2552 Research Article

IntroductionRegulated exocytosis, whereby specialized vesicles are signaledto fuse with the plasma membrane (PM), is a necessary step formany cellular processes. These include the relay of neuronalsignals, the acrosome reaction during sperm maturation, and therepair of torn membranes in a variety of cell types (reviewed byGerasimenko et al., 2001; McNeil and Steinhardt, 2003).Although different from the acrosome reaction, the sessilespermatids of C. elegans must fuse multiple vesicles, calledmembranous organelles (MOs), to their PMs in order to matureinto crawling spermatozoa capable of fertilization. This fusionevent contributes new membrane and proteins to the PM(Chatterjee et al., 2005; Roberts et al., 1986; Ward et al., 1981;Xu and Sternberg, 2003). MOs are specialized, ER-derivedvesicles that have a bi-lobed structure composed of a smallerhead that is separated by an electron dense collar from a largerbody. In fer-1 mutants, MOs do not fuse during spermatozoonmaturation, even though MO heads abut the PM as if they aredocked in preparation for fusion. A short pseudopod forms onfer-1 mutant spermatozoa, which moves ineffectively resultingin non-motile spermatozoa. Consequently, fer-1 spermatozoacannot adhere to the uterine walls and are swept out of thespermatheca in hermaphrodites by the passage of oocytes (Wardet al., 1981; Ward and Miwa, 1978; Ward et al., 1982).

When fer-1 was identified and sequenced, no other proteinshad strong resemblance (Achanzar and Ward, 1997).Subsequently, homologs were found by sequence similarity inhumans and mice, forming a family of similar proteins nowcalled ‘ferlins’ (Bashir et al., 1998). Two of the human

homologs, otoferlin and dysferlin, have mutations that causehuman diseases. Mutations in otoferlin lead to nonsyndromicdeafness DFNB9 (Yasunaga et al., 2000; Yasunaga et al., 1999)and mutations in dysferlin result in the autosomal recessivemuscle diseases limb girdle muscular dystrophy 2B(LGMD2B) and Miyoshi myopathy (Bashir et al., 1998; Liu etal., 1998). Similar to the fer-1 mutant phenotype,LGMD2B/MM patients display defects in vesicle fusion intheir muscle tissue. This results in an accumulation of vesiclesbelow the sarcolemma during membrane repair aftermechanical stress (Cenacchi et al., 2005; Selcen et al., 2001).Immunolocalization studies place normal dysferlin in thesarcolemma and in cytoplasmic vesicles. The PM localizationis eliminated in LGMD2B/MM patients and cytoplasmicvesicle localization is only occasionally observed (Anderson etal., 1999; Bansal et al., 2003; Piccolo et al., 2000).

The ferlin family proteins are characterized by multiple C2domains, a C-terminal transmembrane domain, and most havenested repeat sequences, termed Dysf-N and Dysf-C, of unknownfunction. C2 domains characterized in other proteins such assynaptotagmin, protein kinase C, and phospholipases, have beenfound to bind Ca2+, phospholipids and phosphotyrosine (forreviews, see Bai and Chapman, 2004; Nalefski and Falke, 1996;Rizo and Sudhof, 1998; Sondermann and Kuriyan, 2005).Additionally, C2 domains can mediate protein-proteininteractions. Some of these interactions depend on Ca2+ whereasothers do not require Ca2+ (Chapman et al., 1996; Chapman etal., 1995; Davis et al., 1999; Fukuda and Mikoshiba, 2000;Rickman and Davletov, 2003; Sutton et al., 1999).

FER-1 is required for fusion of specialized vesicles, calledmembranous organelles, with the sperm plasma membraneduring Caenorhabditis elegans spermiogenesis. Toinvestigate its role in membranous organelle fusion, weexamined ten fer-1 mutations and found that they all causethe same defect in membrane fusion. FER-1 and the ferlinprotein family are membrane proteins with four to sevenC2 domains. These domains commonly mediate Ca2+-dependent lipid-processing events. Most of the fer-1mutations fall within these C2 domains, showing that theyhave distinct, non-redundant functions. We found thatmembranous organelle fusion requires intracellular Ca2+

and that C2 domain mutations alter Ca2+ sensitivity. Thissuggests that the C2 domains are involved in Ca2+ sensingand further supports their independent function. Using twoimmunological approaches we found three FER-1 isoforms,

two of which might arise from FER-1 by proteolysis. Byboth light and electron microscopy, these FER-1 proteinswere found to be localized to membranous organellemembranes. Dysferlin, a human homologue of FER-1involved in muscular dystrophy, is required for vesiclefusion during Ca2+-induced muscle membrane repair. Ourresults suggest that the ferlin family members share aconserved mechanism to regulate cell-type-specificmembrane fusion.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/119/12/2552/DC1

Key words: Membrane fusion, Spermatogenesis, fer-1, Calcium,Membranous organelle, C2 domain

Summary

FER-1 regulates Ca2+-mediated membrane fusionduring C. elegans spermatogenesisNicole L. Washington and Samuel Ward*Department of Molecular and Cellular Biology, The University of Arizona, 1007 E. Lowell Street, Life Sciences South 452, Tucson, AZ, 85721, USA*Author for correspondence (e-mail: samward@email.arizona.edu)

Accepted 16 March 2006Journal of Cell Science 119, 2552-2562 Published by The Company of Biologists 2006doi:10.1242/jcs.02980

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2553FER-1 in Ca2+-mediated membrane fusion

The two C2 domains of synaptotagmin have been the mostthoroughly studied and are thought to be the Ca2+ sensors formany types of regulated exocytosis. These C2 domains caninteract with phospholipids in a Ca2+-dependent manner, and canalso interact directly with other proteins of the membrane fusionmachinery such as synaptobrevin and syntaxin (Chapman et al.,1995; Gerona et al., 2000; Rickman and Davletov, 2003). Theseinteractions promote vesicle exocytosis in neurons, andlysosomal exocytosis during PM repair of fibroblasts (Brose etal., 1992; Rao et al., 2004; Reddy et al., 2001). Vesicle fusionduring membrane repair in muscle tissue requires dysferlin andis triggered by a local influx of extracellular Ca2+ near sites ofmembrane disruption (Bansal et al., 2003). This suggests amodel in which dysferlin might facilitate membrane repair byresponding directly to a Ca2+ signal to trigger vesicle fusion(Bansal and Campbell, 2004). The protein sequence similaritiesand the similarity of cellular mutant phenotypes betweendysferlin and FER-1 suggests a common role for these proteinsduring cell-type-specific membrane fusion events.

C. elegans sperm provide an excellent model to study themechanisms involved in ferlin-regulated membrane fusion. C.elegans is easily manipulated genetically, and its spermatidscan be readily isolated for in vitro examination andbiochemical analysis. The MOs of spermatids accumulatebelow the PM and only fuse during spermiogenesis when thespermatids are activated to form spermatozoa in response to anin vivo or in vitro chemical signal. Thus, MO fusion is readilymanipulated, allowing analysis of fusion in real time. Also,since spermatozoa activated in vitro retain fertility, asdemonstrated by artificial insemination, the in vitro processmimics normal spermiogenesis (LaMunyon and Ward, 1994).

