Smith HE et al. (1997) International C. elegans Meeting "SPERM MOTILITY IN C. ELEGANS: STRUCTURE-FUNCTION ANALYSIS OF THE MAJOR SPERM PROTEIN"

Ward S, Smith HE

[International C. elegans Meeting, 1997]

In nematodes, sperm are amoeboid cells which crawl via an extended pseudopod. Unlike other crawling cells, this pseudopod contains no actin or myosin; instead, it utilizes the major sperm protein (MSP). In vivo and in vitro studies of Ascaris suum MSP have demonstrated that motility is derived from the regulated assembly and disassembly of MSP. Filaments composed of MSP dimers are thought to provide the motive force. Mutants defective in MSP-MSP interactions would provide insights into the process of filament formation. Because there are 28 MSP genes expressed in C. elegans, which precludes traditional mutational analysis, the yeast two-hybrid system was used to investigate MSP-MSP interactions. Fusions of MSP to both the lexA DNA binding domain (LEXA-MSP) and a transcriptional activation domain (AD-MSP) exhibit interaction and drive expression of a lacZ reporter construct. A library of AD-MSP mutants was generated via mutagenic PCR and screened for clones which fail to interact with LEXA-MSP. The mutations were mapped to the crystal structure of A. suum MSP. Two classes of mutations predicted by the crystal structure were recovered: changes in residues critical for the overall fold of the protein, and changes in residues in the dimerization interface. In addition, multiple mutations were obtained in the carboxy-terminal beta strand, a region not predicted to be involved in folding or dimer formation. Compensating mutations which restore the interaction also map to this region. The data suggests that the carboxy-terminal beta strand is directly involved in interactions required for MSP filament assembly.

Sung Min Han et al. (2007) International Worm Meeting "Identifying receptors that interact with major sperm protein in Caenorhabditis elegans."

Michael A. Miller, Sung Min Han

[International Worm Meeting, 2007]

An evolutionary conserved feature of oogenesis is that oocytes arrest in meiotic prophase. Oocytes resume meiosis in response to extracellular signals by a process called oocyte meiotic maturation, which prepares the oocyte for fertilization and embryogenesis. The Caenorhabditis elegans major sperm protein (MSP) is the most abundant protein in sperm. It is exported by a budding mechanism from the sperm cytoplasm to the extracellular environment, where it induces oocyte meiotic maturation and ovulation. During evolution, MSP has acquired extracellular signaling and intracellular cytoskeletal functions. Proteins with MSP-related domains have been found in diverse metazoans, raising the possibility that MSP has a conserved signaling function. Previous work using an in situ MSP-FITC binding assay identified the VAB-1 Eph receptor as an MSP receptor. However, vab-1 null gonads still bind to and respond to MSP. These results suggest that MSP binds to multiple receptors that act redundantly to induce oocyte maturation and sheath contraction. To identify additional MSP receptors, I conducted an RNAi-based screened of 58 receptor candidates whose mRNAs are expressed during oogenesis, but not spermatogenesis. Five receptor candidates have been identified that are required for MSP binding. These encode members of evolutionarily conserved receptor classes. Currently, I am testing whether any of five candidates directly binds to MSP and whether mutations in the candidates cause defects in MSP signaling.

Mary E Kosinski et al. (2003) International Worm Meeting "Release of the major sperm protein from spermatids and spermatozoa in vivo"

Jonathan Holder, Ginger Winfrey, David Greenstein, Mary E Kosinski, Gary Olson

[International Worm Meeting, 2003]

