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Nematode sperm locomotion
Abstract
The simplicity and specialization of the amoeboid motility of nematode sperm can give intriguing insights into the molecular mechanisms underlying movement in more conventional actin-based systems. Amoeboid motility in nematode sperm is based on their major sperm protein. Advances over the past year in understanding the assembly of this protein in vivo and in vitro have underlined the importance of vectorial assembly and filament bundling. In this system, it is possible that these two properties may be sufficient to generate motility that closely resembles that seen in conventional actin-based systems.
... Thus, actin and tubulin are cast off into a residual body along with the protein synthesizing machinery. The activated spermatids contain MSP, nuclei, mitochondria and other sperm-specific organelles and, thus, are streamlined for fertilization (Roberts and Stewart, 1995). ...
... Ascaris sperm use their MSP cytoskeleton solely to accomplish movement and therefore, motility can be studied without the complications of a multifunctional cytoskeleton (Roberts and Stewart, 1995). Further, MSP is not homologous to any other cytoskeletal protein, and may inform us of the general mechanisms involved in the process of cell crawling Italiano et al., 2001). ...
... In Ascaris sperm, MSP filaments are organized into long, branched meshworks, called fiber complexes, that span the length of the lamellipod (Sepsenwol et al., 1989). The MSP filament network is assembled preferentially along the leading edge and flows rearward as the cell moves forward, resulting in the characteristic treadmilling of MSP fiber complexes that is easily observed in live cells by light microscopy (Roberts and Stewart, 1995;Roberts and Stewart, 1997). The rate of MSP assembly and disassembly is tightly coupled to the rate of cell movement (Roberts and King, 1991). ...
The amoeboid sperm of Ascaris crawl through a cycle of protrusion, adhesion, and retraction, similar to that seen in conventional actin-based cells. However, instead of actin, these cells power their movement through modulation of the major sperm protein (MSP) cytoskeleton. MSP forms dense filament meshworks that pack the sperm lamellipod. Protrusion is associated with the assembly of MSP filaments at the leading edge of the lamellipod, and retraction is connected with the disassembly of the MSP network at the base of the lamellipod. The motility of Ascaris sperm can be reconstituted in cell-free extracts. In vitro, plasma membrane vesicles are pushed forward by the elongation of fibers constructed from a columnar meshwork of MSP filaments.
This in vitro motility requires components from both the cytosol and the vesicle. LeClaire et al. (2003) recently identified the 48 kDa membrane protein required to orchestrate MSP cytoskeletal assembly at the leading edge of the lamellipod. In this study, I describe the first cytosolic proteins that are components of the MSP locomotory machinery. I fractionated cytosol with a range of biochemical techniques and reconstituted fiber assembly with a limited subset of cytosolic components. Thus, this fraction contains all the cytosolic accessory proteins required to build fibers. Several of the components in this active fraction were used to generate antibodies, which labeled the cytoskeleton in Ascaris sperm and in fibers grown in vitro and thus, identified six proteins, p43, p42, p40, p38, p34, and p16, as part of the MSP cytoskeleton.
Sequence analysis showed that each protein is novel to nematode sperm and has a homolog of unknown function in C. elegans that exhibits sperm-enriched expression (Reinke et al., 2000; Hill et al., 2001). The predicted protein sequence of p43 is based on a tandem array of repeats with serine phosphorylation sites. P38, p40, and p42 appear to be a family of closely related polypeptides. The sequences of p38 and p40 contain a domain duplication as well as proline-rich repeats. P34 shows homology to serine/threonine kinases. The p16 triplet is a family of closely related polypeptides.
Two of the proteins had reciprocal effects on fiber growth: p38 increased fiber growth rate and p16 decreased fiber growth rate. The effects of both p38 and p16 were concentration-dependent and antagonistic. Since the rate-enhancement by p38 was not potentiated by MSP, another cytosolic protein is involved in up-regulation of the rate of MSP elongation. Additionally, p16 altered the number of filaments polymerized at the vesicle surface and thus may regulate MSP nucleation. Immunoprecipitations and affinity chromatography established that these two proteins bind to MSP under conditions that promote in vitro assembly. Based on these data, I present a revised model for the mechanism of membrane-associated MSP polymerization, which proposes that p38 influences the rate of MSP elongation by recruiting MSP to the vesicle surface and that p16 incorporates into MSP filaments and blocks further assembly by acting as a capping protein. My results imply that although the specific components differ, actin-based and MSP-based systems both rely on the interplay of positive and negative regulators of cytoskeletal assembly to maintain the pace of locomotion.
... In addition to providing the basis of cell motility, these structures also function in the process of phagocytosis, movement of vesicles and organelles, surface rearrangement, and cytokinesis. Actin and myosin require sets of accessory proteins for each specific activity creating an interconnected and complex system [12]. It becomes very difficult to untangle this complexity in order to determine what contributes to cell motility in this multi-functional cytoskeleton. ...
... It becomes very difficult to untangle this complexity in order to determine what contributes to cell motility in this multi-functional cytoskeleton. Rather than swimming with the assistance of flagella, the motile sperm of the nematode Ascaris suum crawl like amoebae with the aid of a motile appendage called a lamellipod [12]. These sperm cells, though achieving locomotion through the same cycle of protrusion, adhesion and contraction, have a simplifying characteristic that sets them apart from most crawling cells. ...
... Instead of using an actin/myosin network, the cytoskeleton of Ascaris suum consists of a Major Sperm Protein (MSP) gel network. The MSP cytoskeleton functions only in the process of cell locomotion; therefore, it avoids many of the complications associated with studying actin-based motility [12]. In addition, many cellular components such as the endoplasmic reticulum, the Golgi apparatus, and the usual actin cytoskeleton are left behind in the residual body after meiosis [3]. ...
The nematode sperm cell crawls by recycling major sperm protein (MSP) from dimers into subfilaments, filaments, and filament complexes, as a result of thermal writhing in the presence of hydrophobic patches. Polymerization near leading edges of the cell intercolates MSP dimers onto the tips of growing filament complexes, forcing them against the cell boundary, and extending the cytoskeleton in the direction of motion. Strong adhesive forces attach the cell to the substrate in the forward part of the lamellipod, while depolymerization in the rearward part of the cell breaks down the cytoskeleton, contracting the lamellipod and pulling the cell body forward. The movement of these cells, then, is caused by coordinated protrusive, adhesive and contractile forces, spatially separated across the lamellipod. This paper considers a phenomenological model that tracks discrete elements of the cytoskeleton in curvilinear coordinates. The pseudo-two dimensional model primarily considers protrusion and rotation of the cell, along with the evolution of the cell boundary. General assumptions are that pH levels within the lamellipod regulate protrusion, contraction and adhesion, and that growth of the cytoskeleton, over time, is perpendicular to the evolving cell boundary. The model follows the growth and contraction of a discrete number of MSP fiber complexes, since they appear to be the principle contributors for force generation in cell boundary protrusion and contraction, and the backbone for the dynamic geometry and motion.
... The simplicity and specialisation of the motile machinery of nematode sperm offer unique and powerful advantages for probing the molecular mechanism underlying amoeboid cell motility (for reviews see Roberts & Stewart, 1995;Theriot, 1996). Nematode sperm are unusual amoeboid cells in which motility is not based on actin, but instead on the major sperm protein (MSP). ...
... Whereas actin requires a spectrum of different bundling proteins to control bundling and network formation (for a review see Machesky & Pollard, 1993), the unique structure of MSP filaments ensures that they have intrinsic bundling potential. Vectorial assembly of MSP and its association into networks are intimately involved in nematode sperm amoeboid motility and indeed may be sufficient for pseudopod extension (Roberts & Stewart, 1995), since it appears possible to move membranes in vitro using these properties alone (Italiano et al., 1996). ...
... Polymerization into filaments and their bundling into higher aggregates are the principal biological functions of MSP and are intimately linked to amoeboid locomotion in nematode sperm. MSP polymerization and filament bundling can move membrane vesicles in vitro (Italiano et al., 1996) and appear to be responsible for pseudopod extension in crawling sperm (for reviews see Roberts & Stewart, 1995;Theriot 1996). ...
The major sperm protein (MSP) of Ascaris suum, mediates amoeboid motility by forming an extensive intermeshed system of cytoskeletal filaments analogous to that formed by actin in many amoeboid cells. We have used a combination of biochemical and NMR methods to show that, in contrast to actin, MSP exists in solution as a symmetrical dimer. This result has important implications for the mechanism of both MSP filament assembly and the recognition of different MSP isoforms in vivo. (C) 1996 Academic Press Limited
... For these sperm to function as motile gametes, they must first undergo a process of cellular morphogenesis (spermiogenesis) which transforms them from sessile, apolar spermatids into motile, bipolar spermatozoa. Nematode spermatozoa are unusual in that they lack flagella and instead crawl via a pseupodial projection (Roberts and Stewart, 1995). To test whether the sperm chromatin mass is P. L. Sadler and D. C. Shakesrequired either as a template for transcription or as a cue for polarization during this cell morphogenetic process, we attempted to activate the anucleate spermatids using previously described methods for in vitro sperm activation (Ward et al., 1983). ...