We have investigated the role of FER-1 in MO fusion usingthree approaches. First, we identified and characterized thephenotypes of ten fer-1 mutations. Second, we found that MOfusion is sensitive to intracellular calcium, particularly in fer-1 C2 domain mutants. Third, we showed that there are threeisoforms of FER-1 in sperm and these are localized to the MOmembranes.

Resultsfer-1 mutations and mutant phenotypesFive of the mutations in fer-1 were identified in the initialcloning of the gene before its human homologs and functionaldomains were recognized (Achanzar and Ward, 1997). We

have sequenced the remaining five fer-1 mutant alleles, for atotal of six temperature-sensitive and four non-conditionalalleles, each having a single base-pair mutation in fer-1 thatalters the encoded protein. Table 1 lists, and Fig. 1 shows, thelocation of the predicted protein sequence changes caused byeach mutation in relation to the conserved domains predictedfor several ferlin family members. Two mutants introduce earlystop codons. Seven of the eight missense mutations fall withinpredicted C2 or DYSF domains. The other missense mutation,hc80, is located between the last two C2 domains in a regionof eight consecutive amino acid identities amongst the differenthomologs, which suggests another important functional region.It is striking that seven of the eight missense mutations fall intopredicted domains, suggesting that these regions are moresensitive to amino acid substitutions than the rest of FER-1 andthat each domain has a distinct and essential function.

That each of the C2 domains of FER-1 has distinct functionsis supported by sequence analysis. A BLAST search (Altschulet al., 1990) with FER-1 identified one or more homologs inevery animal genome that has been sequenced, but a strikingabsence in fungi and plants (Fig. 1). All ferlin proteins arecharacterized by the presence of multiple C2 domains,followed by a C-terminal transmembrane domain. To betterunderstand the relationship between the C2 domains found inferlins and in other well-studied proteins, we combined theindividual ferlin C2 domain sequences with the 64 non-ferlin

Table 1. Mutations in fer-1 allelesfer-1 allele Genomic mutation† Amino acid change‡ Domain

hc1 1479 g r a 290 G r Q C2-Chc47 2528 g r a 494 A r * –hc13 3266 c r t 709 A r V DYSFhc91 3494 t r c 785 M r T DYSFeb7 4557 g r a 1081 W r * –hc136 5582 g r a 1345 E r K C2-Eb232 6006 g r a 1486 S r N C2-Ehc80 6568 g r a 1657 E r K –hc82 6904 c r t 1746 H r Y C2-Fhc24 7197 c r t 1817 L r F C2-F

†Genomic position determined from start codon at position 290 inGenBank accession U57652.

‡At indicated amino acid of GenBank accession AAB02243.* indicates STOP codon.

DYSF

DYSF

DYSF

DYSFG. galus

A. mellifera

C. intestinalis

T. nigroviridis

DYSFX. tropicalis

D. melanogaster

T. Castaneum

F. rugripes

hc1ts hc91tshc13ts eb7*

A B C D E FDYSF

DYSF

TM

DYSF

C. elegans

H. sapiens

C. briggsae

DYSF

FER-1

FERL-1

Dysferlin

Myoferlin

Otoferlin

hc47* hc24ts

hc82ts

b232ts hc80

hc136

S. purpuratus (partial)

250 aa

b DYSF

Fig. 1. Ferlin domains and FER-1 mutations. FER-1 and severalhomologs are aligned with their domain composition indicated. Mostferlins have four or more C2 domains, which are shown as coloredshapes reflecting similar positions in the various ferlin proteins.DYSF (striped rectangle) has nested DysfN and DysfC domains; TM(black diamond) transmembrane domain. Predicted protein changesfor all fer-1 alleles are shown here and described in Table 1; *, stopcodon. Letter designations for C2 domains in dysferlin are fromDavis et al. (Davis et al., 2000). Dotted lines below FER-1 indicateantigenic sites for anti-DysfNC (left) and anti-AZ10 (right). Seesupplementary material (Table S1 and Fig. S1) for additionalhomologs.

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C2 domains aligned by Nalfeski and Falke (Nalfeski and Falke,1996). From this new alignment we constructed a phylogenetictree of these C2 domains. Fig. 2 illustrates a summary tree withthe ferlin C2 domain branches color coded (as in Fig. 1) toreflect their position in the ferlin proteins (see supplementarymaterial Fig. S2 for the full tree). Each colored branch in thetree represents individual ferlin C2 domains, which fall intodistinct clades based on their color. This shows for all ferlins

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that a given C2 domain is more similar to others at a similarposition in the protein than it is to the other C2 domains withinthe same protein. This strengthens the argument that each C2domain has a distinct function, and suggests that this functionhas been preserved through ferlin protein evolution.

All fer-1 mutants have a sterile phenotype, but only the hc1tsand hc24ts mutants had been previously examined by electronmicroscopy and found to have temperature-sensitive defects inMO fusion (Ward et al., 1981). To see if fer-1 mutations indifferent domains caused distinguishable phenotypes, wedeveloped a fast quantitative assay to examine MO fusion inlive cells. When applied to spermatids, the lipophilic dye FM1-43 partitions into the outer membrane leaflet and fluoresces,allowing the spermatid PMs to be visualized (Fig. 3A,B).When MOs fuse during the maturation of spermatids intospermatozoa, they leave stable membrane invaginations, whichshow as bright puncta around the cell body periphery in thepresence of FM 1-43 (Fig. 3C,D). Up to five individual MOscan be counted accurately, thereafter the fluorescence mergesand multiple MO fusions make the cell body periphery toobright to distinguish individual MOs. The formation of thesepuncta can be followed in real time since FM 1-43 does notinterfere with spermatozoa development.

No MO fusion was observed in either triethanolamine(TEA)-activated spermatozoa from fer-1 mutants with stopcodons (hc47 and eb7, n>400, each), which have no FER-1(see following section), or in two non-conditional missensemutants (hc80 and hc136), although spermatozoa from allmutants still formed short pseudopods (Fig. 3E,F). Togetherwith the previous observation that fer-1(hc1)/nDf23 has thesame defective phenotype as hc1/hc1 (L’Hernault et al., 1988),this confirms that lack of MO fusion is the null phenotype.

To look for subtle effects that might be caused by thedifferent fer-1 temperature-sensitive mutations in the C2 andDYSF domains, we compared MO fusion in mutant and wild-type TEA-activated spermatozoa at 15°C, 20°C, and 25°C.After 20 minutes, MO fusions in activated spermatozoa fromhc1ts, hc13ts and hc24ts mutants were indistinguishable fromwild-type at the permissive temperature of 15°C (Fig. 3A-D,I).The hc82ts cells had a more severe phenotype with only 6%of cells displaying normal MO fusions. At 20°C, MO fusionsin both hc24ts and hc82ts mutant sperm were reduced. Sincethese mutations both lie in the same domain, MO fusion maybe particularly sensitive to changes in this domain. At 25°C,none of the mutants had normal MOs fusions when comparedto wild type (Fig. 3G,H), although 10% of hc24ts spermunderwent one or two MO fusions, consistent with its leakyphenotype at 25°C (Ward et al., 1981). These results show thatalthough FER-1 contains six predicted C2 domains, for threeof them, just a single amino acid substitution results indefective MO fusion. The mutant phenotypes, together with thesequence analysis, suggest each of the C2 domains isnecessary, so their functions cannot be redundant.

