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First published online 30 May 2006
doi: 10.1242/jcs.02980

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FER-1 regulates Ca²⁺-mediated membrane fusion during C. elegans spermatogenesis

Department of Molecular and Cellular Biology, The University of Arizona, 1007 E. Lowell Street, Life Sciences South 452, Tucson, AZ, 85721, USA

View larger version (41K): [in a new window]   Fig. 1. Ferlin domains and FER-1 mutations. FER-1 and several homologs are aligned with their domain composition indicated. Most ferlins have four or more C2 domains, which are shown as colored shapes reflecting similar positions in the various ferlin proteins. DYSF (striped rectangle) has nested DysfN and DysfC domains; TM (black diamond) transmembrane domain. Predicted protein changes for all fer-1 alleles are shown here and described in Table 1; *, stop codon. Letter designations for C2 domains in dysferlin are from Davis et al. (Davis et al., 2000). Dotted lines below FER-1 indicate antigenic sites for anti-DysfNC (left) and anti-AZ10 (right). See supplementary material (Table S1 and Fig. S1) for additional homologs.

View larger version (14K): [in a new window]   Fig. 2. Ferlin C2 domain phylogeny. A summary phylogenetic tree of C2 domain sequences of one representative ferlin from each species and several other C2 domain-containing proteins is shown. The tree is based on neighbor-joining of C2 domains aligned using ClustalW and refined by hand. Ferlin C2 domains are colored as in Fig. 1, based on their position. The tree shows that the C2 domains with the same color cluster together indicating they are most similar to each other. Non-ferlin C2 domain sequences were previously analyzed by Nalfeski and Falke (Nalfeski and Falke, 1996) (black lines). Bootstrap support indicated along branches for major clades; asterisk (*) indicates <50%. Similar results were obtained with maximum parsimony analysis. Abbreviations: PLCs, phospholipases; Rabs, rabphilins; synapt, synaptotagmins; PKCs, protein kinase C. Additional sequences, together with the individual protein names, are included in supplementary material Fig. S2.

View larger version (47K): [in a new window]   Fig. 3. Temperature sensitivity of MO fusions due to different fer-1 alleles. (A-H) MO fusion assay. Spermatids were activated with 100 mM TEA in the presence of FM 1-43 to visualize PM and MOs upon fusion. Top, DIC images; bottom, FM 1-43 staining. Bar, 5 µm. (A,B) Unactivated spermatids are rounded and only display PM staining. (C,D) Wild-type (shown here) or temperature-sensitive mutants at permissive temperature (15°C) fuse many MOs, which show as bright puncta in the cell body. The PM of the pseudopod is labeled but is devoid of 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 of spermatozoa 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 TEA activation of wild-type or temperature sensitive fer-1 (hc13ts, hc1ts, hc24ts and hc82ts) sperm grown at 15°C, 20°C or 25°C. Cells were activated 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).

View larger version (25K): [in a new window]   Fig. 4. Characterization of FER-1 protein isoforms. (A) Affinity purified polyclonal anti-DysfNC antibody recognizes three proteins in 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) protein extracts. Numbers on right indicate calculated molecular masses, in kDa. (C) Distribution of peptides identified by mass spectrometry from 195 and 180 kDa bands depicted along full-length FER-1 protein (images generated by Tandem at http://www.thegpm.org). Peptides are listed in supplementary material Table S2.

View larger version (62K): [in a new window]   Fig. 5. Immunofluorescent localization of FER-1 in sperm. (A-J) Deconvoluted images of immunostaining performed on spermatids or TEA-activated spermatozoa. Spermatids were dissected from 2-3 day virginized him-5 or fer-1(hc47) males. Cells were stained with anti-AZ10 (green) antibodies to detect FER-1 and with 1CB4 (red) to label MOs. Bar, 2 µm. (A-D) FER-1 is localized to punctate structures and colocalizes with 1CB4. (E-H) FER-1 is localized in the plasma membrane of mature sperm, and partially colocalizes with 1CB4. Arrowheads indicate visible pseudopods. (I-J) fer-1(hc47) sperm, which have a stop codon prior to the AZ10 antigenic site and make no FER-1. (J) No staining is detected in fer-1(hc47) sperm at the same exposure used for normal sperm.

View larger version (63K): [in a new window]   Fig. 6. Distribution of FER-1 and 1CB4 in spermatids and spermatozoa. (A,B) Ultrastructural localization of FER-1 with AZ10 antibodies 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 MO head; 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 the MO membrane and increases in the PM after fusion (G). 1CB4 labels MOs most abundantly (F), which increases two-fold in density along MO membranes and simultaneously increases slightly in the PM after fusion (H). Insets in E and F show average total labeling/cell for spermatids (tid) or spermatozoa (zoa) is not statistically different for either antibody. Insets in G and H show labeling over cell body (CB) or pseudopod (P) plasma membranes, which are not statistically different for either antibody. Graph indicates the average labeling over entire PM. Although nuclear localization was observed at a density similar to that of the MO by immunogold staining with AZ10, we observed no nuclear staining by immunofluorescence and presume this is background staining due to the charged nature of the nucleic acids. Error bars=s.e.m. *Different from unfused body density (P<0.0001); **different from cell body cytoplasm density (P<0.0001); + different from unfused body linear density (P<0.0001); ++ different from spermatid PM linear density (P<0.0001).

View larger version (51K): [in a new window]   Fig. 7. TEA stimulates MO fusion by a Ca2+-dependent mechanism. (A) Spermatids were loaded with 1 mM EGTA and BAPTA-AM for 30 minutes at the indicated concentrations, and the percentage of cells displaying normal MO fusions were counted. Data are representative of three independent experiments. (B) Spermatids treated with EGTA only or with 10 µM BAPTA-AM. Pseudopod formation also appears to be inhibited at high BAPTA-AM concentrations. (C) fer-1 mutant sperm are hypersensitive to internal calcium depletion. Wild-type or fer-1 mutant sperm were incubated in EGTA or 1 µM BAPTA-AM for 30 minutes, then activated in 100 mM TEA in the presence of FM1-43, and scored for normal MO fusion. (D) Sample cells of each of the mutants treated with EGTA (top) or 1 µM BAPTA-AM (bottom). The domain in which the mutation lies is indicated above the images. Arrowheads indicate spermatozoa that have attempted to generate pseudopods, but have no fused MOs. *Significantly different (P<0.01) from EGTA-treated cells with the same mutation. Error bars=s.e.m.

View larger version (27K): [in a new window]   Fig. 8. Model for FER-1-mediated Ca2+-dependent MO fusion. MOs are bi-lobed structures comprising a head and membrane-dense body, separated by an electron dense collar. Spermatids await an `activation' signal, with MO heads docked at the PM prior to fusion. Upon receipt of an activation signal, intracellular Ca2+ is utilized to relay the signal to be received by FER-1 and by others required for MO fusion. When MOs fuse in spermatozoa, the head and a portion of MO body membrane is incorporated into the PM. A fusion pore remains surrounded by the collar, and the glycoprotein contents are spilled from MOs along the membrane surface of cell body. FER-1 and other proteins can move into the plasma membrane, while the 1CB4 antigen and presumably other proteins remain concentrated within the MO cavity. In fer-1 mutants, the MO heads abut the PM but MOs do not fuse.

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