... Providing definitive evidence to support a polarization role for either the sperm centrioles or the sperm asters will require, in part, the identification and analysis of paternal effect mutants which either lack sperm centrioles or are unable to support centriole function. Investigating this role in C. elegans sperm should be easier than in most organisms, since the sperm centrosome is not required for the sperm's unique non-actin, non-tubulin based cell motility (Roberts and Stewart, 1995). Recent studies in our own lab suggest that a subset of our chromosome segregation mutants may, in fact, have defects in the segregation of additional sperm components. ...
It has long been appreciated that spermiogenesis, the cellular transformation of sessile spermatids into motile spermatozoa, occurs in the absence of new DNA transcription. However, few studies have addressed whether the physical presence of a sperm nucleus is required either during spermiogenesis or for subsequent sperm functions during egg activation and early zygotic development. To determine the role of the sperm nucleus in these processes, we analyzed two C. elegans mutants whose spermatids lack DNA. Here we show that these anucleate sperm not only differentiate into mature functional spermatozoa, but they also crawl toward and fertilize oocytes. Furthermore, we show that these anucleate sperm induce both normal egg activation and anterior-posterior polarity in the 1-cell C. elegans embryo. The latter finding demonstrates for the first time that although the anterior-posterior embryonic axis in C. elegans is specified by sperm, the sperm pronucleus itself is not required. Also unaffected is the completion of oocyte meiosis, formation of an impermeable eggshell, migration of the oocyte pronucleus, and the separation and expansion of the sperm-contributed centrosomes. Our investigation of these mutants confirms that, in C. elegans, neither the sperm chromatin mass nor a sperm pronucleus is required for spermiogenesis, proper egg activation, or the induction of anterior-posterior polarity.
... Unlike mammalian sperm, the amoeboid sperm of the nematode Ascaris suum are not swimmers, but crawlers. Much like crawling epithelial gold¢sh keratocytes [40], the sperm extend broad, £at lamellipodia in front of themselves [41,42]. At the leading edge of the lamellipodium, protein is polymerized into a network, and the protein network remains stationary while the cell pulls forward over it. ...
... At the leading edge of the lamellipodium, protein is polymerized into a network, and the protein network remains stationary while the cell pulls forward over it. Although the polymerizing protein in Ascaris sperm is the 14 kDa major sperm protein (MSP), which has no obvious homology to actin [41], the movements of Ascaris worm sperm appear quite similar to those of ¢sh keratocytes. Nature has apparently solved the motility problem with the same blueprint , but di¡erent materials. ...
Listeria monocytogenes and some other infectious bacteria polymerize their host cell's actin into tails that propel the bacteria through the cytoplasm. Here we show that reconstitution of this behavior in simpler media resolves two aspects of the mechanism of force transduction. First, since dilute reconstitution media have no cytoskeleton, we consider what keeps the tail from being pushed backward rather than the bacterium being propelled forward. The dependence of the partitioning of motion on the friction coefficient of the tail is derived. Consistent with experiments, we find that the resistance of the tail to motion is sensitive to its length. That even small tails are stationary in intact cells is attributed to anchoring to the cytoskeleton. Second, the comparatively low viscosity of some reconstitution media magnifies the effects of diffusion, such that a large gap will develop between the bacterium and its tail if they are unattached. At the viscosities of diluted platelet extracts, steady-state gaps of several bacterium lengths are predicted. Since such gaps are not observed, we conclude that Listeria must be attached to their tails. We consider what purposes such attachments might serve under physiological conditions. The implications for related pathogens and amoeboid locomotion are also discussed.
... One exception in eukaryotes of functional rotatory motor called axostyle (not dynein based)) was nevertheless described in some parasitic uni-cells of termites (Yamin & Tam, 1982). Nevertheless, some types of eukaryotic cell have developed alternative forms of motility based on highly different basis: their progression absolutely needs contact with the surface of a substrate and mainly exploit creeping ability of the lower surface of their flat body (Roberts, Stewart, 1995). Quite strikingly, this lower surface mostly use movement principle of a tank with caterpillar tracks; in this case value of velocity barely reaches 70 µm/min in these cells, which is one to two orders of magnitude lower than flagellated cells. ...
Electromagnetic Wave Propagation in a Spatially Inhomogeneous Thin
Film Medium
We present the study of wave propagation in a spatially inhomogeneous thin film medium
carried out here in which for the first case the influence of reflection on the transmitted
wave was neglected. In the second case the Fresnel formula in which case the impact of the
reflection and transmission coefficient on the propagating wave was introduced due to the
interfacial reflection between the anisotropic and isotropic parts of the film medium
occasioned by the film variation in refractive index as manifested in the transmitted wave
for both waves at normal incidence and the one that lies on the plane of incident was
investigated. Further analysis was carried out on the wave incident on the medium at
various angles. This idea was also used in analyzing the reflection and transmission
co-efficient as plotted on the basis of which the reflection and transmitted nature of the thin
film medium was depicted.
... Later work on C. elegans and Ascaris suum demonstrated the role of MSP in the movement of nematode sperm (see above). The presence of actin was therefore not considered of functional sigruficance by most authors studying the MSP cytoskeleton (reviews in Roberts and Stewart, 1995;Roberts and Stewart, 1997;Scott, 1996;Theriot, 1996). ...
... Sperm respond to prostaglandins by increasing speed and directional velocity. Motility is driven by pseudopod translocation, a common feature among nematode species [25]. C. elegans sperm and flagellated sperm from other species share evolutionarily conserved metabolic pathways and cell surface proteins important for fertilization [26][27][28]. ...
The sperm’s crucial function is to locate and fuse with a mature oocyte. Under laboratory conditions, Caenorhabditis elegans sperm are very efficient at navigating the hermaphrodite reproductive tract and locating oocytes. Here, we identify chemosensory and oxygen-sensing circuits that affect the sperm’s navigational capacity. Multiple Serpentine Receptor B (SRB) chemosensory receptors regulate Gα pathways in gustatory sensory neurons that extend cilia through the male nose. SRB signaling is necessary and sufficient in these sensory neurons to influence sperm motility parameters. The neuropeptide Y pathway acts together with SRB-13 to antagonize negative effects of the GCY-35 hyperoxia sensor on spermatogenesis. SRB chemoreceptors are not essential for sperm navigation under low oxygen conditions that C. elegans prefers. In ambient oxygen environments, SRB-13 signaling impacts gene expression during spermatogenesis and the sperm’s mitochondria, thereby increasing migration velocity and inhibiting reversals within the hermaphrodite uterus. The SRB-13 transcriptome is highly enriched in genes implicated in pathogen defense, many of which are expressed in diverse tissues. We show that the critical time period for SRB-13 signaling is prior to spermatocyte differentiation. Our results support the model that young C. elegans males sense external environment and oxygen tension, triggering long-lasting downstream signaling events with effects on the sperm’s mitochondria and navigational capacity. Environmental exposures early in male life may alter sperm function and fertility.
... Nevertheless, some types of eukaryotic cell have developed alternative forms of motility AcademyPublish.org -Wave Propagation 559 based on highly different basis: their progression absolutely needs contact with the surface of a substrate and mainly exploit creeping ability of the lower surface of their flat body (Roberts, Stewart, 1995). Quite strikingly, this lower surface mostly use movement principle of a tank with caterpillar tracks; in this case value of velocity barely reaches 70 µm/min in these cells, which is one to two orders of magnitude lower than flagellated cells. ...
... K, X 3,500. Fibrous bodies of nematode spermatids are known to be a storage organelle for MSP, which is released from these bodies at the spermatozoon stage to make the cytoskeleton of the pseudopods (Hess and Poinar, 1989;Holliday and Roberts, 1995;Italian0 et al., 1996;Kimble and Ward, 1988;Kinget al., 1992;Roberts, 1983;Roberts and King, 1991;Roberts et al., 1986;Roberts and Stewart, 1995;Royal et al., 1995;Sepsenwol et al., 1989;Shakes and Ward, 1989;Ward, 1986;Ward and Klass, 1982;Wolf et al., 1978). We used in our experiments an antibody against C. elegans MSP (Burke and Ward, 1983); this antibody is not species-specific and labels MSP in Neoaplectana (Hess and Poinar, 19891, Heligmosomoides (present study), and other nematodes (unpublished). ...
Nematode spermatozoa are amoeboid cells. In Caernorhabditis elegans and Ascaris suum, previous studies have reported that sperm motility does not involve actin, but, instead, requires a specific cytoskeletal protein, name y major-sperm-protein (MSP). In Heligmosomoides polygyrus, a species with large and elongate spermatids and spermatozoa, cell organelles are easily identified even with light microscopy. Electrophoresis of Heligmosomoides sperm proteins indicates that the main protein band has a molecular weight of about 15 kDa, as MSP in other nematodes, and is specifically labelled by an anti-MSP antibody raised against C. elegans MSP. A minor band at 43 kDa was specifically labelled by an anti-actin antibody. Reaction of anti-actin and anti-MSP antibodies is specific to, and restricted to, their respective targets. Actin and MSP localisation, studied by indirect immunofluorescence in male germ cells of Heligmosomoides polygyrus, are similar: spermatids show rows of dots, corresponding to the fibrous bodies, around an unlabelled central longitudinal core; spermatozoa are labelled strictly in an anterior crescent-shaped cap, at the opposite pole to the nucleus, which contains fibres of the MSP cytoskeleton. Phalloidin labelling shows that F-actin is present in spermatids, but absent in spermatozoa. Tropomyosin shows a distinct pattern in spermatids, but is located in the MSP and actin-containing cap in spermatozoa. It is hypothesized that actin plays a role in the shaping of the cell and in the arrangement of its organelles during nematode spermiogenesis, when MSP is present, in an inactive state, in the fibrous bodies. The concentration of actin and tropomyosin in the anterior cap is not compatible with previous theories about the MSP cytoskeleton which is supposed to act in the absence of actin. © 1996 Wiley-Liss, Inc.