Multiple isoforms of FER-1 protein are found in spermTo study FER-1, we developed two affinity purified rabbitpolyclonal antibodies to regions indicated in Fig. 1. The anti-DysfNC antibody was used for western blotting, and the anti-peptide antibody, AZ10, which did not work for western blots,was used for immunofluorescence studies (see below). As acontrol for antibody specificity we used fer-1(hc47) males,

C2A

synapt.

PLCs

PKCs(���)

PKCs(����)

RasGAPPI3Ks

C2D

C2F

C2E

C2C

C2B

C2de

76

86

62

87

72

100

70

*

*

*

*

*

**

*

*

*

*

*

77

58

100

100

96*

*

*

62

*

*

*

*

*

*

*

*

Fig. 2. Ferlin C2 domain phylogeny. A summary phylogenetic tree ofC2 domain sequences of one representative ferlin from each speciesand several other C2 domain-containing proteins is shown. The treeis based on neighbor-joining of C2 domains aligned using ClustalWand refined by hand. Ferlin C2 domains are colored as in Fig. 1,based on their position. The tree shows that the C2 domains with thesame color cluster together indicating they are most similar to eachother. Non-ferlin C2 domain sequences were previously analyzed byNalfeski and Falke (Nalfeski and Falke, 1996) (black lines).Bootstrap support indicated along branches for major clades; asterisk(*) indicates <50%. Similar results were obtained with maximumparsimony analysis. Abbreviations: PLCs, phospholipases; Rabs,rabphilins; synapt, synaptotagmins; PKCs, protein kinase C.Additional sequences, together with the individual protein names, areincluded in supplementary material Fig. S2.

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2555FER-1 in Ca2+-mediated membrane fusion

which have a stop codon prior to either the anti-DysfNC or theanti-AZ10 antigenic sites, and presumably have no FER-1protein. We compared wild-type male and fer-1(hc47) mutantmale worm extracts because it was difficult to isolate enoughmutant males from which to purify spermatids. On westernblots, three proteins that labeled with anti-DysfNC weredetected in wild-type males, each of which were missing fromfer-1(hc47) males (Fig. 4A). The largest protein migrated aboutthe expected size of FER-1 (230 kDa), but two additional bands

were observed at 195 and 180 kDa in male worm extracts.Since all three bands were missing from fer-1(hc47) males, thissuggested they were likely to be isoforms of FER-1. When weexamined proteins from isolated spermatids, we observed thesame doublet of 195 and 180 kDa proteins, which were absentfrom sperm-less hermaphrodites confirming their spermspecificity (Fig. 4B). Full-length FER-1 was present, but barelydetectable in spermatids. This difference might be due to thepresence of spermatocytes in the males, which are absent inpurified spermatid preparations. If so, this suggests that thefull-length FER-1 protein is present early in spermatogenesisand then becomes less abundant in spermatids, perhapsbecause it is proteolytically processed into the smallerisoforms.

To ensure that the 195 and 180 kDa proteins were derivedfrom FER-1, we immunoprecipitated proteins from wild-typespermatid extracts using anti-DysfNC, separated these by SDS-PAGE and isolated the bands. These were subjected to LC-MS/MS, and peptide masses were matched against the C.elegans proteome. Insufficient protein was obtained to identifyfull length FER-1, but multiple peptides matching FER-1 wereidentified from the 195 and 180 kDa bands confirming that theywere encoded by the fer-1 gene (Fig. 4C; supplementarymaterial Table S2).

The presence of multiple FER-1 isoforms might have arisenby alternative splicing, since multiple splicing variants areobserved for human dysferlin, myoferlin, and otoferlin, andsome of these are known to be translated (Bashir et al., 1998;Ho et al., 2004; Liu et al., 1998; Salani et al., 2004; Yasunagaet al., 2000; Yasunaga et al., 1999). To test this we isolatedadditional fer-1 cDNAs from a sperm-enriched cDNA libraryby PCR. In addition to the full-length 6.2 kb transcript, we

Fig. 3. Temperature sensitivity of MO fusions due to different fer-1 alleles. (A-H) MO fusion assay. Spermatids were activated with 100 mMTEA in the presence of FM 1-43 to visualize PM and MOs upon fusion. Top, DIC images; bottom, FM 1-43 staining. Bar, 5 �m.(A,B) Unactivated spermatids are rounded and only display PM staining. (C,D) Wild-type (shown here) or temperature-sensitive mutants atpermissive temperature (15°C) fuse many MOs, which show as bright puncta in the cell body. The PM of the pseudopod is labeled but is devoidof puncta since the MOs only fuse with the cell body PM. Because the images are taken of live cells that have moving pseudopods, the PM ofspermatozoa does not appear to be as bright as in spermatids. Pseudopod indicated by arrowhead. (E,F) Non-conditional alleles hc47, eb7(shown here), hc80, and hc136 develop pseudopods but never fuse MOs. (G,H) Sperm with temperature sensitive mutations hc1ts, hc13ts(shown here), hc91ts, hc24ts, and hc82ts fuse few to no MOs at the restrictive temperature (25°C). (I) Quantitation of MO fusion during TEAactivation of wild-type or temperature sensitive fer-1 (hc13ts, hc1ts, hc24ts and hc82ts) sperm grown at 15°C, 20°C or 25°C. Cells wereactivated for 20 minutes and scored for normal MO fusions (�5 MO fusions/cell). Results are averages for three worms/temp., in duplicate,with n>2000. Error bars=s.e.m. The significance of differences from wild type are indicated by * (P<0.001) and � (P<0.05).

Fig. 4. Characterization of FER-1 protein isoforms. (A) Affinitypurified polyclonal anti-DysfNC antibody recognizes three proteinsin whole males (wt), which are absent in the stop codon mutant fer-1(hc47). Protein from 250 virginized male worms/lane. (B) Anti-DysfNC western blot of him-5 whole male (�), fem-1 (spermless)hermaphrodites (�), and fem-3(q20) purified spermatid (sp) proteinextracts. Numbers on right indicate calculated molecular masses, inkDa. (C) Distribution of peptides identified by mass spectrometryfrom 195 and 180 kDa bands depicted along full-length FER-1protein (images generated by Tandem at http://www.thegpm.org).Peptides are listed in supplementary material Table S2.

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found only two new cDNA clones, 3.0 and 2.8 kb. These weretoo small to encode proteins corresponding to the 185 and 195kDa bands (see supplementary material Fig. S3). Thus,alternative splicing is unlikely to explain these FER-1isoforms, suggesting they might arise from proteolyticcleavage.