Oocyte meiotic maturation is essential to prepare the oocyte for fertilization and embryonic development. During meiotic maturation, C. elegans oocytes undergo nuclear envelope breakdown, cortical rearrangement, and meiotic spindle assembly in a spatially restricted manner such that the most proximal (-1) oocyte matures, is ovulated, and fertilized. Sperm stimulate oocyte meiotic maturation and gonadal sheath cell contraction using the major sperm protein (MSP) as a signaling molecule. The discovery of MSPs signaling role raised the intriguing question of how sperm release MSP to signal oocytes and sheath cells at a distance. The release of MSP from sperm likely occurs through a non-canonical mechanism since spermatozoa do not possess cellular components required in standard models of protein secretion, such as ribosomes, ER, or Golgi. Moreover, MSP does not have a N-terminal leader sequence, and in vitro release does not involve protein processing. Non-canonical or leaderless secretory pathways are widespread, and in general, poorly understood, despite figuring prominently in cell signaling, disease, and host defense. Using specific antibodies, we detect MSP both inside and outside of spermatids and spermatozoa in vitro. MSP release does not require motility or a pseudopod, and does not involve sperm lysis. We observe extracellular MSP up to approximately 90 m away from spermatozoa in the spermatheca, frequently reaching the -1 oocyte. Examination of extracellular MSP reveals it is localized in a graded distribution with the highest levels of MSP adjacent to spermatozoa and lower levels distally. MSP receptors on the -1 oocyte appear to bind and concentrate MSP resulting in a sharp boundary of staining, which is eliminated when trafficking of oocyte membrane proteins is blocked. Confocal microscopy shows extracellular MSP to be punctate. We observe large (~100-200 nM) MSP-staining puncta near spermatids or spermatozoa in the uterus. We speculate that these puncta are MSP-containing vesicles. Using immuno-EM we examined MSP localization in spermatozoa to pinpoint its localization in the cell. In addition to localization of MSP within the pseudopod, we also found MSP located in the cell body. Interestingly, we found a fraction of MSP associated with the plasma membrane of the spermatozoa. Based on these results we propose a model in which spermatids and spermatozoa shed vesicles containing MSP. In this model, MSP-containing vesicles released by spermatozoa in the spermatheca are unstable, forming a diffusible MSP signal that correlates with meiotic maturation rates and MAP kinase activation. Biochemical tests of this model are underway.

Mary E Kosinski et al. (2005) International Worm Meeting "C. elegans sperm bud vesicles to deliver a meiotic maturation signal to distant oocytes"

David Greenstein, Mary E Kosinski, Joel Schwartz, Ikuko Yamamoto, Kent McDonald

[International Worm Meeting, 2005]

Intercellular signaling facilitates multiple steps of the fertilization process. In many species, sperm prepare the oocyte for fertilization by providing signals for meiotic maturation. Oocyte meiotic maturation is defined by the transition between diakinesis and metaphase I and is accompanied by MAP kinase activation, nuclear envelope breakdown, and meiotic spindle assembly. C. elegans sperm signal oocyte meiotic maturation using the major sperm protein (MSP) as a hormone. Interestingly, the MSP also functions as the central cytoskeletal protein required for the amoeboid motility of nematode sperm. The discovery of MSPs signaling role raised the question of how sperm export MSP to signal oocytes at a distance. MSP lacks a hydrophobic leader sequence and C. elegans sperm lack many standard secretory components, such as ribosomes, ER, or Golgi. We used correlative light and electron microscopy to analyze the mechanism of MSP release from sperm. We will present evidence showing that sperm bud novel MSP vesicles to signal distant oocytes. These 150-300 nm MSP vesicles contain both an inner and an outer membrane, with MSP sandwiched in between. Budding protrusions from the cell body contain MSP, but not the MSD proteins, which counteract MSP filament assembly, suggesting that MSP may generate the protrusive force for its own vesicular export. MSP vesicles are labile structures that generate long-range MSP gradients for signaling at oocyte and sheath cell surfaces. Both spermatozoa and non-motile spermatids bud MSP vesicles, but their stability and signaling properties differ. Spermatozoa generate a long-range, short-acting signal, whereas spermatids generate a long-acting signal. EM results suggest that differential vesicle stability affects the physical and temporal range of signaling.

