Relationship between plasma membrane mobility and substrate attachment in the crawling movement of spermatozoa from Caenorhabditis elegans

Caenorhabditis elegans sperm are nonflagellated cells that lack actin and myosin yet can form pseudopods to propel themselves over solid substrates. Surface-attached probes such as latex beads, lectins, and antimembrane protein monoclonal antibodies move rearward over the dorsal pseudopod surface of sessile cells. Using monoclonal antibodies against membrane proteins of C. elegans sperm to examine the role of localized membrane assembly and rearward flow in crawling movement, we determined that substrates prepared by coating glass with antimembrane protein antibodies, but not naked glass or other nonmembrane-binding proteins, promote sperm motility. Sperm locomotion is inhibited in a concentration-dependent fashion when cells are bathed with soluble antimembrane protein monoclonal antibodies but not with antimouse Ig antibodies or a monoclonal antibody against a sperm cytoplasmic protein. Our results suggest that C. elegans sperm crawl by gaining traction with substrate-attached ligands via their surface proteins and by using the motor that moves those proteins rearward on unattached cells to pull the entire cell forward. Continuous insertion of new proteins at the front of the cell and their subsequent adhesion to the substrate allows this process to continue.     

Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.     

Caenorhabditis elegans spermatozoa assemble membrane proteins onto the surface at the tips of pseudopodial projections

The crawling movement of nematode sperm, like that of many other crawling metazoan cells, is accompanied by movement of membrane components from the leading edge of the cell rearward. We used colloidal gold conjugates of monoclonal antibodies (CGP-ABY) to membrane proteins on Caenorhabditis elegans sperm to examine this surface movement by electron microscopy. Antibody binding sites on fixed sperm are distributed uniformly over the cell surface. However, blocking these sites on live sperm with unlabelled antibody or removing them with protease and then pulse-labelling the cell with CGP-ABY revealed that new antigen is assembled onto the surface at the tips of the stubby projections that stud the pseudopod surface. These proteins then move rearward rapidly so that the pseudopod surface pool of antigen is replaced within 2 min. The same pattern of surface movement was observed when live cells were labelled with CGP-ABY and then washed with buffer before fixation. Bound CGP-ABY was cleared first from the tips of the projections and subsequently from the entire pseudopod surface. These gold particles accumulated at the base of the pseudopod without moving onto the cell body or being internalized. We did, however, detect a pool of antigen in the pseudopod cytoplasm that may be available for assembly onto the pseudopod surface. We propose that the localized assembly of new membrane and its subsequent rearward movement may play an important role in sperm locomotion.     

A marker of acid-secreting membrane movement in rat gastric parietal cells

A monoclonal antibody (mab 146.14) marker of the movement of acid-secreting membranes in rat gastric parital cells has been produced and characterized. Mab 146.14 recognized a 95-kD major component of a purified membrane fraction of rat gastric mucosa, the protein composition of which was similar to that of well characterized porcine H+ -K+ ATPase-enriched membranes, and that presented the characteristic shift of density depending on whether it was purified from resting or stimulated tissues. Further biochemical analysis characterized the antigen as a membranous protein that might be in its native form, part of a higher multimolecular complex. Immunocytochemical localization of the antigen demonstrated that only membranes related to acid secretion in parietal cells expressed the 95-kD antigen. In resting conditions, the 95-kD antigen was diffusely distributed in the cell cytoplasm associated with inactive tubulovesicles. In stimulated cells, by contrast, all the antigen was recovered associated with secretory active microvilli formed by the apical insertion of the previously resting internal tubulovesicles. In conclusion, the 95-kD antigen, presumably a part of the rat gastric proton pump, is a marker of acid-secreting membranes in rat parietal cells. The translocation of antigen and membranes, observed by both light and electron microscopy supports the fusion model of membrane insertion from a cytoplasmic storage pool to the apical surface upon stimulation of acid secretion.     

Membrane and cytoplasmic proteins are transported in the same organelle complex during nematode spermatogenesis

During the development of pseudopodial spermatozoa of the nematode, Caenorhabditis elegans, protein synthesis stops before differentiation is completed. Colloidal gold conjugates of monoclonal antibody SP56, which binds to the surface of spermatozoa, and TR20, which recognizes the major sperm cytoplasmic protein (MSP), were used to label thin sections of testes embedded in Lowicryl K4M in order to follow polypeptides from their synthesis early in spermatogenesis to their segregation to specific compartments of the mature cell. Both antigens are synthesized in primary spermatocytes and are assembled into a unique double organelle, the fibrous body-membranous organelle (FB-MO) complex. However, the antigens are localized in different regions of this FB-MO complex. As described in detail, the assembly of proteins into the FB-MO complex allows both membrane and cytoplasmic components to be concentrated in the spermatids after meiosis. Then, the stepwise disassembly of this transient structure ensures delivery of each component to its final destination in the mature spermatozoan: MSP filaments in the fibrous body depolymerize, releasing MSP into the cytoplasm and the membranous organelles fuse with the plasma membrane, delivering SP56 antigen to the surface.     