FER-1 localizes to the MO in spermatidsSince fer-1 mutants show defects in MO fusion, and FER-1 isa predicted type II (cytoplasmic facing) membrane protein, weanticipated that FER-1 would localize to MO membranes. Thesecond antibody, anti-AZ10, was utilized forimmunolocalization of FER-1, since anti-DysfNC antibodiescross reacted with an additional protein band on western blots(110 kDa) that was not eliminated in the fer-1(hc47) mutant.With anti-AZ10, punctate staining was observed around theperiphery of permeabilized spermatids, confirming that spermcontain FER-1 (Fig. 5B). These puncta were eliminated in fer-1(hc47) mutant sperm (Fig. 5J).

To determine if FER-1 puncta corresponded to MOs,spermatids were co-stained with 1CB4, an antibody that labelsMOs in sperm as well as the sperm PM and other structures ina few other worm cells (Okamoto and Thomson, 1985). Co-localization was only observed on the MOs, but not on the PM(Fig. 5C,D). In mature sperm, 1CB4 still labeled punctatestructures only in the cell body, presumably some fused andunfused MOs, and the PM weakly (Fig. 5G). Anti-AZ10,however, only partially co-localized with these punctatestructures in spermatozoa, with most of the signal observed inthe PM (Fig. 5H) including distinct labeling of the pseudopodmembrane. These antibody labeling experiments indicate thatFER-1 is localized to MO membranes in spermatids and movesinto the PM upon MO fusion during sperm activation. Thispattern is similar to that previously observed for the MO-

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localized Ca2+ channel, TRP-3 (=SPE-41) (Xu and Sternberg,2003), but differs slightly from the tetraspanin SPE-38, whichis also found in the MO using an antibody to an extracellulardomain (Chatterjee et al., 2005).

FER-1 localization by electron microscopyWe utilized quantitative immunoelectron microscopy tolocalize FER-1 more precisely. Isolated spermatids wereactivated with TEA to form spermatozoa, prepared forimmunoelectron microscopy, sectioned, labeled with anti-AZ10 or 1CB4 antibodies and detected with immunogoldlabeled secondary antibodies.

Anti-AZ10 predominately labeled MOs in spermatids, withno significant difference between labeling on the head andbody (Fig. 6A,E). FER-1 localization decreased in MO bodymembranes upon fusion, shown by a reduction ofimmunogold density between unfused and fused MOmembranes (Fig. 6B,G; see Materials and Methods forcalculation). In parallel, the labeling on the PM increasedtwo-fold following MO fusion, supporting the lightmicroscope observations that FER-1 moves into the PM uponfusion. PM labeling was similar between the pseudopod andthe cell body, suggesting that FER-1 is not restricted in itsmovement within the PM (Fig. 6G, inset). In addition, weobserved an increase in localization over the pseudopodcytoplasm, which is higher than in the cell body either beforeor after activation (Fig. 6B,E).

Quantification of 1CB4 antibody labeling revealed itshighest density on MOs in spermatids, with a slight enrichmentin the MO body versus the MO head (Fig. 6C,F). In contrastto FER-1, 1CB4 immunogold density increased along MOmembranes upon fusion (Fig. 6D,H). This increase occursdespite the release of MO luminal contents, indicating that theunidentified antigen recognized by 1CB4 is membrane

Fig. 5. Immunofluorescent localizationof FER-1 in sperm.(A-J) Deconvoluted images ofimmunostaining performed onspermatids or TEA-activatedspermatozoa. Spermatids weredissected from 2-3 day virginized him-5 or fer-1(hc47) males. Cells werestained with anti-AZ10 (green)antibodies to detect FER-1 and with1CB4 (red) to label MOs. Bar, 2 �m.(A-D) FER-1 is localized to punctatestructures and colocalizes with 1CB4.(E-H) FER-1 is localized in the plasmamembrane of mature sperm, andpartially colocalizes with 1CB4.Arrowheads indicate visiblepseudopods. (I-J) fer-1(hc47) sperm,which have a stop codon prior to theAZ10 antigenic site and make no FER-1. (J) No staining is detected in fer-1(hc47) sperm at the same exposureused for normal sperm.

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associated. Thus, different MO proteins can have differentmembrane distributions following MO fusion.

Since the pattern of FER-1 membrane labeling resembledthat of TRP-3 by light microscopy, we used the anti-TRP-3antibody (gift from P. Sternberg, Caltech, CA, USA) toexamine its distribution by electron microscopy. Because thetotal labeling for anti-TRP-3 was much lower than that for anti-AZ10 or 1CB4, we scored for the fraction of gold particles thatlocalized to the MO head versus MO body. MO headmembranes had 23% of TRP-3 labeling (n=83) whereas AZ10and 1CB4 had 16% and 20% labeling of these compartments,respectively. Since the head membrane comprisesapproximately 20% of total MO membranes in these crosssections, all three antigens have a similar distribution in thehead and body MO membranes. This suggests that these MOprotein components are not localized to specific parts of thisorganelle prior to its fusion with the PM.

A Ca2+-dependent mechanism is involved in TEA-stimulated MO fusionMany exocytotic events are stimulated by a rise in Ca2+

concentration. Since FER-1 has six putative C2 domains,which might bind Ca2+, and single missense mutations withinthree of these domains block MO fusion, it is reasonable topredict that Ca2+ triggers MO fusion. Previous experiments,however, showed that external Ca2+ was not the source of thisactivity, since MO fusion still proceeds in the presence of 1mM EGTA (Shakes and Ward, 1989).

Using the membrane-permeable Ca2+ chelator BAPTA-AM,we asked if depletion of intracellular Ca2+ stores would preventMO fusion. Spermatids were incubated with BAPTA-AM for30 minutes at 15°C and triggered to fuse their MOs with TEA.MO fusion was again monitored with FM1-43. No effect wasseen at 1 �M, but the fraction of cells with fused MOs droppedfrom 21% to 4% as the BAPTA-AM concentration increasedfrom 2 to 10 �M (Fig. 7A,B). These results suggest that Ca2+

from an internal source is indeed required for MO fusion.

fer-1 mutants are hypersensitive to intracellular Ca2+

depletionIf MO fusion responds to changes in intracellular Ca2+, and ifat least some FER-1 C2 domains sense Ca2+, we reasoned thatthe single amino acid substitutions found in the fer-1 C2domain mutants might alter the sensitivity of the cell to theCa2+ changes leading to MO fusion. Therefore, we testedsperm from fer-1 temperature-sensitive mutants with C2domain mutations for MO fusion sensitivity to Ca2+ depletion.We did this at permissive temperature, where the protein muststill function sufficiently to allow MO fusion when Ca2+ levelsare normal. With 1 �M BAPTA-AM treatment MO fusion wasnormal in wild-type sperm but was reduced in the four fer-1mutants tested (Fig. 7C,D). All mutants were significantly