Mary Kosinski et al. (2001) International C. elegans Meeting "Control of Oocyte Meiotic Maturation and Gonadal Sheath Cell Contraction by MSP Signaling"

Mary Kosinski, David Greenstein, Michael A Miller

[International C. elegans Meeting, 2001]

Oocyte meiotic maturation and ovulation are essential biological processes required for reproduction. During meiotic maturation, C. elegan s oocytes undergo nuclear envelope breakdown, cortical rearrangement, and meiotic spindle assembly in an assembly-line-like fashion. Recently we found that the major sperm cytoskeletal protein (MSP) is the sperm derived signal that induces both oocyte meiotic maturation and gonadal sheath cell contraction (1). Many questions remain about this newly discovered signaling pathway. For example, how does a cytoskeletal protein function as a signaling molecule? How does MSP signal two distinct responses? To examine MSP signaling in vivo , we analyzed extracellular MSP localization. Previous studies reported the intracellular localization of MSP during spermatogenesis (2). We stained mated fog-2 females with anti-peptide antibodies generated to the N- and C- terminal regions of MSP, as well as previously described antibodies (2). In addition to intracellular staining within spermatozoa, we observed an extracellular MSP gradient, with the highest levels of MSP within the spermatheca and lower levels in the proximal gonad arm. The average extent of the observable MSP gradient is approximately 30 m m. Detection of this extracellular MSP gradient is dependent on the number of sperm present and their position in the spermatheca. By mating females with Mr. Vigorous, we have been able to detect the MSP gradient extending as far as 90 m m. These data suggest that a fraction of MSP is released from sperm in vivo, and the most proximal oocyte receives the highest concentration of released MSP. MSP is a bipartite signal: MSP (106-126) is sufficient to promote gonadal sheath cell contraction, and MSP (1-106) is sufficient to promote oocyte maturation (1). In solution MSP exists as a dimer (3). To determine if dimerization is necessary for signaling, we injected dimerization mutants (4) into females and measured oocyte maturation and sheath cell contraction rates. The dimerization mutants tested signal both oocyte maturation and gonadal sheath cell contraction at rates similar to wild-type MSP. To further identify regions responsible for promoting oocyte maturation and gonadal sheath cell contraction, we tested whether Ascaris suum MSP could signal in C. elegans . Ascaris MSP shares about 80% identity with C. elegans MSP at the amino acid level. Our results indicate that Ascaris MSP can signal maturation and sheath cell contraction in C. elegans at rates comparable to C. elegans MSP. We are currently targeting regions for deletion experiments that are conserved between both C. elegans and Ascaris . 1. Miller et al. (2001). Science 291:2144-2147 2. Ward & Klass. (1982). Dev. Bio. 92:203-208 3. Haaf et al.(1996). J. Mol. Biol. 260:251-260 4. Smith & Ward. (1998). J. Mol. Biol. 279:605-619 We would like to thank T. Roberts and P. Griffin for Ascaris clones and S. Ward and H. Smith for dimerization mutants and antibodies.

McKnight S et al. (1997) International C. elegans Meeting "Identification of proteins that may regulate MSP assembly in spermatozoa."

Ward S, McKnight S

[International C. elegans Meeting, 1997]

MSP (major sperm protein) comprises 15% of the total protein within C. elegans spermatozoa and is the main cytoskeletal component in these cells. The crawling movement of these cells is fueled by the dynamic assembly of MSP filaments within the pseudopod. Regulation of MSP assembly and localization are crucial steps in the differentiation of C. elegans sperm; however proteins regulating MSP assembly have yet to be identified. Presently, we are conducting an interaction trap using the yeast two-hybrid system to screen for genomic proteins that interact with MSP. Proteins identified in this screen will be tested further in biochemical assays for their association with MSP. Once the genes for interacting proteins have been identified, we will be able to determine if the proteins are essential for pseudopod motility in whole cells.