... This gene encoded a putative protein of 216 amino-acids and predicted molecular weight of 23.9 kDa containing a major sperm protein (MSP) domain at the N-terminus and a TMD at the C-terminus (Fig. 2A). MSP is a filament forming protein that mediates amoeboid motility in nematode sperm (Roberts and Stewart 1995). Bioinformatic comparison with 50 other proteins with an N-terminal MSP domain and a C-terminal TMD revealed that the product of locus Tb11.01.4810 encoded a T. brucei orthologue of VAMP-associated proteins (VAPs) (Fig. 3B). ...
Trypanosomes and Leishmanias are important human parasites whose cellular architecture is centred on the single flagellum. In trypanosomes, this flagellum is attached to the cell along a complex flagellum attachment zone (FAZ), comprising flagellar and cytoplasmic components, the integrity of which is required for correct cell morphogenesis and division. The cytoplasmic FAZ cytoskeleton is conspicuously associated with a sheet of endoplasmic reticulum termed the 'FAZ ER'. In the present work, 3D electron tomography of bloodstream form trypanosomes was used to clarify the nature of the FAZ ER. We also identified TbVAP, a T. brucei protein whose knockdown by RNAi in procyclic form cells leads to a dramatic reduction in the FAZ ER, and in the ER associated with the flagellar pocket. TbVAP is an orthologue of VAMP-associated proteins (VAPs), integral ER membrane proteins whose mutation in humans has been linked to familial motor neuron disease. The localisation of tagged TbVAP and the phenotype of TbVAP RNAi in procyclic form trypanosomes are consistent with a function for TbVAP in the maintenance of sub-populations of the ER associated with the FAZ and the flagellar pocket. Nevertheless, depletion of TbVAP did not affect cell viability or cell cycle progression.
... Although nematode sperm contain neither F-actin nor myosin, their locomotion is practically indistinguishable from that of conventional crawling cells (reviewed in Theriot, 1996;Roberts and Stewart, 1997). Sperm motility is driven by a novel locomotory apparatus based on filaments comprised of the 14-kD major sperm protein (MSP) 1 (reviewed in Roberts and Stewart, 1995). In sperm from Ascaris suum , the MSP filaments are arranged into dynamic meshworks, called fiber complexes, that extend the entire length of the lamellipodium (Sepsenwol et al., 1989). ...
The major sperm protein (MSP)-based amoeboid motility of Ascaris suum sperm requires coordinated lamellipodial protrusion and cell body retraction. In these cells, protrusion and retraction are tightly coupled to the assembly and disassembly of the cytoskeleton at opposite ends of the lamellipodium. Although polymerization along the leading edge appears to drive protrusion, the behavior of sperm tethered to the substrate showed that an additional force is required to pull the cell body forward. To examine the mechanism of cell body movement, we used pH to uncouple cytoskeletal polymerization and depolymerization. In sperm treated with pH 6.75 buffer, protrusion of the leading edge slowed dramatically while both cytoskeletal disassembly at the base of the lamellipodium and cell body retraction continued. At pH 6.35, the cytoskeleton pulled away from the leading edge and receded through the lamellipodium as its disassembly at the cell body continued. The cytoskeleton disassembled rapidly and completely in cells treated at pH 5.5, but reformed when the cells were washed with physiological buffer. Cytoskeletal reassembly occurred at the lamellipodial margin and caused membrane protrusion, but the cell body did not move until the cytoskeleton was rebuilt and depolymerization resumed. These results indicate that cell body retraction is mediated by tension in the cytoskeleton, correlated with MSP depolymerization at the base of the lamellipodium.
... The simplicity and specialization of the crawling sperm of nematodes such as Ascaris suum can offer Florida State University Tallahassee, Florida 32306 advantages for investigating the mechanism of amoeboid motility (reviewed by Roberts and Stewart, 1995). † Medical Research Council Laboratory of Molecular Biology Like conventional amoeboid cells, Ascaris sperm crawl by extending a pseudopod that undergoes localized Hills Road Cambridge CB2 2QH ...
We have developed an in vitro motility system from Ascaris sperm, unique amoeboid cells that use filament arrays composed of major sperm protein (MSP) instead of an actin-based apparatus for locomotion. Addition of ATP to sperm extracts induces formation of fibers approximately 2 microns in diameter. These fibers display the key features of the MSP cytoskeleton in vivo. Each fiber consists of a meshwork of MSP filaments and has at one end a vesicle derived from the plasma membrane at the leading edge of the cell. Fiber growth is due to filament assembly at the vesicle; thus, fiber elongation results in vesicle translocation. This in vitro system demonstrates directly that localized polymerization and bundling of filaments can move membranes and provides a powerful assay for evaluating the molecular mechanism of amoeboid cell motility.
... In the spermatozoon, actin (no F-actin detected) and MSP are strictly located in the cap opposite to the nucleus. Fibrous bodies of nematode spermatids are known to be a storage organelle for MSP, which is released from these bodies at the spermatozoon stage to make the cytoskeleton of the pseudopods (Hess and Poinar, 1989; Holliday and Roberts, 1995; Italian0 et al., 1996; Kimble and Ward, 1988; Kinget al., 1992; Roberts, 1983; Roberts and King, 1991; Roberts et al., 1986; Roberts and Stewart, 1995; Royal et al., 1995; Sepsenwol et al., 1989; Shakes and Ward, 1989; Ward, 1986; Ward and Klass, 1982; Wolf et al., 1978). We used in our experiments an antibody against C. elegans MSP (Burke and Ward, 1983); this antibody is not species-specific and labels MSP in Neoaplectana (Hess and Poinar, 19891 , Heligmosomoides (present study), and other nematodes (unpublished ). ...
Nematode spermatozoa are amoeboid cells. In Caenorhabditis elegans and Ascaris suum, previous studies have reported that sperm motility does not involve actin, but, instead, requires a specific cytoskeletal protein, namely major-sperm-protein (MSP). In Heligmosomoides polygyrus, a species with large and elongate spermatids and spermatozoa, cell organelles are easily identified even with light microscopy. Electrophoresis of Heligmosomoides sperm proteins indicates that the main protein band has a molecular weight of about 15 kDa, as MSP in other nematodes, and is specifically labelled by an anti-MSP antibody raised against C. elegans MSP. A minor band at 43 kDa was specifically labelled by an anti-actin antibody. Reaction of anti-actin and anti-MSP antibodies is specific to, and restricted to, their respective targets. Actin and MSP localisation, studied by indirect immunofluorescence in male germ cells of Heligmosomoides polygyrus, are similar: spermatids show rows of dots, corresponding to the fibrous bodies, around an unlabelled central longitudinal core; spermatozoa are labelled strictly in an anterior crescent-shaped cap, at the opposite pole to the nucleus, which contains fibres of the MSP cytoskeleton. Phalloidin labelling shows that F-actin is present in spermatids, but absent in spermatozoa. Tropomyosin shows a distinct pattern in spermatids, but is located in the MSP and actin-containing cap in spermatozoa. It is hypothesized that actin plays a role in the shaping of the cell and in the arrangement of its organelles during nematode spermiogenesis, when MSP is present, in an inactive state, in the fibrous bodies. The concentration of actin and tropomyosin in the anterior cap is not compatible with previous theories about the MSP cytoskeleton, which is supposed to act in the absence of actin.
... Like other ameboid cells, these sperm crawl by extending a pseudopod, and protrusive activity at the leading edge is tightly coupled to localized po-lymerization and centripetal flow of the cytoskeleton. However, these cells contain no F-actin; the polymerizing unit involved in their locomotion is the 14-kD major sperm protein (MSP) 1 (Roberts and Stewart, 1995). MSP and actin have neither sequence nor structural homology, yet the patterns of motility the two proteins produce are so remarkably similar that the physical principles underlying MSP-and actin-based crawling movement must be shared (Theriot, 1996). ...