Fig. 6. Distribution of FER-1 and 1CB4 in spermatids andspermatozoa. (A,B) Ultrastructural localization of FER-1 with AZ10antibodies in wild-type (A) spermatids and (B) spermatozoa.(C,D) Localization of 1CB4 staining in wild-type (C) spermatids and(D) spermatozoa. MO, membranous organelles; M, mitochondrion;LM, laminar membranes; P, pseudopod. Arrowhead indicates MOhead; double arrowhead indicates MO body. Bar, 500 nm.(E-H) Immunogold label was quantitated over cellular compartments(E,F) and along membranes (G,H), for AZ10 (E,G) and 1CB4 (F,H).AZ10 labels MOs at the highest density (E), which decreases in theMO membrane and increases in the PM after fusion (G). 1CB4 labelsMOs most abundantly (F), which increases ~two-fold in densityalong MO membranes and simultaneously increases slightly in thePM after fusion (H). Insets in E and F show average totallabeling/cell for spermatids (tid) or spermatozoa (zoa) is notstatistically different for either antibody. Insets in G and H showlabeling over cell body (CB) or pseudopod (P) plasma membranes,which are not statistically different for either antibody. Graphindicates the average labeling over entire PM. Although nuclearlocalization was observed at a density similar to that of the MO byimmunogold staining with AZ10, we observed no nuclear staining byimmunofluorescence and presume this is background staining due tothe charged nature of the nucleic acids. Error bars=s.e.m. *Differentfrom unfused body density (P<0.0001); **different from cell bodycytoplasm density (P<0.0001); + different from unfused body lineardensity (P<0.0001); ++ different from spermatid PM linear density(P<0.0001).

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different from wild type (P<0.001), and hc1ts, hc24ts andb232ts were all significantly different from their EGTA-treatedcounterpart (P<0.01). Since MO fusion in fer-1 C2 domainmutants is more sensitive to a reduction of internal Ca2+ thanwild type, this supports the argument that FER-1 responds toa Ca2+ signal, and further suggests that the C2 domains areinvolved in this Ca2+ response.

Discussionfer-1 was originally identified in sterile mutants with defectivesperm. Two alleles were found to be defective in the fusion ofsperm MOs with the PM. When the fer-1 gene was identified,its sequence gave little insight into its function. Subsequently,homologs have been found in many animals, including twoproteins associated with human diseases, and this family ofproteins was named ‘ferlin’ after its founding member. Here,we have focused on analyzing the membrane fusion phenotypein order to elucidate the function of FER-1.

Using a simple light microscopic assay to detect MOfusions, we found that all of the fer-1 mutations cause defectsin MO fusion. This includes seven missense mutations, whichfall within predicted functional domains, one missense

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mutation in a conserved region between domains, and twomutants that generate stop codons. Since both western blottingand immunofluorescent staining show FER-1 protein(s) areeliminated in the stop codon mutant fer-1(hc47), the MO fusionphenotype must be the null phenotype, as predicted fromprevious genetic data (L’Hernault et al., 1988). In addition, theonly function of FER-1 appears to be its role in sperm MOfusion since fer-1 mutants have no phenotype other thansterility and expression of fer-1 is only detected duringspermatogenesis (Achanzar and Ward, 1997; Reinke et al.,2000).

Using an antibody specific for FER-1, we found that FER-1 is localized to MOs prior to fusion, and some of it movesinto the PM following fusion. Detailed analysis of FER-1localization by immunoelectron microscopy determined thatFER-1 is not confined to specific membranes within the MOstructure, but has a similar density in the head and bodymembranes of the MO. This is also true for the MO marker1CB4 antigen and for the previously studied Ca2+ channelTRP-3 (Xu and Sternberg, 2003). However, after fusion thedistribution of the 1CB4 antigen differs from that observed forFER-1 and TRP-3. It does not move past the electron dense

Fig. 7. TEA stimulates MO fusion by a Ca2+-dependent mechanism. (A) Spermatids were loaded with 1 mM EGTA and BAPTA-AM for 30minutes at the indicated concentrations, and the percentage of cells displaying normal MO fusions were counted. Data are representative ofthree independent experiments. (B) Spermatids treated with EGTA only or with 10 �M BAPTA-AM. Pseudopod formation also appears to beinhibited at high BAPTA-AM concentrations. (C) fer-1 mutant sperm are hypersensitive to internal calcium depletion. Wild-type or fer-1mutant sperm were incubated in EGTA or 1 �M BAPTA-AM for 30 minutes, then activated in 100 mM TEA in the presence of FM1-43, andscored for normal MO fusion. (D) Sample cells of each of the mutants treated with EGTA (top) or 1 �M BAPTA-AM (bottom). The domain inwhich the mutation lies is indicated above the images. Arrowheads indicate spermatozoa that have attempted to generate pseudopods, but haveno fused MOs. *Significantly different (P<0.01) from EGTA-treated cells with the same mutation. Error bars=s.e.m.

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collar, resulting in a net increase of 1CB4 antigen alongmembranes remaining in the fused MO cavity. These resultsshow that various MO-localized proteins can have differentdistributions following fusion, implying that the MO itself orthe fusion process can regulate membrane protein distributions.

Although the fer-1 mutants were obtained from randommutagenesis screens, the resulting mutations are non-randomlydistributed throughout the fer-1 sequence, with all but onelocated in predicted functional domains. Two mutations liewithin the DysfNC domain, a nested repeat sequence in manyferlins and several other proteins, whose function is unknown.Missense mutations within this domain in dysferlin have beenlinked to Miyoshi myopathy (Aoki et al., 2001; Matsumura etal., 1999) also indicating its importance.

Five fer-1 mutations (hc1ts, hc136, b232ts, hc82ts, hc24ts)fall within three of the predicted C2 domains. This suggeststhat these domains are essential for FER-1 function and areparticularly sensitive to amino acid substitutions. Since singlemissense mutations in these C2 domains result in defective MOfusion, each domain must have distinct, non-overlappingfunctions. This conclusion is further supported by sequenceanalysis of the C2 domains in ferlins from several species,including human dysferlin. Phylogenetic analysis of ferlin C2domains reveals that C2 domains at a similar position indifferent ferlins are more similar to each other than to the otherC2 domains within the same protein. Since most ferlins haveretained the full complement of C2 domains over evolution,this suggests that each must have an important function, andthat the positional order must also be important to overall ferlinfunction, most probably to ensure their orientation in the foldedprotein.

Vesicle fusion in a wide variety of cell types is stimulatedby a Ca2+ increase (Bi et al., 1995; Borgonovo et al., 2002;Eddleman et al., 1997; Reddy et al., 2001; Savina et al., 2003;Steinhardt et al., 1994) and detected by the two C2 domains ofthe protein synaptotagmin, located on the vesicle surface (Baiand Chapman, 2004; Brose et al., 1992). The presence ofmultiple C2 domains in FER-1, as well as its location on theMO membrane, suggests that it might be directly involved inresponding to a Ca2+ signal. Although extracellular Ca2+ is notrequired for MO fusion, we found that the intracellular chelatorBAPTA-AM could block MO fusion in a dose-sensitivemanner when applied to spermatids prior to their activation toform spermatozoa. Thus, like most other membrane fusionprocesses, Ca2+ appears to be essential for MO fusion.