Identification of membrane antigens of goat epididymal spermatozoa

Purified goat sperm plasma membrane was used as antigen to raise the antibody in rabbit. Using this antisera four groups of antigenic membrane polypeptides are determined in caput and cauda epididymal sperm. The immunoresponsiveness of the polypeptides in caput and cauda sperm differs significantly. In case of cauda epididymal sperm, the polypeptides of region A (96KDa, 82KDa, 78KDa, 68KDa) and region D (24KDa, 20KDa, 18KDa) are highly immunoresponsive whereas in case of caput epididymal sperm the same antisera recognized the polypeptides of region B, C and D. By surface labelling with lactoperoxidase iodination and subsequent immunoprecipitation in the iodinated cell extract we demonstrate eight of these above polypeptides (96KDa, 82KDa, 68KDa, 50KDa, 29KDa, 24KDa, 20KDa and 18KDa) as surface antigen. The 96KDa, 82KDa and 68KDa surface polypeptides are highly immunoresponsive than the other lower molecular weight surface antigens in cauda epididymal goat spermatozoa.     

Localization of a maturation-dependent epididymal sperm surface antigen recognized by a monoclonal antibody raised against a 135-kilodalton protein in porcine epididymal fluid.

A specific 135-kDa protein was purified from porcine cauda epididymal fluid. Analysis of its N-terminal amino acid sequence revealed it to be a new protein. Stable clones of hybridomas that produced monoclonal antibodies against the purified 135-kDa protein were established. A clone, B-11, reacting both with epididymal fluid and with sperm plasma membranes was selected and used in this study. Immunoblotting analysis showed that B-11 reacted only with a 135-kDa protein among epididymal fluid proteins. In contrast, B-11 did not recognize a similar 135-kDa sperm protein but did strongly react with a 27-kDa protein among sperm membrane proteins, extracted by NP-40 in the presence of protease inhibitors. B-11 also reacted only with a 27-kDa protein fragment among trypsin digests of the 135-kDa epididymal protein. The 135-kDa protein was first detected, by ELISA or immunoblotting analysis, at the beginning of the corpus epididymis. Maximal levels were reached in the distal corpus and levels were slightly decreased in the cauda epididymis. On the other hand, the surface of caput sperm were found to contain small amounts of antigen(s), the concentration of which gradually increased during epididymal transit. In immunocytochemical studies, the antigen was detectable in the epithelial cells from the initial segment to the corpus of the epididymis but not in the caudal cells. In the lumen, the presence of the 135 kDa protein was apparent in the corpus (at a maximum in the middle and distal corpus) and to a lesser degree in the caudal lumen. The 27-kDa protein was distributed all over the equatorial region of the acrosome of less than 10% of caput epididymal sperm. As sperm passed through the corpus epididymis, the percentage of immunoreactive cells increased and the protein was restricted to specific domains of the sperm head. Thus, on the mature sperm, antigen was localized in a crescent-shaped area of the equatorial segment just behind the anterior part of the acrosome and on the apical rim of the sperm head. This is the first observation of a sperm surface antigen derived from an epididymal protein as a proteolytic fragment that interacts with specific regions of the sperm membrane during the process of spermatozoa maturation.     

Maize mesocotyl plasmodesmata proteins cross-react with connexin gap junction protein antibodies.

Polypeptide present in various cell fractions obtained from homogenized maize mesocotyls were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotted, and screened for cross-reactivity with antibodies against three synthetic polypeptides spanning different regions of the rat heart gap junctional protein connexin43 and the whole mouse liver gap junctional protein connexin32. An antibody raised against a cytoplasmic loop region of connexin43 cross-reacted strongly with a cell wall-associated polypeptide (possibly a doublet) of 26 kilodaltons. Indirect immunogold labeling of thin sections of mesocotyl tissue with this antibody labeled the plasmodesmata of cortical cells along the entire length of the plasmodesmata, including the neck region and the cytoplasmic annulus. Sections labeled with control preimmune serum were essentially free of colloidal gold. An antibody against connexin32 cross-reacted with a 27-kilodalton polypeptide that was present in the cell wall and membrane fractions. Indirect immunogold labeling of thin sections with this antibody labeled the plasmodesmata mainly in the neck region. It is suggested that maize mesocotyl plasmodesmata contain at least two different proteins that have homologous domains with connexin proteins.     

