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Vol. 14, Issue 6, 2530-2542, June 2003
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Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9039
Submitted December 12, 2002;
Revised January 29, 2003;
Accepted February 26, 2003
Monitoring Editor: Paul M. Wassarman
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Wassarman
et al., 2001;
Primakoff and Myles, 2002).
Initial cell–cell interactions between sperm surface proteins and
extracellular matrix molecules on the egg trigger activation of the sperm and
exposure of previously cryptic regions of the plasma membrane proposed to be
involved in gamete fusion. Once the sperm has made its way to the egg plasma
membrane, the membranes of the two gametes interact more intimately, finally
bringing about gamete fusion. Several sperm and egg molecules implicated in
activation of the sperm have been identified, and we know much about the
morphological events that accompany gamete fusion
(Yanagimachi, 1994). On the
other hand, little is known about the molecular mechanisms that underlie the
late steps in fertilization, when the plasma membranes of the interacting
gametes adhere and fuse (for review, see
Primakoff and Myles, 2002). In
the absence of any purely eukaryotic systems in which bona fide fusion
proteins have been identified, the best models for fusion of the external
plasma membrane of cells have come from studies of fusion of viruses with
their eukaryotic target cells (White,
1996; Eckert and Kim,
2001). In these systems, a viral transmembrane surface protein
interacts with a receptor in the host cell plasma membrane, leading to docking
of the virus particle on the cell surface. Subsequent conformational changes
in the interacting proteins finally lead to complete fusion
(Dimitrov, 2000;
Malashkevich et al.,
2001; Mayer,
2001).
A small number of eukaryotic genes has been shown by gene disruption to be
essential for zygote formation and likely to be required for the late step in
fertilization during which the gamete plasma membranes undergo adhesion and
fusion. Mouse CD9, a member of the tetraspanin family of proteins, is an egg
protein that is essential for fertility. The protein is also found in several
nonreproductive cell types in the mouse and is proposed to play a
scaffold-like role during gamete fusion
(Le Naour et al.,
2000; Miller et al.,
2000; Miyado et al.,
2000). Another mouse protein essential for fertility is the
endoplasmic reticulum resident chaperone calmegin, which likely is involved in
proper folding of key sperm proteins
(Watanabe et al.,
1995; Ikawa et al.,
2001; Yamagata et
al., 2002). The best candidate for a fusion protein in yeast
is PRM1p. PRM1p is present at sites of adhesion between a and 
haploid Saccharomyces cerevisiae cells, and when it is disrupted in
both cell types, fusion is inhibited by 50%
(Heiman and Walter, 2000;
White and Rose, 2001).
We have been studying fertilization and gamete fusion in the unicellular,
biflagellated green alga Chlamydomonas reinhardtii. Fertilization in
Chlamydomonas comprises many of the cellular events that typify
fertilization in most organisms. During the Chlamydomonas life cycle,
diploid zygotes undergo meiosis to yield mt+ and mt-
vegetative cells, which undergo gametogenesis when transferred into
nitrogen-free (N-free) medium. When wild-type mt+ and mt-
gametes are mixed, they initially adhere to each other via sex-specific cell
adhesion molecules, agglutinins, on their flagella
(Adair, 1985). Interactions
between the mating type plus and mating type minus
agglutinin molecules on the flagellar membranes activate a gamete-specific
flagellar adenylyl cyclase and the resultant increases in intracellular cAMP
lead to gamete activation (Pasquale and
Goodenough, 1987; Saito et
al., 1993; Zhang and
Snell, 1994). Both of the gametes release their extracellular
matrices (cell walls), they recruit additional agglutinins and a protein
kinase from the cell body to the flagella, and they activate sex-specific
mating structures at their apical ends, which are the sites for cell-cell
fusion (Detmers et al.,
1983; Wilson et al.,
1997; Wilson and Snell,
1998; Pan and Snell,
2000b). The activated minus mating structure is a small
dome-like membrane protrusion 0.3 µm in diameter by 0.2 µm in
height. The activated plus organelle, which is termed the
fertilization tubule, is a more prominent, 0.5 x 3 µm
microvillous-like organelle, filled with 60–80 actin filaments. The
mating structures of both types of gametes display an extracellular coat of
material called fringe (Goodenough et
al., 1982).
Because the mating structures are located at the bases of the two flagella
on each interacting gamete, flagellar adhesion brings the activated fusion
organelles into intimate contact, allowing them to adhere to each other
(Goodenough et al.,
1982) and leading rapidly to fusion of their plasma membranes.
Immediately after mating structure fusion, a cytoplasmic bridge representing
the combined mating structures joins the two gametes to each other. Within
seconds, the bridge shortens and expands and the formerly distinct,
biflagellated gametes merge into a single cell with four flagella, the
quadriflagellated zygote. Fertilization is a rapid process in this organism,
and zygotes occur within minutes after mt+ and mt- gametes
are mixed. Zygote formation is accompanied by inactivation and loss of
flagellar agglutinins (the Chlamydomonas equivalent to a block to
polyspermy) and activation of transcription of new genes as the zygote
developmental pathway commences
(Goodenough, 1991;
Goodenough et al.,
1995b; Pan and Snell,
2000b; Zhao et al.,
2001).