Further evidence that FER-1 is directly involved inresponding to a Ca2+ signal is that fer-1 mutants withtemperature-sensitive missense mutations displayhypersensitivity to a reduction in free Ca2+ with BAPTA-AM,even at the permissive temperature of 15°C. Whereas wild-typesperm activated normally at 15°C, fer-1ts mutant sperm formedpseudopods but failed to fuse MOs, similar to the fer-1 nullphenotype. This was observed in all four of the C2 domainmutants tested. Although it is possible that these C2 domainmissense mutations might act indirectly by altering FER-1structure, because the sperm from each mutant showed a MOfusion defect when free calcium was reduced at the permissivetemperature, this result provides strong evidence that the C2domains of FER-1 directly mediate the FER-1 response to aCa2+ signal to trigger MO fusion.

Vesicle fusion during the repair of human muscle

membranes requires the fer-1 homolog dysferlin, as well asa localized spike in Ca2+ concentration at the sites of fusion,suggesting a role for Ca2+ in this ferlin-mediated fusionprocess (Bansal et al., 2003). In C. elegans sperm, a Ca2+

trigger could promote phospholipid binding or interactionswith other proteins directly involved in membrane fusion,such as SNAREs. In vitro experiments have shown that theN-terminal C2 domain of dysferlin and myoferlin (C2A) iscapable of Ca2+-dependent phospholipid binding, which isdisrupted when a disease-causing mutation is introduced(Davis et al., 2002). Our phylogenetic analysis also showsthat the C2A domain is the only ferlin C2 domain that clusterstogether with the majority of characterized C2 domains,which may explain its unique biochemical behavior. Becausefer-1 does not have this homologous C2 domain, yet issensitive to Ca2+ depletion, it suggests that a Ca2+ signal doesmore than promote phospholipid binding by the remaining C2domains. For example, the C2 domains together might actlike a scaffold to modulate protein-protein interactions,perhaps with SNARE proteins or other membrane fusioncomponents. The conserved order of the different C2 domainsmight specify a spatial organization necessary for theseinteracting proteins.

We found that there were three FER-1 protein isoforms insperm that were missing in the fer-1(hc47) mutant. The smallerproteins were confirmed by mass spectrometry to be fragmentsof FER-1. One explanation for the smaller protein bands is thatthe variants could arise by alternative splicing or alternate startsites. It has been shown that the human homolog OTOF haslong (240 kDa) and short (130 kDa) isoforms, which aregenerated with alternative transcriptional start sites, andmultiple smaller dysferlin transcripts have been identified invarious tissues. We isolated two new fer-1 cDNAs from asperm-enriched library and by RT-PCR, but they were toosmall to encode the observed FER-1 isoforms. Thus, we foundno evidence that the smaller FER-1 proteins arise fromalternative splicing, although we cannot rule out alternativestart sites.

The relative proportion of FER-1 isoforms was differentbetween protein samples prepared from whole males versuspurified spermatid preparations. The 230 kDa (full length)FER-1 was more abundant in males, and reduced inspermatids, whereas the 195 and 180 kDa isoforms wereabundant in both. Unlike males, purified spermatidpreparations include few spermatocytes, so it is likely that thefull-length FER-1 protein is made in spermatocytes and thencleaved to form the smaller isoforms during the maturation ofspermatocytes into spermatids. No matter the source of thesmaller isoforms, their peptides found by mass spectrometryinclude at least the domains between C2C and C2E and fromtheir size must include additional domains, but the exact endsof these isoforms are not known. We do not know the functionof this cleavage, but a possible candidate to cleave FER-1 isthe protein SPE-4, which is also localized to the MO. SPE-4is a divergent member of the presenilin family, which isinvolved in intramembranous proteolysis leading toAlzheimer’s disease (Arduengo et al., 1998; Brunkan andGoate, 2005). It is a surprising coincidence that two proteinsrelated to human diseases are both found in such an unusualorganelle as the nematode sperm MO, so this may suggestsome connection between these human diseases.

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Based on our results, we propose a model whereby FER-1regulates Ca2+-dependent vesicle fusion as depicted in Fig. 8. Inspermatids, MOs are ‘docked’ with their heads abutting the PMas the cells await the activation signal. This localization of theMO is not fer-1 dependent, since MOs appear to be dockedappropriately in fer-1 mutants (Ward et al., 1981). FER-1 proteinis localized uniformly to the MO membrane surface, but becauseof its orientation, or other as yet unidentified head-specificproteins, fusion occurs between the head membrane and PM.Given an activation signal, spermatozoa utilize intracellular Ca2+

to relay this signal, with FER-1 acting to relay a ‘fusion’ signal,which results in fused MOs at the point of contact between theirhead and the PM. As a result, FER-1 and some other MOmembrane proteins mix into the PM, while other membraneproteins remain trapped behind the fusion collar.

This model for FER-1 is similar to the model proposed byBansal and Campbell (Bansal and Campbell, 2004) for the roleof dysferlin in membrane repair. Both proteins reside incytoplasmic vesicles that fuse with the PM in response to aCa2+ signal. Once the vesicles fuse, FER-1/dysferlin is presentin the PM. fer-1 mutants and their Ca2+ sensitivity demonstratethe importance and non-redundancy of the multiple C2domains in the ferlin family. Taking advantage of the C.elegans genetic system, future studies to determine the exactfunction of FER-1 in MO fusion might identify additionalfunctions that are shared with dysferlin and other ferlin familymembers.

Materials and MethodsStrains and geneticsCulture, manipulation of worms and genetic analyses were performed with standardmethods (Brenner, 1974). All strains used in this work were derived from the wild-type C. elegans strain var. Bristol N2. Standard C. elegans nomenclature has beenused throughout this paper (Horvitz et al., 1979). The isolation of fer-1 (hc1ts,hc13ts, hc24ts, hc80, hc91ts, b232ts, hc47, hc82ts) was described previously (Argonand Ward, 1980; L’Hernault et al., 1988). The ethyl methane sulfonate-inducedallele fer-1(eb7) was a gift from S. L’Hernault (Emory University, Atlanta, GA).Genetic markers used were LGI, dpy-5(e61); LGIV, fem-1(hc17ts) (Nelson et al.,1978) and fem-3(q20ts) (Barton et al., 1987); LGV, him-5(e1490) (Hodgkin et al.,1979). Non-conditional alleles hc47, eb7, hc80 and hc136 were marked by linkageto dpy-5, and were maintained by picking Dpy L4 hermaphrodites, checking forsterility, and mating back to wild type. All strains were maintained at 15°C, unlessotherwise stated. The gene T05E8.1 was named ferl-1 (fer-like) based on itssequence similarity to fer-1.