Expression of cytokeratin polypeptides in mouse oocytes and preimplantation embryos

The presence and distribution of intermediate filament proteins in mouse oocytes and preimplantation embryos was studied. In immunoblotting analysis of electrophoretically separated polypeptides, a distinct doublet of polypeptides with Mr of 54K and 57K, reactive with cytokeratin antibodies, was detected in oocytes and in cleavage-stage embryos. A similar doublet of polypeptides, reactive with cytokeratin antibodies, was also detected in late morula-and blastocyst-stage embryos, and in a mouse embryo epithelial cell line (MMC-E). A third polypeptide with Mr of 50K, present in oocytes only as a minor component, was additionally detected in the blastocyst-stage embryos. No cytokeratin polypeptides could be detected in granulosa cells. Immunoblotting with vimentin antibodies gave negative results in both cleavage-stage and blastocyst-stage embryos. In electron microscopy, scattered filaments, 10-11 nm in diameter, were seen in detergent-extracted cleavage-stage embryos. Abundant 10-nm filaments were present in the blastocyst outgrowth cells. In indirect immunofluorescence microscopy (IIF) of oocytes and cleavage-stage embryos, diffuse cytoplasmic staining was seen with antibodies to cytokeratin polypeptides but not with antibodies to vimentin, glial fibrillary acidic protein, or neurofilament protein. Similarly, the inner cell mass (ICM) cells in blastocyst outgrowths showed diffuse cytokeratin-specific fluorescence. We could not detect any significant fibrillar staining in cleavage-stage cells or ICM cells by the IIF method. The first outgrowing trophectoderm cells already had a strong fibrillar cytokeratin organization. These immunoblotting and -fluorescence results suggest that cytokeratin-like polypeptides are present in mouse oocytes and preimplantation-stage embryos, and the electron microscopy observations show that these early stages also contain detergent-resistant 10- to 11-nm filaments. The relative scarcity of these filaments, as compared to the high intensity in the immunoblotting and immunofluorescence stainings, speaks in favor of a nonfilamentous pool of cytokeratin in oocytes and cleavage-stage embryos.     

Identification of the 48-kDa G11 protein from guinea pig testes as sperad

A protein found specifically in the membrane of spermatozoa called G11 has been implicated in sperm-egg binding and fusion. This study describes purification and identification of the G11 antigen. The G11 protein was purified using anion exchange chromatography, immunoaffinity chromatography and preparative SDS-PAGE and was subjected to amino acid microsequencing by tandem mass spectrometry. Internal amino acid sequence data derived from the 48-kDa G11 protein revealed that G11 is the recently discovered guinea pig sperm protein, sperad. Sperad is a transmembrane protein present in the periacrosomal plasma membrane of guinea pig sperm. The cytoplasmic domain of sperad was amplified from a guinea pig testes cDNA expression library by polymerase chain reaction and cloned into a prokaryotic gene expression vector, pGEX-2T. The recombinant glutathione S-transferase fusion protein was immunoblotted with monoclonal antibody G11. The results obtained from this study confirmed the monoclonal antibody G11 epitope location on the cytoplasmic domain of sperad.     

Identification of the p53 protein domain involved in formation of the simian virus 40 large T-antigen-p53 protein complex.