Sperm from nematodes use a major sperm protein (MSP) cytoskeleton in place of an actin cytoskeleton to drive their ameboid locomotion. Motility is coupled to the assembly of MSP fibers near the leading edge of the pseudopod plasma membrane. This unique motility system has been reconstituted in vitro in cell-free extracts of sperm from Ascaris suum: inside-out vesicles derived from the plasma membrane trigger assembly of meshworks of MSP filaments, called fibers, that push the vesicle forward as they grow (Italiano, J.E., Jr., T.M. Roberts, M. Stewart, and C.A. Fontana. 1996. Cell. 84:105-114). We used changes in hydrostatic pressure within a microscope optical chamber to investigate the mechanism of assembly of the motile apparatus. The effects of pressure on the MSP cytoskeleton in vivo and in vitro were similar: pressures >50 atm slowed and >300 atm stopped fiber growth. We focused on the in vitro system to show that filament assembly occurs in the immediate vicinity of the vesicle. At 300 atm, fibers were stable, but vesicles often detached from the ends of fibers. When the pressure was dropped, normal fiber growth occurred from detached vesicles but the ends of fibers without vesicles did not grow. Below 300 atm, pressure modulates both the number of filaments assembled at the vesicle (proportional to fiber optical density and filament nucleation rate), and their rate of assembly (proportional to the rates of fiber growth and filament elongation). Thus, fiber growth is not simply because of the addition of subunits onto the ends of existing filaments, but rather is regulated by pressure-sensitive factors at or near the vesicle surface. Once a filament is incorporated into a fiber, its rates of addition and loss of subunits are very slow and disassembly occurs by pathways distinct from assembly. The effects of pressure on fiber assembly are sensitive to dilution of the extract but largely independent of MSP concentration, indicating that a cytosolic component other than MSP is required for vesicle-association filament nucleation and elongation. Based on these data we present a model for the mechanism of locomotion-associated MSP polymerization the principles of which may apply generally to the way cells assemble filaments locally to drive protrusion of the leading edge.
... Although nematode sperm contain neither F-actin nor myosin, their locomotion is practically indistinguishable from that of conventional crawling cells (reviewed in Theriot, 1996;Roberts and Stewart, 1997). Sperm motility is driven by a novel locomotory apparatus based on filaments comprised of the 14-kD major sperm protein (MSP) 1 (reviewed in Roberts and Stewart, 1995). In sperm from Ascaris suum , the MSP filaments are arranged into dynamic meshworks, called fiber complexes, that extend the entire length of the lamellipodium (Sepsenwol et al., 1989). ...
The major sperm protein (MSP)-based amoeboid motility of Ascaris suum sperm requires coordinated lamellipodial protrusion and cell body retraction. In these cells, protrusion and retraction are tightly coupled to the assembly and disassembly of the cytoskeleton at opposite ends of the lamellipodium. Although polymerization along the leading edge appears to drive protrusion, the behavior of sperm tethered to the substrate showed that an additional force is required to pull the cell body forward. To examine the mechanism of cell body movement, we used pH to uncouple cytoskeletal polymerization and depolymerization. In sperm treated with pH 6.75 buffer, protrusion of the leading edge slowed dramatically while both cytoskeletal disassembly at the base of the lamellipodium and cell body retraction continued. At pH 6.35, the cytoskeleton pulled away from the leading edge and receded through the lamellipodium as its disassembly at the cell body continued. The cytoskeleton disassembled rapidly and completely in cells treated at pH 5.5, but reformed when the cells were washed with physiological buffer. Cytoskeletal reassembly occurred at the lamellipodial margin and caused membrane protrusion, but the cell body did not move until the cytoskeleton was rebuilt and depolymerization resumed. These results indicate that cell body retraction is mediated by tension in the cytoskeleton, correlated with MSP depolymerization at the base of the lamellipodium.
... However, nematode sperm lack the actin machinery typically associated with amoeboid cell motility; instead, their movement is powered by a cytoskeleton built from major sperm protein (MSP) filaments*. MSP is a highly basic 14.5 kDa polypeptide that polymerizes in a hierarchical fashion (Roberts and Stewart, 1995;Roberts and Stewart, 1997). The protein forms symmetrical dimers in solution that polymerize into helical subfilaments, which wind together in pairs to form larger filaments. ...
Sperm of the nematode, Ascaris suum, crawl using lamellipodial protrusion, adhesion and retraction, a process analogous to the amoeboid motility of other eukaryotic cells. However, rather than employing an actin cytoskeleton to generate locomotion, nematode sperm use the major sperm protein (MSP). Moreover, nematode sperm lack detectable molecular motors or the battery of actin-binding proteins that characterize actin-based motility. The Ascaris system provides a simple ‘stripped down’ version of a crawling cell in which to examine the basic mechanism of cell locomotion independently of other cellular functions that involve the cytoskeleton. Here we present a mechanochemical analysis of crawling in Ascaris sperm. We construct a finite element model wherein (a) localized filament polymerization and bundling generate the force for lamellipodial extension and (b) energy stored in the gel formed from the filament bundles at the leading edge is subsequently used to produce the contraction that pulls the rear of the cell forward. The model reproduces the major features of crawling sperm and provides a framework in which amoeboid cell motility can be analyzed. Although the model refers primarily to the locomotion of nematode sperm, it has important implications for the mechanics of actin-based cell motility.
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... MSP isoforms differ by only one to four amino acids. In contrast to the more familiar flagellated sperm, C. elegans sperm are amoeboid in shape and crawl by extending a lamellipod (Roberts and Stewart 1995). Surprisingly, this movement is not dependent on conventional motor proteins , nor is it actin-based, as sperm contain virtually no actin (Nelson et al. 1982). ...
Innate immunity functions as a rapid defense against broad classes of pathogenic agents. While the mechanisms of innate immunity in response to antigen exposure are well-studied, how pathogen exposure activates the innate immune responses and the role of genetic variation in immune activity is currently being investigated. Previously we showed significant survival differences between the N2 and CB4856 Caenorhabditis elegans isolates in response to Staphylococcus epidermidis infection. One of those differences was expression of the mab-5 Hox-family transcription factor, which was induced in N2, but not CB4856, after infection. Here we use survival assays and RNA-sequencing to better understand the role of mab-5 in response to S. epidermidis. We found that mab-5 loss-of-function mutants were more susceptible to S. epidermidis infection than N2 or mab-5 gain-of-function mutants, but not as susceptible as CB4856 animals. We then conducted transcriptome analysis of infected worms and found considerable differences in gene-expression profiles when comparing animals with mab-5 loss-of-function to either N2 or mab-5 gain-of-function. N2 and mab-5 gain-of-function animals showed a significant enrichment in expression of immune genes and C-type lectins, whereas mab-5 loss-of-function mutants did not. Overall, gene expression profiling in mab-5 mutants provided insight into MAB-5 regulation of the transcriptomic response of C. elegans to pathogenic bacteria and helps us to understand mechanisms of innate immune activation and the role that transcriptional regulation plays in organismal health.
Entomopathogenic nematodes, including Heterorhabditis and Steinernema, are parasitic to insects and contain mutualistically symbiotic bacteria in their intestines (Photorhabdus and Xenorhabdus, respectively) and therefore offer opportunities to study both mutualistic and parasitic symbiosis. The establishment of genetic tools in entomopathogenic nematodes has been impeded by limited genetic tractability, inconsistent growth in vitro, variable cryopreservation, and low mating efficiency. We obtained the recently described Steinernema hermaphroditum strain CS34 and optimized its in vitro growth, with a rapid generation time on a lawn of its native symbiotic bacteria Xenorhabdus griffiniae. We developed a simple and efficient cryopreservation method. Previously, S. hermaphroditum isolated from insect hosts was described as first-generation hermaphroditic and second-generation gonochoristic. We discovered that CS34, when grown in vitro, produced consecutive generations of autonomously reproducing hermaphrodites accompanied by rare males. We performed mutagenesis screens in S. hermaphroditum that produced mutant lines with visible and heritable phenotypes. Genetic analysis of the mutants demonstrated that this species reproduces by self-fertilization rather than parthenogenesis and that its sex is determined chromosomally. Genetic mapping has thus far identified markers on the X chromosome and three of four autosomes. We report that S. hermaphroditum CS34 is the first consistently hermaphroditic entomopathogenic nematode and is suitable for genetic model development to study naturally occurring mutualistic symbiosis and insect parasitism.
Entomopathogenic nematodes, including Heterorhabditis and Steinernema , are parasitic to insects and contain mutualistically symbiotic bacteria in their intestines ( Photorhabdus and Xenorhabdus, respectively) and therefore offer opportunities to study both mutualistic and parasitic symbiosis. The establishment of genetic tools in entomopathogenic nematodes has been impeded by limited genetic tractability, inconsistent growth in vitro , variable cryopreservation, and low mating efficiency. We obtained the recently described Steinernema hermaphroditum strain CS34 and optimized its in vitro growth, with a rapid generation time on a lawn of its native symbiotic bacteria Xenorhabdus griffiniae . We developed a simple and efficient cryopreservation method. Previously, S. hermaphroditum isolated from insect hosts was described as first-generation hermaphroditic and second-generation gonochoristic. We discovered that CS34, when grown in vitro, produced consecutive generations of autonomously reproducing hermaphrodites accompanied by rare males. We performed mutagenesis screens in S. hermaphroditum that produced mutant lines with visible and heritable phenotypes. Genetic analysis of the mutants demonstrated that this species reproduces by self-fertilization rather than parthenogenesis and that its sex is determined chromosomally. Genetic mapping has thus far identified markers on the X chromosome and three of four autosomes. We report that S. hermaphroditum CS34 is the first consistently hermaphroditic entomopathogenic nematode and is suitable for genetic model development to study naturally occurring mutualistic symbiosis and insect parasitism.