In studies to delineate the cellular and molecular mechanisms of gamete
fusion in Chlamydomonas, we developed methods for isolating and
characterizing activated plus mating structures, the fertilization
tubules (Wilson et al.,
1997). We showed that the isolated organelles retained their
ability to bind to mating structures on activated mt- gametes. An
important next step in dissecting the molecular mechanisms for fusion in
Chlamydomonas will be to identify proteins on the fertilization
tubule responsible for the functions of the organelle. One candidate for the
molecule responsible for mating structure adhesion and fusion is the protein
encoded by the FUS1 gene. FUS1 is a sex-specific gene that
is located in the mt+ locus, a chromosomal region that contains
several genes involved in sex- and gamete-specific events
(Ferris et al.,
2002).
The fus1-1 mutant was generated in mt+ cells >20 years
ago in a screen for mt+ gametes that were capable of flagellar
adhesion but were unable to fuse when mixed with wild-type mt-
gametes (Goodenough et al.,
1976,
1995a). More extensive
characterization of fus1-1 gametes has shown that they undergo normal
flagellar adhesion and gamete activation, and produce a fertilization tubule
as robust as those produced by wild-type mt+ gametes. On the other
hand, the fus1-1 fertilization tubule fails to fuse with the
activated minus mating structure and the cells in such mixtures
continue to agglutinate for days. Ultrastructural analysis of the
fertilization tubules on fus1 gametes indicated that the organelles
do not contain fringe (Goodenough et
al., 1982). More recently, the FUS1 gene was
identified by its unique presence in the mt+ locus, the
sex-restricted expression of its transcript and by its ability in
transformation experiments to restore fusion competence to several
mt+ mutant strains with lesions in the FUS1 gene
(Ferris et al.,
1996). Analysis of the predicted amino acid sequence suggested
that the Fus1 protein would be an integral membrane protein with a single
transmembrane region and a short cytoplasmic tail at the C termini. These
properties, along with the reappearance of fringe on the mating structures of
the FUS1-rescued mutants, led to the proposal that the protein
encoded by the FUS1 gene is a component of fringe or required for production
of fringe and is involved in mating structure interactions
(Ferris et al.,
1996).
Herein, we set out to test the idea that the FUS1 gene product is located at the site of gamete fusion and to examine its role in fusion. We wanted to reexamine the Fus1 protein sequence for possible clues about its function, to investigate the step in mating structure interactions that requires the FUS1 gene product, to identify the endogenous Fus1 protein, and to determine its cellular location in unactivated and activated gametes. Analysis of the sequence of the Fus1 protein revealed that it shows similarity to bacterial adhesion proteins, the invasins and intimins, and that it has five internal repeats of a 90 amino acid domain. Bioassays with imp12 mt- mutants that are fusion competent, but defective in flagellar adhesion, indicate that Fus1 is required for docking between mt- and mt+ gametes at their activated mating structures. Studies with an anti-Fus1 peptide antibody show that Fus1 is a 95-kDa polypeptide expressed only in mt+ gametes; that it is present in an inactive form on the outer surface of the unactivated plus mating structure; and that it becomes distributed over the surface of the entire fertilization tubule during gamete activation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-tosyl-L-lysine chloromethyl ketone HCl (TLCK),
cold water fish gelatin, and glutathione-agarose beads were from Sigma-Aldrich
(St. Louis, MO). Electron microscopy-grade paraformaldehyde and electron
microscopy-grade glutaraldehyde were from Electron Microscopy Sciences (Ft.
Washington, PA). Alexa 488-phalloidin, Alexa 546-phalloidin, SYTOX-green, and
Alexa 488-conjugated goat anti-rabbit antibody were from Molecular Probes
(Eugene, OR). Kaleidoscope and Precision Prestained molecular weight markers
and horseradish peroxidase-conjugated anti-rabbit antibody were from Bio-Rad
(Hercules, CA). Glutathione S-transferase (GST)-antibody was from
Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were of reagent
grade.
Cells and Cell Culture
Chlamydomonas reinhardtii strains 21gr (wild-type
mt+) (CC-1690), fus1-1 (mt+) (CC-1158),
fus1-2 (mt+) (CC-2062), fus1-3 (mt+)
(CC-2392), 6145C (wild-type mt-) (CC-1691), and
imp12 (mt-) (CC-1149) (available from the
Chlamydomonas Genetic Center, Duke University, Durham, NC) were
cultured vegetatively with aeration at 23°C in medium I of Sager and
Granick on a 13:11-h light/dark cycle. Gametogenesis was induced by
transferring vegetatively growing cells into N-free medium as described
previously (Snell, 1976a;
Pan and Snell, 2000a). Gametes
were activated by incubation with 15 mM dibutyryl cAMP and freshly prepared
0.15 mM papaverine in N-free medium for 45–60 min with vigorous aeration
(Pasquale and Goodenough,
1987). Gamete activation was confirmed by measuring release of
cell walls as described previously (Wilson
et al., 1997). Where indicated, cell walls were removed
from unactivated gametes by incubation with Chlamydomonas g-lysin for
30 min before use (Buchanan et
al., 1989).
Isolation of Fertilization Tubules
Fertilization tubules were isolated from activated wild-type mt+
gametes by differential centrifugation and fractionation on sucrose and
Percoll gradients as described previously
(Wilson et al.,
1997). This method yields an overall purification of fertilization
tubules of 160- to 300-fold. Protein concentrations were determined by use of
a Bio-Rad protein assay kit with bovine serum albumin (Albumin Standard;
Pierce Chemical, Rockford, IL) as a standard.
