating type plus (mt+)1 fertilization tubule, in the genetically tractable organism, The initial stage of fertilization in this unicellular, biflagellated alga occurs when gametes of opposite mating types undergo a signal-transduction cascade, induced by flagellar adhesion, that generates fusion-competent gametes (Goodenough, 1991; Snell, 1993). Cyclic AMP generated during signaling induces activation of the mating structures, specialized regions of the apical plasma membrane where gametic adhesion and fusion will occur (Pasquale and Goodenough, 1987). Activation of the mt+ mating structure results in both the rapid recruitment of membrane and the explosive polymerization of actin to form the 3-m-long, microvillus-like fertilization tubule (Friedmann et al., 1968; Goodenough et al., 1982; Detmers et al., 1985). Ultrastructural analysis has shown that the mt+ mating structure contains an overlying extracellular coat, called fringe, as well.