Youfeng Yang et al. (2008) Development & Evolution Meeting "Role of the PTP-2 tyrosine phosphatase and MAP Kinase Cascade in the MSP Signaling Response"

Michael Miller, Youfeng Yang

[Development & Evolution Meeting, 2008]

In C. elegans, the major sperm protein (MSP) is the most abundant protein in sperm and is the founding member of MSP domain family. MSP functions as an intracellular cytoskeleton protein and a secreted extracellular signal. Secreted MSP binds to the VAB-1 Eph receptor and other receptors on oocyte and ovarian sheath cell surfaces to induce oocyte maturation and sheath contraction. The MSP domain is an evolutionarily conserved immunoglobulin-like structure of about 120 amino acids. C. elegans VPR-1, human VAPB/ALS8, and Drosophila VAP33 are homologous proteins (collectively called VAPs) with an N-terminal MSP domain and a C-terminal transmembrane domain. A P56S missense mutation in the MSP domain of human VAPB/ALS8 causes a dominantly inherited form of Amyotropic Lateral Sclerosis (ALS) and Spinal Muscular Atrophy (SMA). In collaboration with the Hugo Bellen lab at Baylor College of Medicine, we have recently shown that worm, fly, and human VAP MSP domains are secreted ligands for Eph receptors and other receptors. To better understand the MSP domain signaling mechanism, we have been trying to identify downstream effectors. Previous studies have shown that MSP induces the phosphorylation of MPK-1 MAPK in oocytes. Here, we show that the PTP-2 SH2 domain-containing phosphatase acts upstream of let-60 RAS and MPK-1 to induce oocyte maturation. ptp-2 null mutant oocytes progress to diakinesis, yet fail to respond to MSP, lack MPK-1 phosphorylation, and lack histone H3 phosphorylation. The PTP-2/LET-60/MPK-1 cascade is not required for MSP-induced sheath contraction. Taken together, our data indicate that MSP can signal through MAPK-dependent and independent mechanisms. Currently, we are trying to further understand how MSP promotes MPK-1 activation in oocytes and how PTP-2 controls LET-60 activation. We are also testing the hypothesis that VAP MSP domains regulate the PTP-2/LET-60/MPK-1 cascade to control body wall muscle function. These studies may provide insight into mechanisms behind the pathogenesis of ALS and SMA, in addition to providing insight into Eph receptor signaling.

Yang, Y. et al. (2009) International Worm Meeting "Role of the MAP Kinase Cascade in the MSP Signaling Response."

Miller, M.A., Yang, Y.

[International Worm Meeting, 2009]

The MSP domain is an evolutionarily conserved immunoglobulin-like structure of about 120 amino acids. A P56S mutation in the MSP domain of the human vapB gene causes a dominantly inherited form of Amyotropic Lateral Sclerosis (ALS) or sometimes Spinal Muscular Atrophy (SMA) (Nishimura et al., 2004). In C. elegans, MSP is the most abundant protein in sperm and is the founding member of MSP domain family. MSP functions as an intracellular cytoskeleton protein and a secreted hormone. Secreted MSP binds to the VAB-1 Eph receptor and other receptors on oocyte and ovarian sheath cell surfaces to induce oocyte maturation and sheath contraction. We have recently shown that worm, fly, and human VAPB MSP domains are secreted ligands for Eph receptors and other receptors. The human VAPB MSP domain is found in blood serum, consistent with a hormonal function. In fly cells, the P56S mutation prevents secretion of the VAPB MSP domain, suggesting that the signaling function is important for ALS and SMA pathogenesis (Tsuda et al., 2008). To better understand how MSP domains transduce signals, I have been studying the PTP-2 tyrosine phosphatase, an SHP homolog that is thought to couple receptor tyrosine kinase (RTK) activation to the MAPK cascade. Previous studies have shown that MSP promotes MPK-1 MAPK phosphorylation during oocyte maturation (Miller et al., 2001) and that PTP-2 is required for normal fertility (Gutch et al., 1998). I have shown that PTP-2 is expressed in the gonad, where it is required for MSP-induced oocyte maturation and MAPK activation, but not MSP-induced sheath contraction. Expressing GFP::PTP-2 specifically in the germ line can partially rescue the oocyte maturation defects, embryonic lethality, and larval lethality of ptp-2 null mutants. Furthermore, these ptp-2 null defects can be rescued by inactivating multiple RAS GAPs, as well as by a RAS gain of function mutation. MSP binding studies show that MSP receptors are present on the oocyte and sheath plasma membranes in ptp-2 null mutants. These results are consistent with a model in which MSP regulates the activity of a canonical RTK/MAPK pathway to induce oocyte maturation. The identity of the RTK is not known and it is not clear whether MSP regulates RTK activation through a direct interaction (i.e. the RTK is an MSP receptor) or indirectly. VAPB homologs may also regulate the MAPK pathway because PTP-2 is expressed in muscle and loss of PTP-2 causes muscle mitochondrial defects that are similar to loss of worm VAPB (called VPR-1).