Gamete Docking and Fusion Assays
To prepare the fixed gametes for the docking assay, activated gametes were
incubated in 2.5% glutaraldehyde in N-free medium for 10 min, washed twice in
1% glycine in 10 mM phosphate buffered N-free (bN-free) medium for 5 min,
incubated with the live-cell impermeant, nucleic acid fluorochrome SYTOX-green
(1 µM in bN-free medium) for 10 min, and washed twice in N-free medium. To
carry out the docking assay, fixed, SYTOX-labeled mt+ gametes were
mixed with live, activated imp12 mt- gametes and allowed to interact
for 30 min. The mixed samples were placed on a microscope slide, fixed in 2%
paraformaldehyde, covered with a coverslip supported by petroleum jelly posts,
and viewed by fluorescence and differential interference contrast (DIC)
microscopy. Using a fluorescein isothiocyanate long-pass barrier filter
(filter set 09; Carl Zeiss, Thornwood, NY), the fixed mt+ cells
(bright green SYTOX fluorescence) were easily distinguished from the imp12
mt- gametes (low red background autofluorescence). Percentage of docking
was defined as (number of fixed cells docked to a live gamete)/(total number
of fixed cells counted) x 100. At least 100 randomly chosen cells were
counted.
To assay gamete fusion in the absence of flagellar adhesion, activated mt+ and imp12 mt- gametes were mixed at 1:1 ratio, centrifuged at 20,000 x g for 14 s, and resuspended after 20 min. Samples were fixed in 2.5% glutaraldehyde for 1 h after resuspension and the numbers of biflagellated, unfused gametes and quadriflagellated, fused cells were determined by phase-contrast microscopy. Percentage of zygote formation was defined as (number of zygotes x 2)/(number of zygotes x 2 + number of gametes) x 100. At least 100 randomly chosen cells were counted.
Treatment of Cells with Trypsin
Activated or unactivated mt+ gametes were mixed with a freshly
prepared stock solution of trypsin (50 mg/ml in 1 mM HCl) at a concentration
of 5 x 107 cells/ml to yield the final concentrations of
trypsin indicated in the figure legends. Control cells were incubated with
equivalent dilutions of the stock solution buffer. After 20 min at room
temperature, by which time cells had lost their ability to undergo flagellar
agglutination with tester mt- gametes, the cells were diluted 10-fold
with N-free medium, centrifuged, and resuspended in fresh N-free medium
containing 1 mM TLCK (diluted from freshly prepared 10 mM stock in 1 mM HCl).
After a 10-min incubation with TLCK, an aliquot of cells was fixed as
described above for immunofluorescence. For immunoblotting, the remaining
cells were washed twice more with N-free media containing 1 mM TLCK before
analysis by SDS-PAGE and immunoblotting. Both 0.05% and 0.5% trypsin were used
for the protease treatment and yielded similar results for loss of agglutinin
activity, Fus1 immunofluorescence, and Fus1 immunoblotting.
Production of Recombinant Protein
To prepare a GST-tagged, truncated, recombinant Fus1 protein, a polymerase
chain reaction (PCR) product consisting of a FUS1 cDNA fragment
corresponding to amino acids 17–741 (which excludes the putative signal
peptide and transmembrane domain) and 5' and 3' multiple cloning
sites was generated using a full-length FUS1 cDNA plasmid (kindly
provided by Drs. Patrick Ferris and Ursula Goodenough, Washington University,
St. Louis, MO) as the template. The originally obtained, full-length
FUS1 cDNA was modified and sequenced before the PCR by standard
methods to remove an intron and to ensure that the sequence was correct. The
truncated FUS1 PCR product was cloned into the pGEX-2T expression
vector (Amersham Biosciences, Piscataway, NJ) and subsequently transfected
into M15 bacteria (QIAGEN, Valenica, CA) for expression. Protein expression
was induced by adding 1 mM isopropyl 
-D-thiogalactoside at
30°C for 1–3 h. GST-Fus1 protein was purified using
glutathione-agarose beads according to the manufacturer's instructions
(Sigma-Aldrich).
Antibody Production
Anti-Fus1 peptide antibodies were prepared by Biosource International,
Quality Controlled Biochemicals (Hopkinton, MA). The peptide
SDRFTNWIREKSIATQLRVC was synthesized, verified by mass spectrometry, and used
to generate polyclonal antibodies. Antibodies were affinity purified on a
peptide affinity column. For immunoblotting, the affinity-purified antibody
was absorbed against methanol extracted, lyophilized wild-type mt-
gametes to remove nonspecific background staining. To do this,
1010 gametes were extracted twice with ice cold 100% methanol,
resuspended in 1 ml of methanol, and 250-µl aliquots were lyophilized. For
absorption of the antibody, the lyophilized methanol extracts were resuspended
in a 1:10 dilution of the antibody in phosphate-buffered saline and incubated
with gentle agitation at room temperature for 1–3 h. The sample was
cleared by centrifugation at 20, 000 x g at 4°C for 10 min.
Antibodies were stored at 4°C with 0.05% NaN3. For additional
absorption for immunofluorescence, the antibody sample was mixed with
wild-type mt- gametes that had been fixed in 2.5% glutaraldehyde as
described above. After an overnight incubation at 4°C the sample was
cleared by centrifugation. Absorptions with vegetative mt+ cells
yielded similar results.
