The Chlamydomonas Fus1 protein is present on the mating type plus fusion organelle and required for a critical membrane adhesion event during fusion with minus gametes

Abstract

The molecular mechanisms of the defining event in fertilization, gamete fusion, remain poorly understood. The FUS1 gene in the unicellular, biflagellated green alga Chlamydomonas is one of the few sex-specific eukaryotic genes shown by genetic analysis to be essential for gamete fusion during fertilization. In Chlamydomonas, adhesion and fusion of the plasma membranes of activated mt+ and mt- gametes is accomplished via specialized fusion organelles called mating structures. Herein, we identify the endogenous Fus1 protein, test the idea that Fus1 is at the site of fusion, and identify the step in fusion that requires Fus1. Our results show that Fus1 is a approximately 95-kDa protein present on the external surface of both unactivated and activated mt+ gametes. Bioassays indicate that adhesion between mating type plus and mating type minus fusion organelles requires Fus1 and that Fus1 is functional only after gamete activation. Finally, immunofluorescence demonstrates that the Fus1 protein is present as an apical patch on unactivated gametes and redistributes during gamete activation over the entire surface of the microvillous-like activated plus mating structure, the fertilization tubule. Thus, Fus1 is present on mt+ gametes at the site of cell-cell fusion and essential for an early step in the fusion process.

Figure 1.

Alignment of Fus1 domains and the bacterial invasin/intimin Ig-like consensus sequence. The sequence in the first line (INV) is a consensus derived from 25 diverse, invasin-related BID 1, bacterial Ig-like (group 1) domains in proteins with the following accession numbers: 1F00_I, 1CWV_A, gi12513096, gi12516151, gi12519346, gi1706558, gi2125981, gi124714, gi3257750, gi11044949, and gi7462086. The remaining five sequences, whose positions are indicated by the numbers at the left, are from Fus1. The alignment was optimized by visual inspection. Identical and similar amino acids are indicated in bold.

Figure 2.

Use of imp12 gametes to study gamete docking. (A) Phase-contrast micrograph of a pair of interacting wild-type mt+ and mt- gametes showing their adhering, intertwined flagella (arrows). (B) Phase-contrast micrograph of a gamete pair composed of an activated wild-type mt+ gamete and an activated imp12 mt- gamete. The flagella (arrows) are not interacting, and the cells are adherent only via their apical ends at the sites of their mating structures. (C) Phase-contrast micrograph of a quadriflagellated zygote formed from fusion of a live, activated wild-type mt+ gamete and a live, activated imp12 mt- gamete. (D) Low-magnification, fluorescent image of several pairs of docked gametes. The arrows indicate the activated, fixed, SYTOX-labeled wild-type mt+ gametes in the pairs. (E and E′) corresponding DIC (E) and fluorescent micrographs (E′) of an activated, live imp 12 mt- gamete (arrowheads) docked to an activated, fixed, SYTOX-labeled wild-type mt+ gamete (asterisks). (F and F′) Corresponding DIC (F) and fluorescent images (F′) of an activated, fixed, SYTOX-labeled imp12 mt- gamete (asterisks) docked with an activated, live wild-type mt+ (arrowheads) gamete showing a phalloidin-stained fertilization tubule joining them (F′). Gametes are ∼10 μm in diameter.

Figure 3.

Activation is required for gamete docking. Live, activated imp12 mt- mutants were mixed with fixed, SYTOX-labeled wild-type mt+ gametes and the percentage of docked cells was determined as described in MATERIALS AND METHODS. Unactivated mt+ gametes were treated under the following conditions before fixation: A, activated with dibutyryl cAMP and papaverine; UnA→L, cell walls were removed with lysin; L→A, cells treated with lysin followed by activation; and A→L, activation followed by lysin treatment. Triplicate samples were counted for each experiment (bar, SEM).

Figure 4.

fus1-1 mt+ gametes are defective in mating structure docking and fusion. (A) Activated, live imp12 mt- gametes were mixed with activated, fixed, SYTOX-labeled mt+ wild-type or fus1-1 gametes and percent docking was determined as described in MATERIALS AND METHODS (n = 5). (B) Activated wild-type or fus1-1 mt+ gametes were mixed with activated imp12 mt- gametes and scored for quadriflagellated zygote formation (n = 4). (bar, SEM).