Fertilization, the union of an oocyte and a sperm, is a fundamental process that restores the diploid genome and initiates embryonic development. For the sperm, fertilization is the end of a long journey, one that starts in the male testis before transitioning to the female reproductive tract’s convoluted tubule architecture. Historically, motile sperm were thought to complete this journey using luck and numbers. A different picture of sperm has emerged recently as cells that integrate complex sensory information for navigation. Chemical, physical, and thermal cues have been proposed to help guide sperm to the waiting oocyte. Molecular mechanisms are being delineated in animal models and humans, revealing common features, as well as important differences. Exposure to pheromones and nutritional signals can modulate guidance mechanisms, indirectly impacting sperm motility performance and fertility. These studies highlight the importance of sensory information and signal transduction in fertilization.
In both mammals and C. elegans, spermatogenesis is the process where a spermatogonial cell undergoes a series of divisions to produce a highly differentiated cell, the spermatozoon. Spermatogonial cellular divisions are incomplete in mammals so that all subsequent stages occur in a syncitium. The situation is similar in C. elegans, where spermatogonial nuclei initially share a common cytoplasm. Spermatogonial divisions in both mammals and C. elegans are regulated by signaling from gonadal accessory cells. In mammals, this process requires a series of accessory cell types, including Sertoli, peritubular myoid, and Leydig cells (1,2). In contrast, one somatic distal tip cell (Fig. 10.1) regulates exit of spermatogonia from mitosis in C. elegans, and it no longer participates in spermatogenesis after meiosis is initiated (3). Spermatogenesis occurs within a tubular structure in both mammals and C. elegans. Differentiation occurs along the length and across the radius of this tube (the seminiferous tubule) in mammals. The Sertoli accessory cell plays a crucial role in both the linear and radial aspects of mammalian spermatogenesis. At any given time, a single Sertoli cell can be in contact with up to 50 individual, developing germ cells that can be at four different stages of development (4). In each mammalian testis, spermatogenesis occurs within a “ball of yarn” composed of seminiferous tubules that, if unraveled, would be many meters long. The C. elegans gonadal tube is ~400 mm long (Fig. 10.1). and only linear differentiation is observed (5). After initiating meiosis, individual C. elegans spermatogonial cells bud from the syncitial testes and lineally complete development without the aid of accessory cells (6).
Major Sperm Protein (MSP) is one of the important components of nematode sperms and the characters of this protein were widely studied in Caenorhabdit elegans and Ascaris suum. However, no knowledge of homology proteins in Haemonchus contortus was reported. In the present research, the MSP gene of this parasite (HcMSP) was first cloned and characterized. A pair of primer targeted the conserved sequence of nematode MSPs was designed and a partial of HcMSP gene was obtained by reverse transcription PCR (RT-PCR). Then, the 5' and 3' ends of HcMSP gene were cloned by 5' Rapid Amplification of cDNA ends (RACE) and 3' RACE. The full length of cDNA was obtained by overlapping the sequences of 3' and 5' extremities. The Open Reading Frame (ORF) was amplified by RT-PCR and expressed in prokaryotic cell.
This chapter provides a general discussion on sperm motility activation and chemoattraction. Under natural circumstances in sexually reproducing species, sperm motility is critical to fertilization and thus, for the continuation of a species. Several events important for successful fertilization in many species rely on adequate sperm motility—namely, (1) Penetration of the extracellular matrix surrounding eggs, (2) directed motility in response to factors released from the egg or closely associated structures, and, (3) migration through the female reproductive tract or within an environment such as water or seawater. In general, sperm motility is activated at ejaculation as a consequence of changes in intracellular ion concentrations. The external signals that initiate these events, and thus flagellar beating, differ among species according to the environment in which fertilization occurs. Sperm chemoattraction is defined as the movement of spermatozoa in response to a chemical signal toward the source of that signal. This form of motility is reported in many evolutionarily diverse species. This chapter concludes that sperm motility is a complex phenomenon that is responsive to the external cellular environment. Factors regulating motility such as changes in extracellular ion concentrations and secreted products from the male and/or female reproductive systems, activate sperm cell signaling involving changes in cyclic nucleotides, calcium, and protein phosphorylation/dephosphorylation in diverse species. In the conceptual model of sperm motility, the aspects describing both the plasma membrane and the final axoneme are poorly defined.
The soybean cyst nematode (SCN), Heterodera glycines, is the major disease-causing agent limiting soybean production in the USA. The current management strategy to reduce yield loss by SCN involves the deployment of resistant soybean cultivars and rotation to non-host crops. Although this management scheme has shown some success, continued yearly yield loss estimates demonstrate the limitations of these techniques. As a result, new control strategies are needed to complement the existing methods. Reported here is a novel method of SCN control that utilises RNA interference (RNAi). Transgenic soybeans were generated following transformation with an RNAi expression vector containing inverted repeats of a cDNA clone of the major sperm protein (MSP) gene from H. glycines. The accumulation of MSP-specific short interfering RNA (siRNA) molecules were detected by northern blot analysis of transgenic soybeans. T0 plants displaying MSP siRNA accumulation were deployed in a bioassay to evaluate the effects of MSP interfering molecules on H. glycines reproduction. Bioassay data has shown up to a 68% reduction in eggs g-1 root tissue, demonstrating that MSPi transgenic plants significantly reduced the reproductive potential of H. glycines. An additional bioassay evaluating progeny nematodes for maintenance of reproductive suppression indicated that progeny were also impaired in their ability to successfully reproduce, as demonstrated by a 75% reduction in eggs g -1 root tissue. The results of this study demonstrate the efficacy of an RNAi-based strategy for control of the soybean cyst nematode. In addition, these results may have important implications for the control of other plant parasitic nematodes.
The Free Tropospheric Experiment (FREETEX'98) was conducted at the Jungfraujoch Observatory in the Swiss Alps (3580 m above sea level) during the well-documented spring maximum in ozone. In spring the Jungfraujoch frequently lies in the free troposphere but can also be influenced by air from the planetary boundary layer. Measurements of NOx, NOy, peroxyacetylnitrate (PAN), HCHO, O3, CO, nonmethane hydrocarbons, peroxy radicals, j(O1D),j(NO2), and a variety of other tropospheric constituents crucial to ozone photochemical cycles were made over a 1-month period. Two independent measurements of NOx, NOy, and PAN showed good agreement. Average free tropospheric daytime NO levels were about 50 pptv, sufficient to sustain photochemical ozone formation. Although high mixing ratios were encountered, PAN decomposition did not contribute to NOx production during FREETEX'98. Ozone production efficiencies (EN) derived from observed DeltaO3/(NOz) ratios in free tropospheric air were 20-30 molecules of O3 produced per NOx molecule oxidized and agreed well with a photochemical model. A much lower ozone production efficiency of 4 was determined in a photochemically aged air mass arriving from southern Europe, in line with other measurements and calculations in regimes containing high levels of oxidized nitrogen. Model simulations indicated that by sequestering NOx and HO2, low-temperature formation of peroxynitric acid (PNA) decreased ozone production by 20% and instantaneous ozone production efficiencies by 40%, whereas PAN formation had little effect. The model reproduced well the observed sharp transformation from ozone production to ozone destruction (defined as DeltaO3/Delta(NOz)=0) at 20-25 pptv NO. The observed and calculated strong dependence of EN on NOx concentration in the low-NOx regime highlights the difficulty in assigning a single O3 production efficiency value to remote regions, where most of the CO and CH4 in the atmosphere are oxidized.
Introduction: The Nematoda are widely distributed invertebrates. Although they are fundamentally bisexual, certain species show a wide variety of reproductive phenomena, including parthenogenesis, hermaphroditism, and pseudogamy. Adaptation to parasitism has led certain species to display enormous reproductive outputs in the number of eggs. The male reproductive system generally comprises a testis (rarely two), a seminal vesicle, and a vas deferens. The male copulatory organs generally include two spicules (sometimes' one) which act as a guide to sperm during copulation within the vulva of the female. Internal fertilization through female genital ducts is the rule in nematodes, but a few cases of traumatic insemination have been reported. Our knowledge of sperm structure in the Nematoda is paradoxically unbalanced. On the one hand, the number of species studied for sperm ultrastructure (55 genera, 72 species, Tables 4-6), although important, may be considered small for a large phylum, and observations are completely lacking, or restricted to a single species, for several large groups; on the other hand, modern studies of the motile cellular system of nematode sperm, restricted to two model species, have attained the highest current levels of technology and sophistication. A review of nematode sperm should thus encompass observations made with a variety of techniques, from basic light microscope observations of spermatozoa sometimes reported within works of basic systematics, to a report on three-dimensional structure of proteins of nematode spermatozoa.