SDS-PAGE and Immunoblot Analysis
Cells harvested by centrifugation were resuspended in buffer containing 10
mM NaCl, 10 mM HEPES, pH 7.2, and 1 x Plant Protease Inhibitor Cocktail;
sonicated on ice three times for 10 s each; mixed with an equal volume of 2
x SDS sample buffer (0.125 M Tris, pH 6.8, 20% glycerol, 4% SDS, 0.2 M
dithiothreitol, and 0.05% bromphenol blue); and boiled for 5 min. In some
experiments, detergent extracts of cells were used for immunoblotting. To
prepare the detergent extracts, cells were resuspended in 10 mM HEPES, pH 7.2,
150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1 x Plant
Protease Inhibitor Cocktail at a concentration of 8 x 108
cells/ml and sonicated as described above. After 30 min on ice samples were
centrifuged at 10,000 rpm for 10 min at 4°C and the supernatant was used
for immunoblotting. Control experiments indicated that essentially all of Fus1
protein was extracted into the supernatant with the detergent solution.
The samples were subjected to electrophoresis on 6% polyacrylamide
mini-slab gels at 100 V in buffer containing 25 mM Tris, 192 mM glycine, and
0.1% SDS. Typically, each well was loaded with 50 µl of sample containing
2 x 107 cell equivalents. After SDS-PAGE, proteins were
transferred to a nitrocellulose membrane (Protran; Schleicher & Schuell,
Keene, NH) for anti-Fus1 immunoblotting. For the GST and
Chlamydomonas aurora-like protein kinase (CALK) immunoblots a
polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA) was
used (Pan and Snell, 2000). Transfer was carried out overnight at 36 V at
4°C in buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol. For
detection of Fus1, the membrane was rinsed several times with 25 mM
KPO4 buffer, pH 7.0 and fixed with 0.2% glutaraldehyde for 45 min,
followed by rinsing twice with TBST (20 mM Tris, pH 7.6, 137 mM NaCl, and
0.05% Tween 20) (Hulen et al.,
1991; Wilson et al.,
1997). The membrane was blocked with 5% Carnation dry milk
(Nestle) in TBST for 2 h and incubated with the primary antibody at a final
dilution of 1:1000 in 3% Carnation dry milk in TBST. After 1 h, the membrane
was washed 3 x for 7 min each with TBST, followed by incubation for 30
min with a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody
diluted 1:10,000 in TBST containing 3% Carnation dry milk. The membrane was
washed as before and incubated in enhanced chemiluminescence immunoblotting
reagents (Pierce Chemcial) for 1 min as described by the manufacturer, exposed
to Hyperfilm ECL (Amersham Biosciences), and the film was developed in an
automatic film processor. For GST and CALK immunoblots, the procedure was
similar except the fixation step was omitted and the primary antibody was used
at a dilution of 1:1,000 and 1:5,000, respectively.
Microscopy
Samples for microscopy were fixed with either 2.5% glutaraldehyde in
bN-free medium for visible light microscopy or 2% paraformaldehyde in bN-free
medium for fluorescence microscopy. Cells were affixed to eight-well glass
slides coated with 0.1% polyethylenimine
(Sanders and Salisbury, 1994).
Fertilization tubules were visualized by fluorescence microscopy by using the
actin-specific fluorochromes Alexa 488-phalloidin or Alexa 546-phalloidin.
Samples were incubated in 5 U of fluorochrome/ml in bN-free medium for 15 min
and processed as described by Wilson
et al., 1997. Samples for immunofluorescence were
prepared as follows: paraformaldehyde-fixed cells were permeabilized in a cold
acetone series (80%, 100%, 6 min each), blocked for 30 min at 37°C in
blocking buffer (1% cold water fish gelatin, 0.1% bovine serum albumin, 5%
glycerol, 30 mM NaCl, and 2 mM sodium phosphate buffer, pH 7.3)
(Sanders and Salisbury, 1994),
incubated in absorbed primary antibody (1:200 dilution in blocking buffer) at
37°C for 1 h, washed three times in phosphate-buffered saline and
incubated in absorbed secondary antibody (Alexa 488-conjugated goat
anti-rabbit, 1:2000) for 30 min at 37°C.
Microscopy was performed using either an Axioplan2 or a III RS microscope (Carl Zeiss) equipped with epifluorescence and DIC or phase-contrast optics. Images were acquired using Hammamatsu Orca digital cameras and Openlab (Improvision) image acquisition software. Final composite images were constructed using Adobe Photoshop (Adobe Systems, San Jose, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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90 Amino Acids). More recent analysis, however, indicates that Fus1 protein
has sequence similarity to members of the invasin/intimin family of bacterial
proteins. The BLAST-link feature on the National Center for Biotechnology
Information Entrez PubMed Web site indicates 22% identity and 35% similarity
between Fus1 and enteropathogenic Escherichia coli invasin (accession
number BAA15799
[GenBank]
) protein >405 amino acids. This region of invasin contains
contiguous repeats of an 90 amino acid domain found in all members of the
invasin/intimin family of proteins
(Oelschlaeger, 2001), which
are used for bacterial adhesion to their mammalian host cells
(Kalman et al., 1999;
Vallance and Finlay,
2000).