PCR and sequencing of fer-1 mutationsGenomic DNA from temperature-sensitive mutants was obtained by washing offmixed-stage populations with buffer and adding PCR lysis buffer (50 mM KCl, 10mM Tris-HCl, pH 8.2, 2.5 mM MgCl2, 0.45% NP-40, 0.45% Tween 20, 0.1 mg/mlgelatin). For hc47 and eb7 non-conditional strains, dumpy hermaphrodite progenyof fer-1 dpy-5/++ were transferred to individual 6 mm plates and scored 24 hourslater for self-sterility. Sterile worms were picked directly into a 5 �l drop of PCRlysis buffer. Worms were frozen at –20°C and a worm lysate was prepared (Barsteadand Waterston, 1991). For DNA sequencing, 20 overlapping fer-1 gene segmentswere amplified from worm lysate by PCR with fer-1-specific primers, inquadruplicate, using Taq DNA polymerase (Promega Corp., Madison, WI, USA).Mutations were identified by sequencing PCR fragments in both directions(Genomic Analysis and Technology Core Sequencing Facility, The University ofArizona, USA). Only one mutation was found in each strain.

Sequence analysisSequences were obtained from Ensembl (www.ensembl.org) and analyzed withVectorNTI Advance 9.0. A table of accession numbers for ferlin protein sequencesis provided in supplementary material Table S1. Approximate C2 domainboundaries were initially determined by SMART (smart.embl-heidelberg.org) andadjusted by eye. Ferlin C2 domain sequences were aligned to an existing alignmentof non-ferlin C2 domains generated by Nalfeski and Falke (Nalfeski and Falke,1996) using ClustalW with default parameters, followed by adjustment by eye. Atree search was conducted using neighbor-joining algorithm in PAUP* 4.0(Swofford, 2003). Bootstrap values were obtained with 1000 replicates, with 10random sequence additions per replicate. The tree represents majority-ruleconsensus.

Fusion protein, peptides and antisera preparationFor antisera preparation one series of rabbits was immunized with a partial FER-1fusion protein representing amino acids 723-957, consisting of the DysfN-DysfCdomains fused in-frame after Schistosoma japonicum glutathione S-transferase(GST) in the pGEXM7 bacterial expression vector (gift from David Drechsel, MaxPlanck Institute for Cell Biology and Genetics, Dresden, Germany). Soluble proteinwas purified from E. coli extracts using a glutathione-Sepharose fast flow affinitycolumn according to the manufacturer’s instructions (Amersham Biosciences).Purified GST-DysfNC protein was used to inoculate rabbits and serum from thesewas selected by ELISA assay (Eurogentec, Herstal, Belgium). The same fusionprotein was covalently coupled to CNBr-activated Sepharose (Amersham) to createan affinity column. Anti-DysfNC antisera were first cleared of any anti-GST andanti-E. coli antibodies by passage through GST and E. coli affinity columns,followed by affinity purification over the GST-DysfNC column following themanufacturer’s instructions. Purified anti-DysfNC antibodies were combined andconcentrated for a final stock concentration of 2 mg/ml.

A second set of rabbits was immunized (Cocalico Biologicals, Reamstown, PA)with the synthetic peptide CKSMKGDFDDPEEKEK corresponding to residues1334-1348 with an N-terminal cysteine residue [Macromolecular StructuresFacility, Arizona Research Laboratories (W. E. Achanzar, Analysis of a generequired for membrane fusion during nematode spermiogenesis. PhD Thesis, TheUniversity of Arizona, 1996.)]. Antiserum (anti-AZ10) was purified by peptideaffinity chromatography and concentrated as above. Final stock concentration ofresulting anti-AZ10 peptide antiserum was 4 mg/ml.

fused

spermatidactivation

Ca2+

spermazoon

FER-1 1CB4 other MO-proteins Calcium

head

body

collar

unfused

PM

FER-1Fig. 8. Model for FER-1-mediated Ca2+-dependent MOfusion. MOs are bi-lobed structures comprising a head andmembrane-dense body, separated by an electron densecollar. Spermatids await an ‘activation’ signal, with MOheads docked at the PM prior to fusion. Upon receipt of anactivation signal, intracellular Ca2+ is utilized to relay thesignal to be received by FER-1 and by others required forMO fusion. When MOs fuse in spermatozoa, the head and aportion of MO body membrane is incorporated into the PM.A fusion pore remains surrounded by the collar, and theglycoprotein contents are spilled from MOs along themembrane surface of cell body. FER-1 and other proteinscan move into the plasma membrane, while the 1CB4antigen and presumably other proteins remain concentratedwithin the MO cavity. In fer-1 mutants, the MO heads abutthe PM but MOs do not fuse.

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Western blot analysisSpermatids were isolated from fem-3(q20) hermaphrodites or him-5(e1490) malesas previously described (Nelson et al., 1982). For western analysis using handpickedmales, individual L4-virgin him-5 or fer-1(hc47) dumpy male worms were pickedonto plates and allowed to accumulate sperm without mating for 2-3 days at 20°C.These were then picked and frozen at –20°C. Worms and/or sperm proteinpreparations for SDS-PAGE were resuspended in sperm medium, pH 7.8 [SM;Nelson and Ward (Nelson and Ward, 1980)], + 10 mg/ml glucose [SMG; Machacaet al. (Machaca et al., 1996)], sonicated, and TCA precipitated. Protein pellets weretaken up in 1� sample buffer, boiled, and electrophoresed on 15�20 cm 5-20%(0.4:1 acrylamide:PDA crosslinker) gradient gels and blotted onto Hybond-Pmembranes. Blots were probed with anti-DYSFNC 1:10,000, followed by HRP-conjugated donkey anti-rabbit (Pierce) at 1:50,000, and visualized with ECL-PlusChemiluminescent Reagent (Amersham). The anti-AZ10 antibody does not workon western blots, although it works properly by indirect immunofluorescence oncells.

Immunoprecipitation and mass spectrometry analysisFor immunoprecipitation from spermatids, 1.5�108 frozen isolated cells werehomogenized by sonication in 500 �l IP buffer (300 mM NaCl, 10 mM NaHPO4

pH 7.5, 5 mM EGTA, 0.2 mM EDTA, 1 mM MgCl2, 0.02% azide) with proteaseinhibitors (Complete, Roche) [adapted from Fowler et al. (Fowler et al., 1993) andGregorio and Fowler (Gregorio and Fowler, 1995)]. To lysates, an equal volume ofboiling IP buffer containing 0.4% (w/v) SDS was added, and samples boiled for 2minutes. After cooling to room temperature, 100 �l/ml 20% Triton X-100 wasadded and gently vortexed. Insoluble material was sedimented at 66,000 g for 1hour at 4°C, and soluble extracts were transferred to prepared beads.

Washed protein A-Tris-acryl beads (20 �l, packed; Pierce) were incubated with50 �g antibody for 1 hour with constant mixing. Immunoprecipitations were carriedout overnight at 4°C with end-over-end rotation. In parallel, control beads withoutbound antibodies were also incubated with clarified spermatid extract. Afterbinding, beads were washed four times with IP buffer (without SDS). Washed beadswere resuspended in 2� SDS sample buffer, boiled, and separated by SDS-PAGE(Laemmli, 1970). For mass spectrometry, gels were stained with silver and proteinbands were excised, trypsin digested and eluted [adapted from Blum et al. (Blumet al., 1987) and Shevchenko et al. (Shevchenko et al., 1996)]. Peptides wereidentified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) by L. Breci at The University of Arizona Proteomics Facility. Peptidemasses were searched using TANDEM (http://www.thegpm.org), with WormPeprelease WS140 (May 2005).

Immunofluorescent localizationSperm from him-5 males were dissected into 10 �l SMG on a slide coated with 1mg/ml poly-L-lysine (Sigma), fixed, and permeabilized as described previously(Nelson and Ward, 1980). For activation, selected slides were treated with 100 mMtriethanolamine (TEA, Sigma), pH 7.8 for 15 minutes. Nonspecific binding wasblocked with BSA blocking solution (1% BSA + 0.01% Tween 20 in PBS) for 1hour. Primary antibodies diluted in this blocking solution were added to slides for1 hour at room temperature (or 4°C overnight), followed by washing and thensecondary antibodies were added for 30 minutes. 4,6-diamidino-2-phenylindole(DAPI, 1 mg/ml in BSA-block) was added for 15 minutes, and quickly rinsed off.Samples were mounted in Prolong Antifade mounting medium (Molecular Probes).

Affinity purified anti-AZ10 peptide antibodies were used at a dilution of 1:100for immunofluorescence. The cell line that secretes the monoclonal antibody 1CB4(Okamoto and Thomson, 1985) was provided by J. Ahringer and J. Hodgkin, andculture supernatant was prepared by C. Heilman and A. I. Levey (Department ofNeurology, Emory University School of Medicine, Atlanta, GA). 1CB4 hybridomaculture supernatant was used for immunofluorescence at 1:5,000.

Mouse monoclonal primary antibodies were visualized by using Alexa Fluor 647-conjugated goat anti-mouse IgG secondary antibodies (Molecular Probes). Rabbitpolyclonal antibodies were detected with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Molecular Probes). All microscopy employed a LeicaDMRXA microscope fitted with a 100� Plan Apo objective and 1.6� Optivar.Micrographs were taken through appropriate filters (Cy5 for Alexa Fluor 647, FITCfor Alexa Fluor 488, and DAPI; Chroma Technology Corp., Brattleboro, VT) witha Retiga EX digital camera. Z-series stacks of cell images were captured,deconvolved, and reconstructed with MetaMorph 6.2 software.

Electron microscopySpermatids were isolated in bulk and activated with 100 mM TEA for 10 minutes.Cells were fixed in 4% paraformaldehyde + 0.2% gluteraldehyde in SM, pH 7.8,and embedded in LR White. Blocks were prepared by the Electron MicroscopyFacility at The Max Planck Institute for Cell Biology and Genetics (Dresden,Germany). Ultrathin sections were mounted on nickel-coated grids. All incubationswere performed at room temperature in a humid chamber, with grids submerged in10 �l drops of medium on Parafilm. Grids were blocked on both sides with 0.5%BSA in PBS for 30 minutes, followed by incubation with AZ10 (1:10) or 1CB4(1:500) antibodies for 1 hour. Washing was performed by transfer of grids through

three 0.1% BSA + 0.05% Tween 20 in PBS drops, 5 minutes each. Primaryantibodies were detected with 10 nm immunogold goat anti-rabbit or goat anti-mouse secondary antibodies (diluted 1:20; Electron Microscopy Sciences, Hatfield,PA), incubated for 1 hour, followed by washing as above. Antibody-labeled gridswere fixed with 4% paraformaldehyde + 0.2% gluteraldehyde in SM for 2.5 minutes,rinsed in water, stained with saturated uranyl acetate (10 minutes), rinsed with waterand dried. Images were captured on a Phillips 420 transmission electron microscopewith Kodak film. Negatives were digitized, and distance/area measurements wereacquired with Metamorph software.

Because membranes are not well visualized and cannot be measured accuratelyin samples prepared for immunogold labeling, we determined the amount ofmembrane present within the compacted MO using sections from the same spermpreparation fixed in parallel with OsO4. Using images of spermatids with well-preserved membranes, we measured the lengths of membranes invaginated into theMOs, the surrounding membrane and the membrane forming the MO head, togetherwith their corresponding areas. These membrane lengths were plotted against thecorresponding area of the structure. The resulting data were fitted by least squares(r2=0.9) resulting in the following relationship:

Length = 0.41 �m + 22 �m–1 � Area . (1)

Equation 1 was then used to estimate the corresponding membrane length forunfused MOs in immunogold-labeled samples, since the area of the MO was readilymeasured. Similarly, we used equation 1 to determine the length of membraneremaining invaginated within the MO cavity following fusion, given the areaoccupied by electron-dense material. The results obtained from these membranemeasurements were similar to those reported previously based on line-interceptmorphometry (Roberts et al., 1986).

MO fusion assayFor temperature-sensitivity assays, picked him-5 hermaphrodites were allowed tolay eggs at 15°C, 20°C, or 25°C. Resulting L4 virgin males were picked ontoseparate plates to prevent mating. Spermatids were dissected from these males ontopoly-L-lysine-coated slides into 50 �l Ca2+-free SMG + 10 mM EGTA, pH 7.8(SMGE). For BAPTA-AM experiments, spermatids were incubated in SMGEcontaining 0.001% (v/v) Pluronic-127 with or without BAPTA-AM at varyingconcentrations for 30 minutes then washed for 10 minutes at 15°C in SMGE. A flowchamber was created by raising the coverslip with a thin application of Vaseline on2 parallel sides (Shakes and Ward, 1989). Cells were mounted in SMGE with 2 �MN-(3-triethylammonium-propyl)-4-[4-(dibutylamino)styryl]pyridinium dibromide(FM1-43; Molecular Probes) to visualize membranes. Spermatids were activated atroom temperature by flowing 100 mM TEA in SMGE with FM1-43 through thechamber. DIC and FITC filtered images were captured every 20 seconds for 20minutes. Longer times did not lead to additional fusions. Surrounding fields of cellswere collected and counted. Up to five individual MO fusions could be scored easily,thereafter the MO fluorescence merged so that individual MOs could not bedistinguished. Spermatozoa with �5 fused MOs were scored as ‘normal’, althoughwild-type cells have an average of 18-25 MOs, and approximately 70% of them fuseat 25°C (40% fuse at 16°C) (Ward et al., 1981). Lysed cells, which were identifiedby the entire contents of the cell fluorescing, were not counted.

We thank Sarah McCarthy for help with DNA sequencing, DavidDreschel, Bill Achanzar and Michael Galligan for antibodypreparation, and David Bentley for help with electron microscopy. Wealso thank Asher Cutter and Matthew Terry for help with statistics andphylogenetic analysis. We thank Carol Dieckman, Johnny Fares,Carol Gregorio and Lisa Nagy for advice, members of the Fares andNagy laboratories for helpful discussions, and an anonymous reviewerfor comments on the text. N.W. was supported by NIH training grantsT32-CA09213 and T32-A09213. This work was supported in part byNIH grant R01 GM 25243.

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