The Saccharomyces cerevisiae SCS2 gene has been cloned as a suppressor of inositol auxotrophy of CSE1 and hac1/ire15 mutants (J. Nikawa, A. Murakami, E. Esumi, and K. Hosaka, J. Biochem. 118:39-45, 1995) and has homology with a synaptobrevin/VAMP-associated protein, VAP-33, cloned from Aplysia californica (P. A. Skehel, K. C. Martin, E. R. Kandel, and D. Bartsch, Science 269:1580-1583, 1995). In this study we have characterized an SCS2 gene product (Scs2p). The product has a molecular mass of 35 kDa and is C-terminally anchored to the endoplasmic reticulum, with the bulk of the protein located in the cytosol. The disruption of the SCS2 gene causes yeast cells to exhibit inositol auxotrophy at temperatures of above 34 degrees C. Genetic studies reveal that the overexpression of the INO1 gene rescues the inositol auxotrophy of the SCS2 disruption strain. The significant primary structural feature of Scs2p is that the protein contains the 16-amino-acid sequence conserved in yeast and mammalian cells. The sequence is required for normal Scs2p function, because a mutant Scs2p that lacks the sequence does not complement the inositol auxotrophy of the SCS2 disruption strain. Therefore, the Scs2p function might be conserved among eukaryotic cells.
Cell locomotion in amoeboid nematode sperm is generated by the vectorial assembly and bundling of filaments of the major sperm protein (MSP). MSP filaments are constructed from two helical subfilaments and here we describe the structure of putative MSP subfilament helices determined by X-ray crystallography at 3.3 A resolution. In addition to establishing the interfaces involved in polymerization, this structural model shows that the MSP helices are constructed from dimers and have no overall polarity, suggesting that it is unlikely that molecular motors play a direct role in the generation of protrusive force in these amoeboid cells.
Although the nematode Caenorhabditis elegans produces self-fertile hermaphrodites, it descended from a male/female species, so hermaphroditism provides a model for the
origin of novel traits. In the related species C. remanei, which has only male and female sexes, lowering the activity of tra-2 by RNA interference created XX animals that made spermatids as well as oocytes, but their spermatids could not activate without the addition of male seminal
fluid. However, by lowering the expression of both tra-2 and swm-1, a gene that regulates sperm activation in C. elegans, we produced XX animals with active sperm that were self-fertile. Thus, the evolution of hermaphroditism in Caenorhabditis probably required two steps: a mutation in the sex-determination pathway that caused XX spermatogenesis and a mutation that allowed these spermatids to self-activate.
The ultrastructure of spermatozoa in the free-living marine nematode Halalaimus dimorphus was studied with transmission electron microscopy. Spermatozoa in the posterior testis of the male had a large cavity filled with cellular processes, which contained a variable number of small tubules. Mitochondria and small tubules were the only cell structures observed in the cytoplasm. The spermatozoa had a bipolar structure. The anteriorly situated nucleus, which was electron-dense and homogeneous, was surrounded by a single membrane. The size of the small tubules in the cytoplasm (diam. 12-13 nm) and their relatively thick wall structure suggested that they were not normal microtubules (diam. 25 nm). The material of the small tubules was assumed to be major sperm protein (MSP). The cavity appeared to open on the surface of the spermatozoon at the posterior extremity of the cell, and also medially, at the level of the anterior end of the cavity. The pores apparently were closed by a special plug-like structure, which was an evagination of the cell. The wall of the cavity was characterized by longitudinal folds, which were mushroom-shaped in transverse section. Spermatids in the anterior testis of H. dimorphuswere characterized by fibrous bodies packed with small tubules and by cellular processes also containing small tubules. H. dimorphus sperm seem to perform swimming movements based on liquid currents commonly present in turbin-like systems. Spermatogenesis resembled that found in ticks.
Particles, 30 nm in diameter, were found in Triton X-100-extracted pseudopodial remnants of spermatozoa of Ascaris suum. SDS-PAGE analysis of particles isolated by differential centrifugation and detergent extraction indicated that they are composed of proteins with molecular weights of 33,000 D, 42,000 D and a group between 52,000 and 56,000 D. Antibodies against particle-associated proteins, used in immunoblots, showed that they are sperm-specific in Ascaris, and that all of the particle-associated proteins are related. Immunofluorescence labeling confirmed that particle-associated proteins are present in pseudopods of Ascaris spermatozoa. These data are consistent with the particles being elements of the motility apparatus of nematode spermatozoa.
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Actin filaments and microtubules form the cytoskeleton of all eukaryotic cells, and they are responsible for organizing the cytoplasm and supporting motile processes. Both polymers are highly dynamic, and their polymerization dynamics are central to their organization. Though their evolutionary origins appear to be distinct, actin and tubulin have a similar mechanism for promoting polymerization dynamics in which the energy of nucleotide triphosphate hydrolysis during polymerization is used to weaken the bonds between subunits, thus promoting subsequent depolymerization. The evolutionary origins of actin and tubulin are unclear. It is likely that motile mechanisms driven by reversible polymerization, termed thermal ratchets, are older than those based on ATPase motor proteins. Such mechanisms are still important in modern eukaryotes, and may have powered early versions of the critical motile processes of phagocytosis and chromosome segregation in primitive cells. Thus evolution of dynamic cytoskeletal polymers may have been one of the earliest and most important steps leading to the evolution of eukaryotes. Plausible evolutionary pathways can be constructed leading from simple enzymes to dynamic cytoskeletal polymers.
We explicitly acknowledge key intellectual contributions from Jody Rosenblatt (depolymerization) and Julie Theriot (protrusion, Listeria motility). Owing to space restrictions, we were unable to cite all relevant publications. We attempted to fairly represent different laboratories, systems, and opinions and apologize to authors whose papers we did not include. We thank Matt Welch and Jody Rosenblatt for helpful comments on the manuscript. This work was funded by grant GM48027 from the National Institutes of Health and a fellowship from the Packard Foundation to T. J. Mitchison, and a senior postdoctoral fellowship from the American Cancer Society to L. P. Cramer.
We have obtained two new crystal forms of the Ascaris major sperm protein (MSP) that mediates amoeboid cell motility in nematode sperm. We obtained crystals with C2 symmetry from bacterially expressed alpha-MSP with a = 216.5 A, b = 38.6 A, c = 32.5 A, gamma = 93.1 degrees and also crystals with P2(1) symmetry from native beta-MSP with a = 63.1 A, b = 91.7 A, c = 72.5 A, gamma = 91.3 degrees. A full native data set has been collected for each crystal form using synchrotron radiation. Both crystal forms diffract to 2 A and are suitable for high-resolution structural investigation.
HIS minireview discusses recent information about the septin family of proteins, which suggests that the septins may be elements of a new filament system that functions in all or most eukaryotic cells. Septins are found in a wide variety of eukaryotes, are important for cytokinesis, and may compose or regulate a ubiquitous fil- ament system that has not been previously recognized. A more extensive review of septins was recently published (26). This minireview also presents a new comparison of septin sequences. Actin filaments, microtubules, intermediate filaments, and myosin thick filaments, have been extensively studied over the past several decades, using methodologic ad- vances in electron and light microscopy, detergent extrac- tion of cells, and purification of proteins. This collection of filaments has been assumed to represent a fairly complete picture of the cytoskeletal filaments commonly found in eukaryotes. However, recent work suggests that cells aug- ment these common cytoskeletal elements with additional elements, at least one of which, the septins, appears to be widely expressed and used for essential cell processes.
We have determined the structure of the Ascaris major sperm protein (MSP) to 2.5 A resolution using X-ray crystallography. The MSP polypeptide chain has an immunoglobulin-like fold based on a seven-stranded beta sandwich. In two strands, cis-proline residues impart distinctive kinks, and overall the structure most closely resembles that of the N-terminal domain of the bacterial chaperonin, PapD. In the C2 crystal form which we have solved here, two MSP chains are tightly associated in the asymmetric unit and are related by a non-crystallographic 2-fold rotation axis. This arrangement almost certainly represents the MSP dimer that is present in solution. Additionally, the arrangement of two MSP dimers at one of the crystallographic 2-fold axes in the 215 A unit cell suggests a possible mode for the assembly of MSP into the filaments which promote cell movement. This dimer-dimer association is based on a beta sheet extension mechanism between adjoining MSP monomers which resembles the interaction between PapD and its protein substrate.
Myosin II is required for normal amoeboid locomotion. In order to understand how myosin II elicits its effects on locomotive behavior, we have mapped myosin II-cytoskeleton interactions in locomoting endothelial cells. Bovine microcapillary endothelial cells were microinjected with fluorescently labeled myosin II, and the distribution of myosin II was imaged in the living cells by fluorescence microscopy. The same cells were then permeabilized with Triton X-100 and imaged again. The second set of images showed only myosin II that was associated with detergent-insoluble cytoskeleton. Dividing the image of retained myosin II by that of total myosin II produced a map of the extent to which myosin II was associated with the detergent-resistant cytoskeleton at any point in the cell. In cells migrating at the edge of a scrape wound, myosin II was preferentially retained in a region approximately 10 microm wide located just behind the cells' leading lamellipodia. Relatively little myosin II was retained in perinuclear cytoplasm. A vector representation of the distribution of total versus retained myosin II demonstrated that myosin II retention was sharply polarized with respect to locomotion, favoring the front of migrating cells. Myosin II-enriched cytoskeleton in this region may help polarize protrusive activity and/or move cytoplasmic bulk forward. Patches of myosin II retention were also observed in adherent tails of many cells, consistent with a role in pulling the rear of the cell forward.