Further BLAST analysis and visual inspection of the Fus1 sequence indicated
that it contained five internal repeats of 90 amino acids. Interestingly,
these regions bore some resemblance to a repeating domain in
invasins/intimins. Figure 1
shows an alignment of an invasin/intimin domain consensus sequence and the
five repeats of Fus1. These similarities between Fus1 and the bacterial
adhesion proteins were consistent with the possibility that Fus1 plays a role
in adhesion of Chlamydomonas mt+ and mt- gametes during
gamete fusion.
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Gamete Activation and a Functional FUS1 Gene Are Essential for Gamete
Docking
To learn more about the role of the FUS1 gene product in gamete
fusion, we developed methods for identifying and characterizing the properties
of mating structures at discrete phases of fertilization. To do this required
a system in which flagellar adhesion did not interfere with our ability to
examine mating structure interactions. We bypassed flagellar adhesion and
focused directly on the interactions between mating structures by exploiting
two features of the Chlamydomonas system: the availability of mutant
mt- gametes (imp12) that do not express functional flagellar
agglutinins (Pasquale and Goodenough,
1987; Goodenough,
1991; Goodenough et
al., 1995a) and the ability to activate gametes of a single
mating type experimentally by incubating them in dibutyryl cAMP
(Pjist et al., 1984;
Pasquale and Goodenough, 1987;
Wilson et al., 1997).
As shown in Figure 2A,
flagellar adhesion between wild-type mt+ and mt- gametes is
characterized by close interactions between the flagella and accompanied by
intimate contacts between the cell bodies at the sites of the activated mating
structures. On the other hand, and confirming previous reports
(Pasquale and Goodenough,
1987), no cellular interactions were observed when imp12
mt- gametes and wild-type mt+ gametes were mixed (our
unpublished data), unless both types of gametes were activated by incubation
in dibutyryl cAMP and papaverine. The activated mt- and mt+
gametes still did not interact with each other via their flagella
(Figure 2B). Rather, the random
collisions that occurred as a consequence of the high motility of these cells
brought their activated mating structures into contact
(Figure 2B), an interaction
that was followed by fusion to form a quadriflagellated zygote
(Figure 2C).
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This system made it possible to establish a gamete docking assay, in which gametes of opposite mating type would adhere to each other via their mating structures without fusing. To do this, we activated wild-type mt+ gametes, fixed them briefly with glutaraldehyde, and mixed them with live, activated imp12 mt- gametes. As expected, we could find no evidence for fusion, nor did we observe large aggregates of cells. Rather, we detected numerous pairs of gametes (Figure 2D). Within a pair, no flagellar adhesion was detected; each cell was oriented with its apical end directed toward the apical end of the other cell and the sites of their activated mating structures were juxtaposed (Figure 2, E and F). Similar results were obtained when activated, fixed imp12 mt- gametes were mixed with live activated wild-type mt+ gametes. In both circumstances, the interactions were stable when prepared for examination by phase contrast microscopy, and the docked gametes remained as pairs even as the live member of the pair propelled its fixed partner rapidly through the medium. On the other hand, if the samples were vigorously pipetted or otherwise subjected to strong agitation, the pairs came apart, indicating that the gamete mating structure interactions were easily disrupted.
In addition to determining that the cells interacted at their apical ends, we documented that the pairs were composed exclusively of one mt+ gamete and one imp12 mt- gamete adherent to each other at their mating structures. No pairs were observed in suspensions of activated cells of a single mating type, only one cell in a pair was fixed (Figure 2, D and E'), and only one cell in a pair contained a fertilization tubule, which was localized at the site of cell adhesion (Figure 2F').
With these tools in hand it became possible to examine whether gamete
activation was required for docking and to identify the particular step in
fertilization that was abrogated in fus1 mutants. To examine the
requirement for gamete activation, we mixed unactivated, SYTOX-labeled,
glutaraldehyde-fixed, wild-type mt+ gametes with activated, live
imp12 mt- gametes and assessed pair formation by using the docking
assay. Before mixing, we removed the extracellular matrix (cell wall) that
encloses unactivated gametes by use of the wall-degrading
Chlamydomonas collagenase, g-lysin
(Buchanan and Snell, 1988;
Kinoshita et al.,
1992). We found that only activated gametes were capable of
docking (Figure 3). Thus,
although 70% of the control, activated wild-type mt+ gametes formed
pairs with activated imp12 mt- gametes
(Figure 3A), unactivated,
lysin-treated wild-type mt+ gametes were unable to form pairs with
the activated imp12 mt- gametes (UnA-L). The lysin treatment did not
interfere with docking, as wild-type mt+ gametes that were activated
after lysin treatment still formed pairs in the assay (L-A), and activated
gametes subsequently treated with the lysin preparation (A–L) also
retained functional mating structure adhesion molecules. Thus, the results
indicated that gamete activation was required for gamete docking and suggested
that molecules involved in docking either were stored intracellularly or were
present at the cell surface in an inactive form.
|
Although it was known that the FUS1 gene product was essential for gamete fusion, we could now ask whether the gene was also required for gamete docking. We activated fus1-1 gametes by incubating them in dibutyryl cAMP and papaverine and assayed for their docking ability as described above. As shown in Figure 4, not only were the activated fus1-1 gametes incapable of gamete fusion (Figure 4B), but also docking was abrogated (Figure 4A). Thus, the FUS1 gene product is essential for a key membrane adhesion event during gamete fusion.