Winek, Jessica L et al. (2011) International Worm Meeting "Unraveling the VAP MSP secretion mechanism."

Winek, Jessica L, Miller, Michael

[International Worm Meeting, 2011]

VAPs are evolutionarily conserved proteins with an N-terminal MSP (major sperm protein) domain and C-terminal transmembrane domain. A P56S substitution in the VAPB/ALS8 MSP domain is associated with amyotrophic lateral sclerosis (ALS) and late-onset spinal muscular atrophy (Nishimura et al., 2004). We have shown that VAP MSP domains are cleaved from the transmembrane domain and secreted into the extracellular environment (Tsuda et al., 2008). The P56S mutation inhibits secretion. Therefore, MSP secretion may play a critical role in ALS pathogenesis and understanding this secretion mechanism could lead to novel therapeutic strategies. MSP domains do not contain a signal peptide and are secreted by an unconventional mechanism (Kosinski et al., 2005). The goal of this project is to elucidate the mechanism by which neurons secrete VAP MSP domains. Recent work in yeast has shown that the Acb1 protein is secreted by a novel mechanism that depends on autophagy genes (Duran et al., 2010; Manjithaya et al., 2010). We are testing the hypothesis that this mechanism is essential for VAP MSP secretion. Our initial data suggests that MSP domains may have a similar secretion mechanism to the Acb-1 protein in Saccharomyces cerevisiae.

Mary Kosinski et al. (2004) East Coast Worm Meeting "A Vesicle-Budding Model for the Release of MSP from C. elegans Sperm"

Mary Kosinski, David Greenstein, Jay Jerome, Kent McDonald

[East Coast Worm Meeting, 2004]

Oocyte meiotic maturation is essential to prepare the oocyte for fertilization and embryonic development. C. elegans sperm stimulate oocyte meiotic maturation and gonadal sheath cell contraction using major sperm protein (MSP) as a signaling molecule. MSP promotes meiotic maturation and activates MAP kinase in oocytes in part by binding the VAB-1 Eph receptor protein-tyrosine kinase. The discovery of MSP's signaling role raised the question of how sperm release MSP to signal oocytes and sheath cells at a distance. MSP is a cytoskeletal protein that nematode sperm utilize for motility and it does not possess a hydrophobic leader sequence. In addition, C. elegans sperm lack many standard secretory components, such as ribosomes, ER, or Golgi. Thus, the release of MSP may depend on a novel mechanism. Using specific antibodies, we detect MSP as far as 90 microns outside of spermatids and spermatozoa in vivo, consistent with its function as an extracellular signal. Labeling with vital dyes and sperm specific antibodies rules out sperm lysis as a potential mechanism. