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.     

Posttranslational insertion of a membrane protein on Caenorhabditis elegans sperm occurs without de novo protein synthesis

We have examined the mechanism of membrane protein insertion in the ameboid spermatozoa of Caenorhabditis elegans using two monoclonal antibodies which recognize the same set of eight sperm-specific polypeptides. Previous electron microscopic studies demonstrated that these antibodies label surface and cytoplasmic populations of antigen. Cells whose surface antigen had been removed by proteolysis were able to localize new membrane protein insertion at the tips of pseudopodial projections. C. elegans sperm do not contain the protein synthesizing machinery needed for delivery of new membrane to the cell surface. It has, therefore, been of interest to determine how localized membrane assembly occurs. Here we have determined the subcellular location of each of these eight polypeptides. A closely positioned doublet of bands around 97 kD (comprising 40% of the total antigen in sperm) represents surface (larger member of doublet) and cytoplasmic (lower member) forms of protein. Proteolysis of live cells eliminated this surface form from immunoblots but did not affect the cytoplasmic protein. When cells were allowed to reinsert new protein following removal of the enzyme, this surface form was regenerated. Since sperm are unable to synthesize new protein, this higher molecular weight species may arise from a posttranslational modification of proteins in the cytoplasmic pool. We present evidence suggesting that the surface protein is generated from this cytoplasmic pool by addition of fatty acid. Fatty acid acylation would account for both the observed decrease in electrophoretic mobility of the surface form and provide increased hydrophobicity to the protein which may allow for its insertion into the lipid bilayer.     

A unique cytoskeleton associated with crawling in the amoeboid sperm of the nematode, Ascaris suum

Nematode sperm extend pseudopods and pull themselves over substrates. They lack an axoneme or the actin and myosins of other types of motile cells, but their pseudopods contain abundant major sperm protein (MSP), a family of 14-kD polypeptides found exclusively in male gametes. Using high voltage electron microscopy, a unique cytoskeleton was discovered in the pseudopod of in vitro-activated, crawling sperm of the pig intestinal nematode Ascaris suum. It consists of 5-10-nm fuzzy fibers organized into 150-250-nm-thick fiber complexes, which connect to each of the moving pseudopodial membrane projections, villipodia, which in turn make contact with the substrate. Individual fibers in a complex splay out radially from its axis in all directions. The centripetal ends intercalate with fibers from other complexes or terminate in a thickened layer just beneath the pseudopod membrane. Monoclonal antibodies directed against MSP heavily label the fiber complexes as well as individual pseudopodial filaments throughout their length. This represents the first evidence that MSP may be the major filament protein in the Ascaris sperm cytoskeleton. The large fiber complexes can be seen clearly in the pseudopods of live, crawling sperm by computer-enhanced video, differential-interference contrast microscopy, forming with the villipodia at the leading edge of the sperm pseudopod. Even before the pseudopod attaches, the entire cytoskeleton and villipodia move continuously rearwards in unison toward the cell body. During crawling, complexes and villipodia in the pseudopod recede at the same speed as the spermatozoon moves forward, both disappearing at the pseudopod-cell body junction. Sections at this region of high membrane turnover reveal a band of densely packed smooth vesicles with round and tubular profiles, some of which are associated with the pseudopod plasma membrane. The exceptional anatomy, biochemistry, and phenomenology of Ascaris sperm locomotion permit direct study of the involvement of the cytoskeleton in amoeboid motility.     

In vitro induction of crawling in the amoeboid sperm of the nematode parasite, Ascaris suum

In a highly synchronous process, the immotile spermatids of Ascaris suum extend pseudopods and become rapidly crawling sperm when treated with an extract from the glandular vas deferens of the male under strict anaerobic conditions. Within 9-12 min, a pseudopod develops, elongates rapidly, and exhibits a continuous flow of membrane specializations, the villipodia, from tip toward base. When attached to acid-washed glass, the pseudopod pulls the cell body along at speeds exceeding 70 microns/min. The pseudopod length remains constant while retrograde flow of villipodia proceeds at the same rate as the sperm's forward movement. Cohorts of about 15 villipodia form at the leading edge, move rearward together, and disappear at the junction of pseudopod and cell body. These are the terminations of branched, refringent fibers, which extend the length of the pseudopod. The latter are the fiber complexes that form its cytoskeleton (Sepsenwol et al.: Journal of Cell Biology 108:55-66, 1989). Locomoting cells sometimes change direction when another crawls by and follow each other. When cells are exposed to air, forward movement ceases in a predictable pattern: the forward extension of the leading edge ceases, the pseudopod shortens from the base, and the cell body continues to be pulled forward. These data contribute to a model for Ascaris sperm amoeboid motility in which independent processes of continuous extension at the leading edge and continuous shortening at the base of the pseudopod act to propel the cell forward.     