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The Endogenous Fus1 Protein is 95 kDa and Is Enriched in
Preparations of Isolated Fertilization Tubules
Although the genetic data and the docking experiments were consistent with
the idea that Fus1 plays a role in adhesion and fusion of mating structures,
the endogenous protein had not been identified or characterized. To study
endogenous Fus1, we used a polyclonal antibody raised against a 19 amino acid
peptide located near the N terminus of the polypeptide
(Figure 5A). The immunoblots in
Figure 5B show that the
antibody recognized a bacterially expressed GST-Fus1 fusion protein (left,
anti-Fus1-peptide antibody; right, anti-GST antibody). The arrow indicates the
full-length GST-Fus1, whose identity was confirmed by mass spectrometry (our
unpublished data). The lower molecular mass bands presumably were GST-Fus1
fragments, some of which contained only the N-terminally positioned GST.
|
Immunoblot analysis of wild-type mt+ Chlamydomonas
gametes showed reactivity with a protein of 95 kDa, which was not present
in wild-type mt- gametes (Figure
6A). (An unidentified, weakly staining, cross-reactive band of
higher molecular mass was present in both samples.) Consistent with the
localization of the FUS1 gene exclusively at the mt+ locus
and the previous report that the FUS1 transcript was not detected in
vegetative mt+ cells (Ferris
et al., 1996), immunoblot analysis of mt+ and
mt- gametes and vegetative cells showed that Fus1 was expressed only
after gametogenesis and only in wild-type mt+ cells
(Figure 6B). Moreover, as
expected, gametes of three fus1 mutant strains, each with unique
lesions in the FUS1 gene, also failed to express the protein
(Figure 6C). The observation
that the observed 95-kDa molecular mass of endogenous Fus1 was close to the
88-kDa mass predicted by its peptide sequence suggested that the
expressed protein may not be heavily glycosylated and is mostly
polypeptide.
|
We used cell fractionation to determine if endogenous Fus1 was enriched in
isolated fertilization tubules. Fertilization tubules were isolated from
activated wild-type mt+ gametes as described previously
(Wilson et al., 1997)
and the following three fractions obtained during purification were analyzed
by immunoblotting: the starting cells, partially purified fertilization
tubules from the sucrose gradient, and more highly purified fertilization
tubules harvested from the final Percoll gradient.
Figure 7A shows activated
wild-type mt+ gametes stained for actin with fluorescent phalloidin.
Figure 7B shows similarly
stained, isolated fertilization tubules from the Percoll gradient. Immunoblot
analysis of equal amounts of protein from each of the three samples from the
purification procedure showed that Fus1 became highly enriched during the
purification of fertilization tubules
(Figure 7C).
|
Fus1 Is on the External Surface of the Activated plus
Mating Structure
Having shown by cell fractionation and immunoblotting that Fus1 was present
in fertilization tubules isolated from activated mt+ gametes, we also
used immunolocalization methods to determine the cellular distribution of
Fus1. To do this, wild-type mt+ gametes were activated as described
above and Fus1 location was assessed by indirect immunofluorescence. As shown
in Figure 8, the protein was
distributed over the entire surface of the fertilization tubules.
Figure 8A shows a
low-magnification view of a field of activated wild-type mt+ gametes
stained only with the anti-Fus1 peptide antibody. Almost every cell displayed
an antibody-reactive structure with the morphology of a fertilization tubule.
Figure 8B shows a higher
magnification view of several activated wild-type mt+ gametes with
their fertilization tubules brightly stained by the antibody.
Figure 8B' is a
corresponding image of the same cells, which were also stained for actin with
fluorescent phalloidin. In each case, the structures that stained with the
anti-Fus1 antibody were also stained with phalloidin. Thus, the Fus1 protein
is present at fertilization tubules and distributed along their length. As
expected, although activated fus1-1 mutant mt+ gametes
erected prominent fertilization tubules that could be visualized with
fluorescent phalloidin, their fertilization tubules did not stain with the
Fus1 antibody (Figure 8, C and
C') because the cells do not express the Fus1 protein
(Figure 6).
|
We tested the idea that Fus1 is a cell surface protein by using three independent methods. In one approach, we carried out immunolocalization studies in which the antibody would have access only to surface-exposed molecules by using cells that had been fixed, but not permeabilized. The indirect immunofluorescence images shown in Figure 8 were obtained from samples of cells that had been fixed with paraformaldehyde and permeabilized with acetone or methanol before incubation with the antibody. Consistent with the predicted topology, Fus1 was accessible to the antibody in the nonpermeabilized sample (Figure 9A).
|
In the second approach, the surface localization of Fus1 was assessed by
use of the protease trypsin, which, based on the amino acid sequence of Fus1,
would cleave the protein at multiple sites. To do this, live, activated
wild-type mt+ gametes were incubated with trypsin for 20 min and then
prepared for indirect immunofluorescence. Consistent with previous reports
(Snell, 1976b;
Hunnicutt et al.,
1990), the trypsin-treatment did not have any effects on the
morphology or motility of the cells, although flagellar adhesion and gamete
fusion were blocked (our unpublished data). On the other hand, although the
control samples showed typical Fus1 staining
(Figure 9B), Fus1 staining was
eliminated by the trypsin treatment (Figure
9C). The higher magnification views in the insets show that the
fertilization tubules on control samples were stained for Fus1 and actin
(Figure 9B, inset), whereas
only actin staining remained in the trypsin-treated samples
(Figure 9C, inset).
Finally, these indirect immunofluorescence experiments demonstrating that
Fus1 was on the external surface of the fertilization tubules were also
confirmed by immunoblotting; Figure
9D). Control, activated wild-type mt+ gametes exhibited
typical levels of Fus1, but the protein was almost completely absent from the
trypsin-treated cells (Figure
9D). Immunoblot analysis with an antibody against the CALK
(Pan and Snell, 2000a) showed
that this cytoplasmic protein was not accessible to the trypsin in this
experiment (Figure 9D),
although CALK was sensitive to trypsin if cells were sonicated before the
trypsin treatment (our unpublished data). Finally, and consistent with our
previous studies, assays for docking (our unpublished data) documented that
the trypsin treatment eliminated mating structure adhesion.
Fus1 Is on the External Cell Surface of Unactivated Gametes in a
Patch at the Mating Structure
Having shown that Fus1 was localized on the surface of activated
plus mating structures, we also determined its location on
unactivated gametes. One interpretation of the failure of unactivated
wild-type mt+ gametes to adhere to activated imp12 mt-
gametes in the docking assay (Figure
3) was that Fus1 might not be on the cell surface before
activation. Analysis of unactivated wild-type mt+ gametes by indirect
immunofluorescence, however, showed that Fus1 was present as an apical patch
at the site of the unactivated mating structure
(Figure 9E).
Figure 9E', which shows
the corresponding cells stained for actin, documents the expected absence of
actin in the unactivated mating structures
(Goodenough et al.,
1982; Detmers et al.,
1983). Indirect immunofluorescence analysis of unactivated
fus1 gametes again documented the specificity of the antibody, as
these cells do not contain Fus1 protein and did not stain with the antibody
(Figure 9F).
Surface localization experiments similar to those carried out with activated gametes demonstrated that, like the Fus1 on activated gametes, Fus1 on unactivated gametes also was on the external surface of the cell. The protein was accessible to the antibody on nonpermeabilized, unactivated gametes (Figure 9G), and trypsin treatment of live gametes eliminated Fus1 as assessed both by indirect immunofluorescence (Figure 9H, control cells; 9I, trypsin-treated cells) and by immunoblotting (Figure 9J). Thus, the results indicated that the Fus1 protein is present on the surface of unactivated mating structures in an inactive form. Furthermore, the results strongly suggest that all of the Fus1 that covers the fully formed fertilization tubule is derived from the Fus1 present on the surface of the inactive mating structure.
|
DISCUSSION |
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|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Gamete Docking Requires the Fus1 Protein
Even though it is likely that fusion proceeds through a mating structure
adhesion step as originally proposed by Friedman et al.
(1968), fusion is such a rapid
process in Chlamydomonas that it has been difficult experimentally to
identify membrane adhesion, in large part because of the inability to
determine whether cell pairs are adherent via their mating structures or via
their flagella. Rare images of adherent mating structures were obtained in
mixtures of wild-type cells in which the mt+ gametes had been treated
with cytochalasin to disrupt the actin filaments in the fertilization tubules
(Goodenough et al.,
1982; Detmers et al.,
1983). Presumably, the absence of actin filaments within the
fertilization tubules slowed the process of fusion. Related studies with the
pseudo-plus fertilization mutant, imp11, which is
genotypically mt- and has a lesion in the master sex-determining gene
mid, also provided suggestive evidence for docking
(Ferris and Goodenough, 1997).
After being mixed with wild-type mt- gametes, imp11 cells
transformed with the FUS1 gene underwent flagellar adhesion and
formed pairs of cells that seemed to be adherent also via their mating
structures. Attempts to determine whether the pairs were adherent via their
mating structures or their flagella by deflagellating the interacting gametes
with a pH shock led to fusion of the gametes. Although intriguing, such a
system did not lend itself to a good method for assaying mating structure
adhesion. In other studies, fusion-defective mt- mutants, including
the gam-1 mutant, were reported to bind to mt+ gametes via
their mating structures (Forest,
1987). Those experiments are difficult to interpret, though,
because the genes disrupted in the mt- mutants are unknown and the
mt- mutants exhibited normal flagellar adhesion, making it impossible
to determine whether the cells were adherent via their mating structures or
via their flagella. Moreover, the gam-1 mutant is reported to be
defective in gamete activation (Forest
et al., 1978) and, therefore, the mating structures of
the gam-1 cells would not have been activated in those
experiments.
More direct observation of mating structure adhesion came from previous
studies with isolated fertilization tubules. We showed that fertilization
tubules isolated from activated, wild-type mt+ gametes bound to the
mating structures of wild-type mt- gametes. Only a single
fertilization tubule bound to each mt- gamete and organelles isolated
from trypsin-treated wild-type mt+ gametes did not bind
(Wilson et al.,
1997). In the experiments reported herein, we examined docking by
use of activated imp12 mt- gametes. These cells contained activated
mating structures, as evidenced by their ability to fuse with activated
wild-type mt- gametes, but they did not express functional flagellar
agglutinins. Therefore, we were able experimentally to detect mating structure
adhesion without the interference of flagellar adhesion. Our results that
activated fus1-1 gametes failed to form pairs with activated
imp12 gametes (Figure
4) directly demonstrated that the FUS1 gene product is
required for adhesion between the plasma membranes of plus and
minus mating structures during gamete fusion.
The observation that Fus1 is required for membrane adhesion does not
address the question of whether it is also required for the next stage in cell
fusion, the actual merging of the lipid bilayers of the two adherent
membranes. During fusion of intracellular membranes in the secretory pathway,
the proteins involved in vesicle adhesion are also strongly implicated in the
subsequent fusion event. In this case, transmembrane proteins on both
interacting membranes are proposed to participate directly in bilayer fusion
(Jahn and Sudhof, 1999). In
several viral systems, the virus adheres to its target cell via interactions
between a viral transmembrane protein and a receptor protein on the target
cell. After undergoing a conformational change, which exposes a socalled
fusion peptide that inserts into the target cell membrane, the viral protein
participates directly in bilayer fusion
(Doms and Moore, 2000;
Eckert and Kim, 2001).
The analysis of the Fus1 sequence offers only limited insights into its
role in fertilization, especially because no cell-cell fusion proteins have
been identified in eukaryotes. The resemblance of Fus1 to bacterial adhesion
proteins described above and the absence of an obvious "fusion
peptide" (Ferris et al.,
1996) or other domains found in viral or vesicle fusion proteins,
suggest that Fus1 might be involved only in adhesion. On the other hand,
because we do not yet have an even rudimentary understanding of the molecular
mechanisms of fusion initiated at the external surfaces of plasma membranes in
any eukaryotic system, it is too early to establish whether Fus1 has more than
one role in bilayer fusion. Future studies, in which intact FUS1
constructs and FUS1 constructs with selected domains deleted are used
to transform fus1 mutants, should provide new insights about whether
Fus1 also participates directly in membrane fusion.
The Fus1 Protein Is in the Right Place at the Right Time for a Direct
Role in Docking and Fusion
The identification and characterization of the endogenous Fus1 protein
document that Fus1 is in the right place at the right time to play a direct
role in mating structure interactions. The results of our adhesion bioassays
and results from previous studies (Ferris
et al., 1996) were consistent with the idea that Fus1 is
on the surface of the fertilization tubule. On the other hand, Fus1 could have
been an intracellular membrane protein with only an indirect role in gamete
fusion. For example, the protein calmegin is essential for normal fertility in
mouse, but calmegin is an endoplasmic resident chaperone and not expressed at
the cell surface (Watanabe et
al., 1995; Ikawa et
al., 2001; Yamagata
et al., 2002). The result that Fus1 was localized to the
external surface of wild-type plus mating structures was exciting
because it placed the protein in the proper cellular compartment to be
directly involved in interactions between the membranes of the mating
structures. Moreover, the surface localization experiments revealed that not
just a portion of total cellular Fus1 was on the surface of unactivated
gametes; essentially all detectable Fus1 was surface localized
(Figure 9).
It will be interesting to learn the molecular mechanisms that underlie this
striking localization to such a restricted area of the gamete surface. It is
likely that the molecular mechanisms that target Fus1 to the specialized
microvillus in Chlamydomonas will be similar to microvillus targeting
mechanisms in the gametes of multicellular organisms. Adhesion and fusion in
mouse eggs occurs in the region of the egg surface that is replete with
microvilli; and the microvillous-like acrosomal extension in the sperm of many
invertebrates is specialized for membrane fusion (for review, see
Wilson and Snell, 1998). The
sequence analysis of Fus1 predicts that <10 amino acids are in the
cytoplasm, and this short region does not contain obvious features, such as
protein interaction domains, that might provide clues about how it is
localized. The presence of the protein on the unactivated organelles
was also surprising, because docking assays showed that unactivated mating
structures are incapable of adhering
(Figure 3). We should note that
there is a precedent for the existence of inactive forms of cell surface
adhesion molecules in Chlamydomonas. Flagellar agglutinins are
present in an inactive form on the external surface of the cell body plasma
membrane and become active only after delivery to the flagella
(Hunnicutt et al.,
1990). Cell surface integrins in mammalian cells also exist in
active and inactive forms under the regulation of signal transduction pathways
(Hughes and Pfaff, 1998). One
explanation of our results is that that signals generated during gamete
activation render Fus1 active for docking and possibly for fusion. For
example, Fus1 alone could undergo posttranslational, activating modifications.
Or, gamete activation might release inhibitory Fus1-associated proteins, or a
second protein could become available or competent to interact with Fus1 to
form an active complex.
Another facet of Fus1 demonstrated by the localization studies is that the mechanisms that constrain its location to the mating structure before gamete activation likely persist in some form after activation. Thus, the protein did not spread over the cell body plasma membrane after activation. On the other hand, it did not remain at the base of the mating structure after activation, nor did it all occur as a patch at the tip of the fertilization tubule. Thus, although its location still is restricted, it can be mobilized. Given its distribution along the length of the organelle, it will be important to learn whether the sides of the fertilization tubules are competent for adhesion and fusion. In addition, future studies that identify putative plus gamete proteins that interact with Fus1 should provide insights into the mechanisms that underlie the remarkable preactivation localization and subsequent activation-induced redistribution over the entire surface of the fertilization tubule.
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Footnotes |
|---|
-tosyl-L-lysine chloromethyl ketone HCl. * Corresponding author. E-mail address: william.snell{at}UTSouthwestern.edu.
|
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L, cell walls were removed with lysin; L