Figure 5.

A polyclonal antibody directed against a synthetic Fus1 peptide recognizes recombinant Fus1 protein. (A) Amino acid sequence of the Fus1 protein with the antigenic peptide underlined. (B) Immunoblot of a recombinant Fus1-GST fusion protein probed with the anti-Fus1 polyclonal antibody or an anti-GST polyclonal antibody. Migration of prestained molecular weight markers is indicated on the left. The arrow indicates the full-length GST-Fus1 fusion protein.

Figure 6.

Endogenous Fus1 protein migrates as a ∼95-kDa protein and is expressed only in wild-type mt+ gametes. (A) Wild-type mt+ (+G) and mt- (-G) gametes were analyzed by SDS-PAGE and immunoblotting. The arrow indicates the endogenous Fus1 protein. (B) Anti-Fus1 immunoblot analysis of detergent extracts of 2 × 10⁷ cell equivalents of wild-type mt+ gametes (+G), mt- gametes (-G), mt+ vegetative cells (+V), and mt- vegetative cells (-V). (C) Anti-Fus1 immunoblot analysis of gametes of wild-type mt+ (wt) and three fus1 mutant strains.

Figure 7.

Fus1 is enriched in isolated fertilization tubules. (A) Alexa 488-phalloidin staining of activated mt+ gametes. The activated gametes display a prominent, actin-rich fertilization tubule. (B) Percoll gradient fraction of isolated fertilization tubules were stained with Alexa 488-phalloidin. (C) Samples (5 μg each) of homogenized cells (HC), and sucrose fractions (SP), and Percoll gradient fractions were analyzed by immunoblotting with the anti-Fus1 antibody. Bar, 5 μm.

Figure 8.

Indirect immunofluorescence localization of Fus1 to the fertilization tubule of activated mt+ gametes. (A) Anti-Fus1 indirect immunofluorescence of wild-type mt+ gametes. (B and B′) Corresponding micrographs of wild-type mt+ gametes dual-labeled with the Fus1 polyclonal antibody (B, green) and the filamentous actin-specific fluorochrome Alexa 546-phalloidin (B′, red). (C and C′) Corresponding micrographs of fus1-1 mt+ gametes dual-labeled with the Fus1 polyclonal antibody (C) and the actin-specific fluorochrome Alexa 546-phalloidin (C′). The arrowheads point to the position of fertilization tubules.

Figure 9.

Fus1 is on the external surface of fertilization tubules in activated wild-type mt+ gametes and in a discrete patch on the external surface of unactivated wild-type mt+ gametes. (A) Fluorescence micrograph of activated gametes not permeabilized before immunolocalization with the Fus1 antibody. (B and C) Activated wild-type mt+ gametes incubated with 0% (B) or 0.5% trypsin (C). The insets for B and C each show control (B) and trypsin-treated (C) samples dual labeled with anti-Fus1 antibody (green) and Alexa 546-phalloidin (red). (D) Activated wild-type mt+ gametes incubated with 0.05% trypsin were analyzed for Fus1 by immunoblotting (top). Identical samples also were immunoblotted for CALK, an intracellular protein (bottom). In each lane, 2 × 10⁷ cells were loaded. (E and E′) Corresponding micrographs of unactivated wild-type mt+ gametes dual labeled with the anti-Fus1 antibody (E, green) and the actin-specific fluorochrome, Alexa 546-phalloidin (E′, red). (F) Unactivated fus1 mt+ gametes incubated with the anti-Fus1 antibody. (G) Indirect immunofluorescence image of unactivated nonpermeabilized wild-type mt+ gametes stained with the anti-Fus1 antibody. (H and I) Anti-Fus1 indirect immunofluorescence images of control (H) and 0.5% trypsin-treated (I) unactivated wild-type mt+ gametes. (J) Anti-Fus1 immunoblot of unactivated wild-type mt+ gametes treated with 0.05% trypsin (top). The lower panel shows an anti-CALK immunoblot of identical samples.