Caenorhabditis elegans spermatozoan locomotion: amoeboid movement with almost no actin

The pseudopods of Caenorhabditis elegans spermatozoa move actively causing some cells to translocate when the sperm are dissected into a low osmotic strength buffered salts solution. On time-lapse video tapes, pseudopodial projections can be seen moving at 20-45 micrometers/min from the tip to the base of the pseudopod. This movement occurs whether or not the cell is attached to a substrate. Translocation of the cell is dependent on the substrate. Some spermatozoa translocate on acid-washed glass, but a better substrate is prepared by drying an extract of Ascaris uteri (the normal site of nematode sperm motility) onto glass slides. On this substrate more than half the spermatozoa translocate at a velocity (21 micrometers/min) similar to that observed in vivo. Translocating cells attach to the substrate by their pseudopodial projections. They always move toward the pseudopod; changes in direction are caused by changes in pseudopod shape that determine points of detachment and reattachment of the cell to the substrate. Actin comprises less than 0.02% of the proteins in sperm, and myosin is undetectable. No microfilaments are found in the sperm. Immunohistochemistry shows that some actin is localized in patches in the pseudopod. The movement of spermatozoa is unaffected by cytochalasins, however, so there is no evidence that actin participates in locomotion. Fertilization-defective mutants in genes fer-2, fer-4, and fer-6 produce spermatozoa with defective pseudopodial projections, and these spermatozoa are largely immotile. Mutants in the spermatozoa do not translocate. Thus pseudopod movement is correlated with the presence of normal projections. Twelve mutants with defective muscles have spermatozoa with normal movement, so these genes do not specify products needed for both muscle and nonmuscle cell motility.     

Preparing to move: assembly of the MSP amoeboid motility apparatus during spermiogenesis in Ascaris

We exploited the rapid, inducible conversion of non-motile Ascaris spermatids into crawling spermatozoa to examine the pattern of assembly of the MSP motility apparatus that powers sperm locomotion. In live sperm, the first detectable motile activity is the extension of spikes and, later, blebs from the cell surface. However, examination of cells by EM revealed that the formation of surface protrusions is preceded by assembly of MSP filament tails on the membranous organelles in the peripheral cytoplasm. These organelle-associated filament meshworks assemble within 30 sec after induction of spermiogenesis and persist until the membranous organelles are sequestered into the cell body when the lamellipod extends. The filopodia-like spikes, which are packed with bundles of filaments, extend and retract rapidly but last only a few seconds before giving way to, or converting into, blebs. Coalescence of these blebs, each supported by a dense mesh of filaments, often initiates lamellipod extension, which culminates in the formation of the robust, dynamic MSP fiber complexes that generate sperm motility. The same membrane phosphoprotein that orchestrates assembly of the fiber complexes at the leading edge of the lamellipod of mature sperm is also found at all sites of filament assembly during spermiogenesis. The orderly progression of steps that leads to construction of a functional motility apparatus illustrates the precise spatio-temporal control of MSP filament assembly in the developing cell and highlights the remarkable similarity in organization and plasticity shared by the MSP cytoskeleton and the actin filament arrays in conventional crawling cells.     

Centripetal flow and directed reassembly of the major sperm protein (MSP) cytoskeleton in the amoeboid sperm of the nematode, Ascaris suum.

The cytoskeleton of the amoeboid spermatozoa of Ascaris suum consists of major sperm protein (MSP) filaments arranged into long, branched fiber complexes that span the length of the pseudopod and treadmill rearward continuously due to assembly and disassembly at opposite ends of the complexes (Sepsenwol et al., Journal of Cell Biology 108:55-66, (1989)). Examination by video-enhanced microscopy showed that this cytoskeletal flow is tightly coupled to sperm locomotion. The fiber complexes treadmilled rearward at the same rate (10-50 microns/min) as the cell crawled forward. Only fiber complexes with their plasmalemmal ends within a limited sector along the leading edge of the pseudopod underwent continuous assembly. Thus, the location of this sector, which occupies about 50% of the pseudopod perimeter, determined the direction of sperm locomotion. Treatment of sperm with agents that lower intracellular pH, such as weak acids and protonophores, caused the fiber complexes to disassemble completely in 4-5 sec. Removal of these compounds resulted in reassembly of the cytoskeleton in a pattern that mimicked treadmilling in intact sperm. The fiber complexes were reconstructed by assembly at their plasmalemmal ends so that within 30-60 sec the entire filament system reformed and the cell resumed locomotion. Both cytoskeletal reassembly and treadmilling required exogenous HCO3-. These results suggest that variation in intracellular pH may help regulate cytoskeletal treadmilling and thereby play a significant role in sperm locomotion.     