The major sperm protein (MSP) of Ascaris suum mediates amoeboid motility by forming an extensive intermeshed system of cytoskeletal filaments analogous to that formed by actin in many other amoeboid cells. MSP is a dimeric molecule that polymerizes to form non-polar filaments constructed from two helical subfilaments that wind round one another. Moreover, MSP filaments can interact with one another to form higher-order assemblies without requiring the range of accessory proteins usually employed in actin-based systems. A knowledge of how MSP polymerizes and forms the hierarchical series of helical MSP macromolecular assemblies is fundamental to understanding locomotion in these cells. Here we describe the solution structure of MSP dimers determined by NMR spectroscopy under conditions where MSP does not polymerize to form filaments. The solution structure is indistinguishable from that observed in putative MSP subfilament helices by X-ray crystallography, indicating that MSP polymerization is not accompanied by a major conformational change. We also show that the rate of MSP polymerization associated with movement of vesicles in an in vitro motility assay is enhanced by the presence of magnesium and manganese ions and use NMR to show that the primary residues that bind these ions are 24-25 and 83-86. These residues are distant from the interface formed between MSP dimers in subfilament helices, and so are probably not involved in this level of polymerization. Instead the manganese and magnesium ion binding appears to be associated with the assembly of subfilaments into filaments and their subsequent aggregation into bundles.
Tight junctions create a regulated intercellular seal between epithelial and endothelial cells and also establish polarity between plasma membrane domains within the cell. Tight junctions have also been implicated in many other cellular functions, including cell signaling and growth regulation, but they have yet to be directly implicated in vesicle movement. Occludin is a transmembrane protein located at tight junctions and is known to interact with other tight junction proteins, including ZO-1. To investigate occludin's role in other cellular functions we performed a yeast two-hybrid screen using the cytoplasmic C terminus of occludin and a human liver cDNA library. From this screen we identified VAP-33 which was initially cloned from Aplysia by its ability to interact with VAMP/synaptobrevin and thus was implicated in vesicle docking/fusion. Extraction characteristics indicated that VAP-33 was an integral membrane protein. Antibodies to the human VAP-33 co-localized with occludin at the tight junction in many tissues and tissue culture cell lines. Subcellular fractionation of liver demonstrated that 83% of VAP-33 co-isolated with occludin and DPPIV in a plasma membrane fraction and 14% fractionated in a vesicular pool. Thus, both immunofluorescence and fractionation data suggest that VAP-33 is present in two distinct pools in the cells. In further support of this conclusion, a GFP-VAP-33 chimera also distributed to two sites within MDCK cells and interestingly shifted occludin's localization basally. Since VAP-33 has previously been implicated in vesicle docking/fusion, our results suggest that tight junctions may participate in vesicle targeting at the plasma membrane or alternatively VAP-33 may regulate the localization of occludin.
Nematode sperm are amoeboid cells that use a major sperm protein (MSP) cytoskeleton in place of a conventional actin cytoskeleton to power their amoeboid motility. In these simple, specialized cells cytoskeletal dynamics is tightly coupled to locomotion. Studies have capitalized on this feature to explore the key structural properties of MSP and to reconstitute motility both in vivo and in vitro. This review discusses how the mechanistic properties shared by the MSP machinery and actin-based motility systems lead to a "push-pull" mechanism for amoeboid cell motility in which cytoskeletal assembly and disassembly at opposite ends of the lamellipodium are associated with independent forces for protrusion of the leading edge and retraction of the cell body.
Angiotensin IV (Ang IV), the 3-8 fragment of angiotensin II (Ang II), binds to a distinct receptor designated the AT(4) receptor. The peptide elicits a range of vascular and central actions including facilitation of memory retention and retrieval in several learning paradigms. The aim of this study was to characterize the AT(4) receptor in a human cell line of neural origin. Receptor binding studies indicate that the human neuroblastoma cell line SK-N-MC cells express a high-affinity Ang IV binding site with a pharmacological profile similar to the AT(4) receptor: (125)I]-Ang IV and (125)I]-Nle(1)-Ang IV bind specifically to the SK-N-MC cell membranes (K(d) = 0.6 and 0.1 nM) in a saturable manner (B(max) = 1.2 pmol/mg of protein). AT(4) receptor ligands, Nle(1)-Ang IV, Ang IV and LVV-haemorphin 7 (LVV-H7), compete for the binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to the SK-N-MC cell membranes with rank order potencies of Nle(1)-Ang IV > Ang IV > LVV-H7 with IC(50) values of 1.4, 8.7 and 59 nM ([(125)I]-Ang IV) and 1.8, 20 and 168 nM ([(125)I]-Nle(1)-Ang IV), respectively. The binding of [(125)I]-Ang IV or [(125)I]-Nle(1)-Ang IV to SK-N-MC cell membranes was not affected by the presence of GTP gamma S. Both Ang IV and LVV-H7 stimulated DNA synthesis in this cell line up to 72 and 81% above control levels, respectively. The AT(4) receptor in the SK-N-MC cells is a 180-kDa glycoprotein; under non-reducing conditions a 250-kDa band was also observed. In summary, the human neuroblastoma cell line, SK-N-MC, expresses functional AT(4) receptors that are responsive to Ang IV and LVV-H7, as indicated by an increase in DNA synthesis. This is the first human cell line of neural origin shown to express the AT(4) receptor.
Immature spermatids from Caenorhabditis elegans are stimulated by an external activation signal to reorganize their membranes and cytoskeleton to form crawling spermatozoa. This rapid maturation, termed spermiogenesis, occurs without any new gene expression. To better understand this signal transduction pathway, we isolated suppressors of a mutation in the spe-27 gene, which is part of the pathway. The suppressors bypass the requirement for spe-27, as well as three other genes that act in this pathway, spe-8, spe-12, and spe-29. Eighteen of the suppressor mutations are new alleles of spe-6, a previously identified gene required for an early stage of spermatogenesis. The original spe-6 mutations are loss-of-function alleles that prevent major sperm protein (MSP) assembly in the fibrous bodies of spermatocytes and arrest development in meiosis. We have isolated the spe-6 gene and find that it encodes a predicted protein-serine/threonine kinase in the casein kinase 1 family. The suppressor mutations appear to be reduction-of-function alleles. We propose a model whereby SPE-6, in addition to its early role in spermatocyte development, inhibits spermiogenesis until the activation signal is received. The activation signal is transduced through SPE-8, SPE-12, SPE-27, and SPE-29 to relieve SPE-6 repression, thus triggering the formation of crawling spermatozoa.
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.
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.
Taking advantage of conditions that allow spermatogenesis in vitro, the timing and sequence of morphological changes leading from the primary spermatocyte to the spermatozoon is described by light and electron microscopy. Together with previous studies, this allows a detailed description of the nuclear, cytoplasmic, and membrane changes occurring during spermatozoan morphogenesis. By comparison with wild type, abnormalities in spermatogenesis leading to aberrant infertile spermatozoa are found in six fertilization-defective (fer) mutants. In fer-1 mutant males, spermatids appear normal, but during spermiogenesis membranous organelles (MO) fail to fuse with the sperm plasma membrane and a short, though motile. pseudopod is formed. In fer-2, fer-3, and fer-4 mutants, spermatids accumulate 48-nm tubules around their nuclei where the centriole and an RNA containing perinuclear halo would normally be. In all three mutants, spermatids still activate to spermatozoa with normal fusion of their MOs, but the pseudopods formed are aberrant in most fer-2 and fer-4 spermatozoa and in some fer-3 spermatozoa. In fer-5 mutant males, spermatozoa do not form. Instead, defective spermatids with crystalline inclusions and abnormal internal laminar membranes accumulate. In fer-6 mutant males, only a few spermatozoa form and these have defective pseudopods. These spermatozoa retain their fibrous bodies, a structure which normally disassembles in the spermatid. The time of appearance of developmental abnormalities in all of these mutants correlates with the temperature-sensitive periods for development of infertility. The observation that each of these mutants has a different and discreet set of morphological defects, a structure which normally disassembles in the spermatid. The time of appearance of developmental abnormalities in all of these mutants correlates with the temperature-sensitive periods for development of infertility. The observation that each of these mutants has a different and discreet set of morphological defects, a structure which normally disassembles in the spermatid. The time of appearance of developmental abnormalities in all of these mutants correlates with the temperature-sensitive periods for development of infertility. The observation that each of these mutants has a different and discreet set of morphological defects shows that the strict sequence of morphogenetic events that occurs during wild-type spermatogenesis cannot arise because each event is dependent on previous events. Instead, spermatozoa, like bacteriophages, must be formed by multiple independent pathways of morphogenesis.