An expression vector utilizing the enhancer and promoter region of the simian virus 40 (SV40) DNA regulating a murine p53 cDNA clone was constructed. The vector produced murine p53 protein in monkey cells identified by five different monoclonal antibodies, three of which were specific for the murine form of p53. The murine p53 produced in monkey cells formed an oligomeric protein complex with the SV40 large tumor antigen. A large number of deletion mutations, in-frame linker insertion mutations, and linker insertion mutations resulting in a frameshift mutation were constructed in the cDNA coding portion of the p53 protein expression vector. The wild-type and mutant p53 cDNA vectors were expressed in monkey cells producing the SV40 large T antigen. The conformation and levels of p53 protein and its ability to form protein complexes with the SV40 T antigen were determined by using five different monoclonal antibodies with quite distinct epitope recognition sites. Insertion mutations between amino acid residues 123 and 215 (of a total of 390 amino acids) eliminated the ability of murine p53 to bind to the SV40 large T antigen. Deletion (at amino acids 11 through 33) and insertion mutations (amino acids 222 through 344) located on either side of this T-antigen-binding protein domain produced a murine p53 protein that bound to the SV40 large T antigen. The same five insertion mutations that failed to bind with the SV40 large T antigen also failed to react with a specific monoclonal antibody, PAb246. In contrast, six additional deletion and insertion mutations that produced p53 protein that did bind with T antigen were each recognized by PAb246. The proposed epitope for PAb246 has been mapped adjacent (amino acids 88 through 109) to the T-antigen-binding domain (amino acids 123 through 215) localized by the mutations mapped in this study. Finally, some insertion mutations that produced a protein that failed to bind to the SV40 T antigen appeared to have an enhanced ability to complex with a 68-kilodalton cellular protein in monkey cells.     

Role of the mature protein sequence of maltose-binding protein in its secretion across the E. coli cytoplasmic membrane

Secretion of amber fragments of an E. coli periplasmic protein, the maltose-binding protein, was studied to determine if the mature portion of the protein is required for its export across the cytoplasmic membrane. A fragment lacking 25–35 amino acid residues at the C terminus is secreted at normal levels, suggesting that this sequence is not required for secretion. This is in contrast to the results obtained with the periplasmic protein β-lactamase. In studying another fragment of one-third the molecular weight of the intact protein, we found that the majority of the fragment is not recovered from the periplasmic fraction. However, a small amount of secretion of this polypeptide was observed. This fragment is synthesized as a larger molecular weight form when cells are induced for the synthesis of a maltose-binding protein-β-galactosidase hybrid protein, which was previously shown to block the proper localization and processing of envelope proteins. This result is consistent with the idea that the larger form is a precursor with an unprocessed signal sequence, whereas in the absence of the hybrid protein the fragment is a processed mature form. Thus secretion of the smaller fragment may be occurring up to the point where the signal sequence is removed. That this fragment has passed through the cytoplasmic membrane is further supported by its accessibility to externally added trypsin. We suggest that the fragment may be secreted to the periplasm, but cannot assume a water-soluble conformation; the majority of the polypeptide may be associated with the external surface of the cytoplasmic membrane. Thus the mature sequence of maltose-binding protein, at least its C-terminal two thirds, may not be required for its export across the cytoplasmic membrane.     

A localized surface protein of guinea pig sperm exhibits free diffusion in its domain

Using the technique of fluorescence redistribution after photobleaching, we are studying the cellular mechanisms involved in localizing surface molecules to particular domains. A number of antigens localized to discrete surface regions have been identified with monoclonal antibodies on guinea pig sperm cells ( Primakoff , P., and D. G. Myles , 1983, Dev. Biol., 98:417-428). One of these monoclonal antibodies, PT-1, binds exclusively to the posterior tail region of the sperm cell surface. PT-1 recognizes an integral membrane protein that in complex with n-octyl-beta-D-glucopyranoside has a sedimentation coefficient of 6.8S in sucrose density gradients. Fluorescence redistribution after photobleaching measurements reveal that within its surface domain the PT-1 antigen diffuses rapidly (D = 2.5 X 10(-9) cm2/s) and completely (greater than 90% recovery after bleaching). These results rule out for this membrane protein all models that invoke immobilization as a mechanism for maintaining localization. We propose that the mechanism for localization of the PT-1 antigen may be a barrier to diffusion at the domain boundary.     

Changes in sperm surface membrane and luminal protein fluid content during epididymal transit in the boar

The surface membrane protein of boar sperm and the proteins in the fluid surrounding the gametes were analyzed during epididymal transit. The present study demonstrated that sequential dramatic changes occur in protein composition of the sperm membrane and epididymal fluid during epididymal transit. The maturation process of the boar sperm surface was characterized by a complex sequential evolution of the composition and orientation of macromolecules in the sperm membrane. Epididymal maturation resulted in the progressive disappearance of most of the surface testicular compounds, which were either renewed or masked by new permanent or transient low molecular weight polypeptides on the boar sperm surface membrane. In the fluid surrounding the spermatozoa, composition of the luminal proteins was altered throughout the epididymal transit and several new compounds were characterized. Very few proteins were correlated either with blood plasma or sperm surface compounds.     