Dissection of the Ascaris sperm motility machinery identifies key proteins involved in major sperm protein-based amoeboid locomotion

Although Ascaris sperm motility closely resembles that seen in many other types of crawling cells, the lamellipodial dynamics that drive movement result from modulation of a cytoskeleton based on the major sperm protein (MSP) rather than actin. The dynamics of the Ascaris sperm cytoskeleton can be studied in a cell-free in vitro system based on the movement of plasma membrane vesicles by fibers constructed from bundles of MSP filaments. In addition to ATP, MSP, and a plasma membrane protein, reconstitution of MSP motility in this cell-free extract requires cytosolic proteins that orchestrate the site-specific assembly and bundling of MSP filaments that generates locomotion. Here, we identify a fraction of cytosol that is comprised of a small number of proteins but contains all of the soluble components required to assemble fibers. We have purified two of these proteins, designated MSP fiber proteins (MFPs) 1 and 2 and demonstrated by immunolabeling that both are located in the MSP cytoskeleton in cells and in fibers. These proteins had reciprocal effects on fiber assembly in vitro: MFP1 decreased the rate of fiber growth, whereas MFP2 increased the growth rate.     

Centripetal flow of pseudopodial surface components could propel the amoeboid movement of Caenorhabditis elegans spermatozoa

Latex beads and wheat germ agglutinin (WGA) were used to examine the movement of membrane components on amoeboid spermatozoa of Caenorhabditis elegans. The behavior of beads attached to the cell revealed continuous, directed movement from the tip of the pseudopod to its base, but no movement on the cell body. Lectin receptors are also cleared from the pseudopod (4). Blocking preexisting lectin receptors with unlabeled WGA followed by pulse-labeling wih fluorescent WGA showed that new lectin receptors are continuously inserted at the tip of the pseudopod. Like latex beads, these new lectin receptors move continuously over the pseudopod surface to the cell body-pseudopod junction where they are probably internalized. Mutants altering the rate of membrane flow, and eliminating its topographical asymmetry, have been identified. Together with the observation that fluorescent phospholipids are cleared from the pseudopod of developing spermatozoa at the same rate as lectin receptors (25), these results show that there is bulk membrane flow over the pseudopod with assembly at the tip and apparent disassembly at the base. There are no vesicles visible at either the pseudopodial tip or base, so these spermatozoa must have a novel mechanism for insertion and uptake of membrane components. This membrane flow could provide the forward propulsion of spermatozoa attached to a substrate by their pseudopods.     

Comparative behavior of membrane protein-antibody complexes on motile fibroblasts: implications for a mechanism of capping

A characteristic feature of fibroblast locomotory activity is the rearward transport across the leading lamella of various materials used to mark the cell surface. The two processes most frequently invoked as explanations for this transport phenomenon, called capping, are (a) retrograde membrane flow arising from directed membrane insertion and (b) rearward cortical cytoskeletal flow arising from cytoskeletal assembly and contraction. The retrograde lipid flow hypothesis, the most current form of the membrane flow scheme, makes explicit predictions about the movement of membrane proteins subjected to the postulated rearward lipid flow. Several of these predictions were tested by comparing the behavior of four membrane proteins, Pgp-1, Thy- 1, H-2, and influenza HA0, identified by fluorescent antibodies. With the exception of Pgp-1, these proteins were uniformly distributed under nonaggregated conditions but were capped when aggregated into patches. In contrast, Pgp-1 was capped in similar time frames in both nonaggregated and aggregated states where the lateral diffusion coefficients were very different. Furthermore, the capping behavior of two tagged membrane proteins was markedly different yet both had similar diffusion coefficients. The results from these tests disprove the bulk membrane flow hypothesis and are at odds with explicit predictions of the retrograde lipid flow hypothesis for the mechanism of capping. This work, therefore, supports the alternative cytoskeletal- based mechanism for driving capping. Requirements for coupling cytoskeletal movement to membrane components are discussed.     