Sperm of the nematode, Ascaris suum, are amoeboid cells that do not require actin or myosin to crawl over solid substrata. In these cells, the role usually played by actin has been taken over by major sperm protein (MSP), which assembles into filaments that pack the sperm pseudopod. These MSP filaments are organized into multi-filament arrays called fiber complexes that flow centripetally from the leading edge of the pseudopod to the cell body in a pattern that is intimately associated with motility. We have characterized structurally a hierarchy of helical assemblies formed by MSP. The basic unit of the MSP cytoskeleton is a filament formed by two subfilaments coiled around one another along right-handed helical tracks. In vitro, higher-order assemblies (macrofibers) are formed by MSP filaments that coil around one another in a left-handed helical sense. The multi-filament assemblies formed by MSP in vitro are strikingly similar to the fiber complexes that characterize the sperm cytoskeleton. Thus, self-association is an intrinsic property of MSP filaments that distinguishes these fibers from actin filaments. The results obtained with MSP help clarify the roles of different aspects of the actin cytoskeleton in the generation of locomotion and, in particular, emphasize the contributions made by vectorial assembly and filament bundling.
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.
The major sperm proteins (MSPs) are a family of closely related, small, basic proteins comprising 15% of the protein in Caenorhabditis elegans sperm. They are encoded by a multigene family of more than 50 genes, including many pseudogenes. MSP gene transcription occurs only in late primary spermatocytes. In order to study the genomic organization of transcribed MSP genes, probes specific for the 3′ untranslated regions of sequenced cDNA clones were used to isolate transcribed genes from genomic libraries. These and other clones of MSP genes were located in overlapping cosmid clones by DNA fingerprinting. These cosmids were aligned with the genetic map by overlap with known genes or in-situ hybridization to chromosomes. Of 40 MSP genes identified, 37, including all those known to be transcribed, are organized into six clusters composed of 3 to 13 genes each. Within each cluster, MSP genes are not in tandem but are separated by at least several thousand bases of DNA. Pseudogenes are interspersed among functional genes. Genes with similar 3′ untranslated sequences are in the same cluster. The six MSP clusters are confined to only three chromosomal loci; one on the left arm of chromosome II and two near the middle of chromosome IV. Additional sperm-specific genes are located in one cluster of MSP genes on chromosome IV. The multiplicity of MSP genes appears to be a mechanism for enhancing MSP synthesis in spermatocytes, and the loose clustering of genes could be a result of the mechanism of gene duplication or could play a role in regulation.
Ascaris sperm are amoeboid cells that crawl by extending pseudopods. Although amoeboid motility is generally mediated through an actin-based cytoskeleton, Ascaris sperm lack this system. Instead, their major sperm protein (MSP) forms an extensive filament system that appears to fulfil this function. Because their motility appears to be essentially the same as that of their actin-rich counterparts, Ascaris sperm offer a simple alternative system for investigation of the molecular mechanism of amoeboid movement. To examine the structure and composition of the cytoskeleton, we stabilized the extremely labile native MSP filaments by detergent lysis of sperm in the presence of either glutaraldehyde or polyethylene glycol (PEG). Biochemical analysis showed that the cytoskeleton contained two isoforms of MSP, designated alpha- and beta-, that we purified and sequenced. Both contain 126 amino acids and have an acetylated N-terminal alanine, but differ at four residues so that alpha-MSP is 142 Da larger and 0.6 pH unit more basic than beta-MSP. Neither isoform shares sequence homology with other cytoskeletal proteins. In ethanol, 2-methyl-2,4-pentanediol (MPD), and other water-miscible alcohols each isoform assembled into filaments 10 nm wide with a characteristic substructure repeating axially at 9 nm. These filaments were indistinguishable from native fibers isolated from detergent-lysed sperm. Pelleting assays indicated a critical concentration for assembly of 0.2 mM for both isoforms in 30% ethanol, but alpha-MSP formed filaments at lower solvent concentration than beta-MSP. When incubated in polyethylene glycol, both isoforms formed thin, needle-shaped crystals that appeared to be constructed from helical fibers, with a 9 nm axial repeat that matched that seen in isolated filaments. These crystals probably contained a parallel array of helical filaments, and may enable both the structure of MSP molecules and their mode of assembly into higher aggregates to be investigated to high resolution.
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.
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.
The DNA from a number of free-living and parasitic nematode species was examined to determine the genomic number and distribution of DNA sequences encoding two evolutionarily conserved proteins; the major sperm protein (MSP) and nematode actin. Ascaris and Caenorhabditis MSP cDNA sequences and Ascaris genomic actin sequences were used to probe Southern blots of Eco RI and Hin d III digested nematode DNA. The number of MSP genes varied widely between the 1 MSP gene in Ascaris and the 60 MSP genes in Caenorhabditis. Filarial nematodes appeared to have 1-4 MSP genes while the plant and insect parasitic species showed from 5-12 MSP-hybridizing restriction fragments. Mammalian intestinal parasites showed between 1 and 13 bands hybridizing with the MSP probes. Blots probed to estimate the number of actin genes showed that, with the exception of Ascaris which contains more than 20 germ line sequences that encode actin, all of the nematodes tested had between 3 and 9 bands that hybridized to the Ascaris genomic actin probe. The possible use of highly conserved sequences such as MSP and actin to differentiate between nematode species in diagnostic and taxonomic studies is discussed.
DNA fragments corresponding to genes encoding the MSP of Caenorhabditis elegans sperm have been isolated by recombinant DNA techniques. Analyses of individual genomic clones suggest that there are multiple MSP genes that are dispersed in the genome. From restriction enzyme digests of genomic DNA fractionated and hybridized with an MSP complementary DNA probe, there appear to be more than 30 MSP genes in the genome. Despite the occurrence of this large dispersed multigene family, the MSP messenger RNA from both males and hermaphrodites is homogene in size. There are at least three different proteins of identical molecular weight but different isoelectric point that cross-react with anti-MSP antisera. Each protein is a primary translation product with no detectable post-translational modifications, suggesting that at least three of the MSP genes are expressed.
The amoeboid motility nematode sperm is mediated by cytoskeletal filaments composed of major sperm protein (MSP). We have used electron microscopy and image processing to show that MSP filaments are constructed from two subfilament strands which are themselves formed from a helical arrangement of subunits. The subfilaments are based on left-handed helices of pitch 9 nm that then coil along right-handed helical tracks of pitch 22.5 nm to form filaments. The subfilaments appear to be indistinguishable from the helices present in orthorhombic crystals of MSP. Because in filaments the subfilaments are themselves helical, not all subunits are able to participate in protein-protein interactions between different strands. One consequence of this interaction geometry is that the same molecular interactions that function to assemble subfilaments into filaments can also be used to assemble filaments into larger supramolecular assemblies such as the macrofibres formed in vitro and the fibre bundles found in vivo in sperm pseudopods. These results indicate the importance of filament bundling in addition to vectorial filament assembly in amoeboid cell motility.
The development and locomotion of the amoeboid sperm of the nematode, Ascaris suum, depend on precise control of the assembly of their unique major sperm protein (MSP) filament system. We used fluorescence ratio imaging of cells loaded with BCECF to show that intracellular pH (pHi) is involved in controlling MSP polymerization in vivo. Spermatogenesis is marked by a cycle of MSP assembly-disassembly-reassembly that coincides with changes in pHi. In spermatocytes, which contain MSP in paracrystalline fibrous bodies, pHi was 6.8, 0.6 units higher than in spermatids, which disassemble the fibrous bodies and contain no assemblies of MSP filaments. Activation of spermatids to complete development resulted in rapid increase in pHi to 6.4 and reappearance of filaments. Treatment of spermatocytes with weak acids caused the fibrous bodies to disassemble whereas incubation of spermatids in weak bases induced MSP assembly. The MSP filaments in spermatozoa are organized into fiber complexes that flow continuously rearward from the leading edge of the pseudopod. These cells established a pseudopodial pH gradient with pHi 0.15 units higher at the leading edge, where fiber complexes assemble, than at the base of the pseudopod, where disassembly occurs. Acidification of these cells caused the MSP cytoskeleton to disassemble and abolished the pH gradient. Acid removal resulted in reassembly of the cytoskeleton, re-establishment of the pH gradient, and re-initiation of motility. MSP assembly in sperm undergoing normal development and motility and in cells responding to chemical manipulation of pHi occurs preferentially at membranes. Thus, we propose that filament assembly in sperm is controlled by pH-sensitive MSP-membrane interaction.
We have obtained orthorhombic crystals (space group P2(1)2(1)2(1), a = 72 A, b = 78 A, c = 452 A) of the major cytoskeletal protein associated with the amoeboid motility of Ascaris sperm. These crystals diffract past 3.5 A and appear to be constructed from arrays of helical fibres of the major sperm protein. The fibres within the crystals appear to be closely analogous to those seen in vitro and in vivo.
Cells crawl in response to external stimuli by extending and remodeling peripheral elastic lamellae in the direction of locomotion.
The remodeling requires vectorial assembly of actin subunits into linear polymers at the lamella's leading edge and the crosslinking
of the filaments by bifunctional gelation proteins. The disassembly of the crosslinked filaments into short fragments or monomeric
subunits away from the leading edge supplies components for the actin assembly reactions that drive protrusion. Cellular proteins
that respond to lipid and ionic signals elicited by sensory cues escort actin through this cycle in which filaments are assembled,
crosslinked, and disassembled. One class of myosin molecules may contribute to crawling by guiding sensory receptors to the
cell surface, and another class may contribute by imposing contractile forces on actin networks in the lamellae.
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