Differential Glycosylation of the 5A11/HT7 Antigen by Neural Retina and Epithelial Tissues in the Chicken

Abstract: The 5A11/HT7 antigen, a member of the immunoglobulin supergene family, has been implicated in heterotypic cell-cell interactions during retina development. Immunopurified 5A11 antigen isolated from Nonidet P-40-solubilized retina membranes had two components as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 45.5-kDa doublet and a 69-kDa polypeptide. Immunoreactive bands of 46-50 kDa were recognized following SDS-PAGE of detergent-solubilized membrane proteins from liver, kidney, and erythrocytes. Treatment with N-glycosidase F (EC 3.2.2.18) converted the 45.5–50-kDa immunoreactive polypeptides from all tissues to 32 kDa, indicating that the observed differences in molecular mass were due to differences in glycosylation. N-Glycosidase Ftreatment also converted the 69-kDa form from retina to 46 kDa, indicating a different polypeptide core than the 32-kDa species. Treatment with endo-β-N-acetylglucosaminidase H (EC 3.2.1.96) resulted in modest increases in electrophoretic mobility due to hydrolysis of high mannose or hybrid oligosaccharides and lack of hydrolysis of complex oligosaccharides resistant to endo-β-N-acetylglucosaminidase H digestion. Immunoreactivity was retained after deglycosylation. Much of the difference in molecular weight could be attributed to variations in sialylation. The higher molecular mass species of the 45.5-kDa doublet from retina and the polypeptides from other tissues were susceptible to neuraminidase (EC 3.2.1.18) and O-glycosidase (endo-α-N-acetylgalactosaminidase; EC 3.2.1.97) digestion. Labeling with elderberry bark lectin (specific for α2, 6-linked sialic acid) was confined to the higher molecular mass species of the 45.5-kDa doublet and was considerably greater in antigen derived from epithelia rather than neural retina. In paraffin sections of chick retina, elderberry bark lectin staining was confined to the retinal pigmented epithelium, photoreceptor cells, and bipolar cells with no staining of the Müller cells, which bear the bulk of the 5A11 antigen. These results indicate tissue-specific posttranslational modifications, particularly differences in sialylation of antigen-bearing polypeptides.     

Detection of a sperm-coating antigen in the semen of Bubalus bubalis.

1. Surface antigens of B. bubalis spermatozoa were solubilized by Triton X-100 and EDTA; the sperm extract was used to raise antibodies in rabbits. 2. Two major polypeptides, immunoprecipitated from the seminal plasma by the antibodies against the sperm extract, exhibited the same electrophoretic mobilities of two immunorelated sperm surface antigens. 3. The two polypeptides were isolated from the seminal plasma, by a multi-step chromatographic procedure, and found subunits of a single protein (MW 30,000), called SP 30. 4. The SP 30 protein bound in vitro to the postacrosomal region of homologous spermatozoa from cauda epididymis. 5. The localization of the sperm-coating antigen on the cell surface is compatible with a role in the fertilization process.     

The carboxyl terminus of Dictyostelium discoideum protein 1I encodes a functional glycosyl-phosphatidylinositol signal sequence

The 1I gene is expressed in the prespore cells of culminating Dictyostelium discoideum. The open reading frame of 1I cDNA encodes a protein of 155 amino acids with hydrophobic segments at both its NH(2)- and COOH-termini that are indicative of a glycosyl-phosphatidylinositol (GPI)-anchored protein. A hexaHis-tagged form of 1I expressed in D. discoideum cells appeared on Western blot analysis as a doublet of 27 and 24 kDa, with a minor polypeptide of 22 kDa. None of the polypeptides were released from the cell surface with bacterial phosphatidylinositol-specific phospholipase C, although all three were released upon nitrous acid treatment, indicating the presence of a phospholipase-resistant GPI anchor. Further evidence for the C-terminal sequence of 1I acting as a GPI attachment signal was obtained by replacing the GPI anchor signal sequence of porcine membrane dipeptidase with that from 1I. Two constructs of dipeptidase with the 1I GPI signal sequence were constructed, one of which included an additional six amino acids in the hydrophilic spacer. Both of the resultant constructs were targeted to the surface of COS cells and were GPI-anchored as shown by digestion with phospholipase C, indicating that the Dictyostelium GPI signal sequence is functional in mammalian cells. Site-specific antibodies recognising epitopes either side of the expected GPI anchor attachment site were used to determine the site of GPI anchor attachment in the constructs. These parallel approaches show that the C-terminal signal sequence of 1I can direct the addition of a GPI anchor.