Integrin-cytoskeletal interactions in migrating fibroblasts are dynamic, asymmetric, and regulated

We have used laser optical trapping and nanometer-level motion analysis to investigate the cytoskeletal associations and surface dynamics of beta 1 integrin, a cell-substrate adhesion molecule, on the dorsal surfaces of migrating fibroblast cells. A single-beam optical gradient trap (laser tweezers) was used to restrain polystyrene beads conjugated with anti-beta 1 integrin mAbs and place them at desired locations on the cell exterior. This technique was used to demonstrate a spatial difference in integrin-cytoskeleton interactions in migrating cells. We found a distinct increase in the stable attachment of beads, and subsequent rearward flow, on the lamellipodia of locomoting cells compared with the retracting portions. Complementary to the enhanced linkage of integrin at the cell lamellipodium, the membrane was more deformable at the rear versus the front of moving cells while nonmotile cells did not exhibit this asymmetry in membrane architecture. Video microscopy and nanometer-precision tracking routines were used to study the surface dynamics of integrin on the lamellipodia of migrating cells by monitoring the displacements of colloidal gold particles coated with anti-beta 1 integrin mAbs. Small gold aggregates were rapidly transported preferentially to the leading edge of the lamellipod where they resumed diffusion restricted along the edge. This fast transport was characterized by brief periods of directed movement ("jumps") having an instantaneous velocity of 37 +/- 15 microns/min (SD), separated by periods of diffusion. In contrast, larger aggregates of gold particles and the large latex beads underwent slow, steady rearward movement (0.85 +/- 0.44 micron/min) (SD) at a rate similar to that reported for other capping events and for migration of these cells. Cell lines containing mutated beta 1 integrins were used to show that the cytoplasmic domain is essential for an asymmetry in attachment of integrin to the underlying cytoskeletal network and is also necessary for rapid, intermittent transport. However, enhanced membrane deformability at the cell rear does not require integrin-cytoskeletal interactions. We also demonstrated that posttranslational modifications of integrin could potentially play a role in these phenomena. These results suggest a scheme for the role of dynamic integrin-mediated adhesive interactions in cell migration. Integrins are transported preferentially to the cell front where they form nascent adhesions. These adhesive structures grow in size and associate with the cytoskeleton that exerts a rearward force on them. Dorsal aggregates more rearward while those on the ventral side remain fixed to the substrate allowing the cell body to move forward. Detachment of the cell rear occurs by at least two modes: (a) weakened integrin- cytoskeleton interactions, potentially mediated by local modifications of linkage proteins, which lead to weakened cell-substratum interactions and (b) ripping of integrins and the highly deformable membrane from the cell body.     

Structure of MFP2 and its function in enhancing MSP polymerization in Ascaris sperm amoeboid motility

The simplicity and specialization of the cell motility machinery of Ascaris sperm provides a powerful system in which to probe the basic molecular mechanism of amoeboid cell motility. Although Ascaris sperm locomotion closely resembles that seen in many other types of crawling cell, movement is generated by modulation of a cytoskeleton based on the major sperm protein (MSP) rather than the actin present in other cell types. The Ascaris motility machinery can be studied conveniently in a cell-free in vitro system based on the movement of plasma membrane vesicles by fibres constructed from bundles of MSP filaments. In addition to ATP, MSP and a plasma membrane protein, reconstitution of MSP motility in this cell-free extract requires cytosolic proteins to orchestrate the site-specific assembly and bundling of MSP filaments that generates locomotion. One of these proteins, MFP2, accelerates the rate of movement in this assay. Here, we describe crystal structures of two isoforms of MFP2 and show that both are constructed from two domains that have the same fold based on a novel, compact beta sheet arrangement. Patterns of conservation observed in a structure-based analysis of MFP2 sequences from different nematode species identified regions that may be putative functional interfaces involved both in interactions between MFP2 domains and also with other components of the sperm motility machinery. Analysis of the growth of fibres in vitro in the presence of added MFP2 indicated that MFP2 increases the rate of locomotion by enhancing the effective rate of MSP filament polymerization. This observation, together with the structural data, suggests that MFP2 may function in a manner analogous to formins in actin-based motility.     

A Ser/Thr kinase required for membrane-associated assembly of the major sperm protein motility apparatus in the amoeboid sperm of Ascaris

Leading edge protrusion in the amoeboid sperm of Ascaris suum is driven by the localized assembly of the major sperm protein (MSP) cytoskeleton in the same way that actin assembly powers protrusion in other types of crawling cell. Reconstitution of this process in vitro led to the identification of two accessory proteins required for MSP polymerization: an integral membrane phosphoprotein, MSP polymerization-organizing protein (MPOP), and a cytosolic component, MSP fiber protein 2 (MFP2). Here, we identify and characterize a 34-kDa cytosolic protein, MSP polymerization-activating kinase (MPAK) that links the activities of MPOP and MFP2. Depletion/add-back assays of sperm extracts showed that MPAK, which is a member of the casein kinase 1 family of Ser/Thr protein kinases, is required for motility. MPOP and MPAK comigrated by native gel electrophoresis, coimmunoprecipitated, and colocalized by immunofluorescence, indicating that MPOP binds to and recruits MPAK to the membrane surface. MPAK, in turn, phosphorylated MFP2 on threonine residues, resulting in incorporation of MFP2 into the cytoskeleton. Beads coated with MPAK assembled a surrounding cloud of MSP filaments when incubated in MPAK-depleted sperm extract, but only when supplemented with detergent-solubilized MPOP. Our results suggest that interactions involving MPOP, MPAK, and MFP2 focus MSP polymerization to the plasma membrane at the leading edge of the cell thereby generating protrusion and minimizing nonproductive filament formation elsewhere.     

Analysis of gamete membrane dynamics during double fertilization of Arabidopsis

Angiosperms have a unique sexual reproduction system called “double fertilization.” One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.     

Cell Surface Topography Is a Regulator of Molecular Interactions during Chemokine-Induced Neutrophil Spreading

Adhesive interactions between neutrophils and endothelium involve chemokine-induced neutrophil spreading and subsequent crawling on the endothelium to sites of transmigration. We investigated the importance of cell topography in this process using immunofluorescence, scanning electron microscopy, and live-cell imaging using total internal reflectance microscopy to observe redistribution of key membrane proteins, both laterally and relative to surface topography, during neutrophil spreading onto glass coated with interleukin 8. During formation of the lamellipod, L-selectin is distributed on microvilli tips along the top of the lamellipodium, whereas the interleukin 8 receptors CXCR1 and CXCR2 and the integrin LFA-1 (α_Lβ₂) were present at the interface between the lamellipodium and the substrate. Total internal reflection fluorescence imaging indicated that LFA-1 and both chemokine receptors redistributed into closer contact with the substrate as the cells spread onto the surface and remodeled their topography. A geometric model of the surface remodeling with nonuniform distribution of molecules and a realistic distribution of microvilli heights was matched to the data, and the fits indicated a 1000-fold increase in the concentration of chemokine receptors and integrins available for bond formation at the interface. These observations imply that topographical remodeling is a key mechanism for regulating cell adhesion and surface-induced activation of cells.     

Cell Surface Topography Is a Regulator of Molecular Interactions during Chemokine-Induced Neutrophil Spreading

Adhesive interactions between neutrophils and endothelium involve chemokine-induced neutrophil spreading and subsequent crawling on the endothelium to sites of transmigration. We investigated the importance of cell topography in this process using immunofluorescence, scanning electron microscopy, and live-cell imaging using total internal reflectance microscopy to observe redistribution of key membrane proteins, both laterally and relative to surface topography, during neutrophil spreading onto glass coated with interleukin 8. During formation of the lamellipod, L-selectin is distributed on microvilli tips along the top of the lamellipodium, whereas the interleukin 8 receptors CXCR1 and CXCR2 and the integrin LFA-1 (α_Lβ₂) were present at the interface between the lamellipodium and the substrate. Total internal reflection fluorescence imaging indicated that LFA-1 and both chemokine receptors redistributed into closer contact with the substrate as the cells spread onto the surface and remodeled their topography. A geometric model of the surface remodeling with nonuniform distribution of molecules and a realistic distribution of microvilli heights was matched to the data, and the fits indicated a 1000-fold increase in the concentration of chemokine receptors and integrins available for bond formation at the interface. These observations imply that topographical remodeling is a key mechanism for regulating cell adhesion and surface-induced activation of cells.     

Mobility of GPIIb-IIIa receptors within membranes of surface- and suspension-activated platelets does not depend on assembly and contraction of cytoplasmic actin

Investigations using fibrinogen coupled to colloidal gold (Fgn/Au) have shown that glycoprotein IIb-IIIa (GPIIb-IIIa) receptors are mobile and undergo centripetal reorganization on spread platelets following surface activation. The assembly of cytoplasmic actin and its constriction into an inner filamentous zone have been proposed as the mechanism driving the Fgn/Au receptor complexes across the plasma membrane. The present study has used cytochalasin B (CB), an agent known to inhibit actin assembly and cause breakdown of newly formed actin filaments to test that hypothesis. At a concentration which inhibited pseudopod formation and spreading, CB did not block movement of Fgn/Au receptor complexes toward platelet centers or into the open canalicular system (OCS). Channels of the OCS filled with Fgn/Au receptor complexes were evident in over 90% of the CB-treated, surface-activated platelets. The findings do not support the concept that assembly of cytoplasmic actin and its contraction move receptor-ligand complexes on the platelet plasma membrane.     

Zona pellucida protein binding ability of porcine sperm during epididymal maturation and the acrosome reaction

In many mammals, the first interaction between gametes during fertilization occurs when sperm contact the zona pellucida surrounding the egg. Although porcine sperm first contact the zona pellucida via their plasma membrane, the regions of the sperm surface that display zona receptors have not been determined. We have used the Alexa 488 fluorophore conjugated to solubilized porcine zona pellucida proteins to observe zona receptors on live boar sperm. Zona proteins bound live, acrosome-intact sperm on the anterior portion of the sperm head, concentrated in a thin band over the acrosomal ridge. When sperm membranes were permeabilized by fixation or acrosome reactions induced by the ionophore A23187, zona binding was extended to a broad area covering the entire acrosomal region. Zona binding proteins were present in the acrosomes of sperm from all regions of the epididymis. In contrast, zona binding sites were found on the plasma membrane of most sperm from the corpus and cauda epididymis, but on only 6% of caput epididymal sperm. In conclusion, acrosome-intact boar sperm exhibit concentrated zona protein binding over the acrosomal ridge and acquire this binding in the corpus region of the epididymis, correlating with the developmental stage at which sperm gain the ability to fertilize oocytes.     

A Role for the Membrane in Regulating Chlamydomonas Flagellar Length

Flagellar assembly requires coordination between the assembly of axonemal proteins and the assembly of the flagellar membrane and membrane proteins. Fully grown steady-state Chlamydomonas flagella release flagellar vesicles from their tips and failure to resupply membrane should affect flagellar length. To study vesicle release, plasma and flagellar membrane surface proteins were vectorially pulse-labeled and flagella and vesicles were analyzed for biotinylated proteins. Based on the quantity of biotinylated proteins in purified vesicles, steady-state flagella appeared to shed a minimum of 16% of their surface membrane per hour, equivalent to a complete flagellar membrane being released every 6 hrs or less. Brefeldin-A destroyed Chlamydomonas Golgi, inhibited the secretory pathway, inhibited flagellar regeneration, and induced full-length flagella to disassemble within 6 hrs, consistent with flagellar disassembly being induced by a failure to resupply membrane. In contrast to membrane lipids, a pool of biotinylatable membrane proteins was identified that was sufficient to resupply flagella as they released vesicles for 6 hrs in the absence of protein synthesis and to support one and nearly two regenerations of flagella following amputation. These studies reveal the importance of the secretory pathway to assemble and maintain full-length flagella.     

Evidence for phosphorylation in the MSP cytoskeletal filaments of amoeboid spermatozoa

Nematode spermatozoa are highly specialized cells that lack flagella and, instead, extend a pseudopod to initiate motility. Crawling spermatozoa display classic features of amoeboid motility (e.g. protrusion of a pseudopod that attaches to the substrate and the assembly and disassembly of cytoskeletal filaments involved in cell traction and locomotion), however, cytoskeletal dynamics in these cells are powered exclusively by Major Sperm Protein (MSP) rather than actin and no other molecular motors have been identified. Thus, MSP-based motility is regarded as a simple locomotion machinery suitable for the study of plasma membrane protrusion and cell motility in general. This recent focus on MSP dynamics has increased the necessity of a standardized methodology to obtain C. elegans sperm extract that can be used in biochemical assays and proteomic analysis for comparative studies. In the present work we have modified a method to reproducibly obtain relative high amounts of proteins from C. elegans sperm extract. We show that these extracts share some of the properties observed in sperm extracts from the parasitic nematode Ascaris including Major Sperm Protein (MSP) precipitation and MSP fiber elongation. Using this method coupled to immunoblot detection, Mass Spectrometry identification, in silico prediction of functional domains and biochemical assays, our results indicate the presence of phosphorylation sites in MSP of Caenorhabditis elegans spermatozoa.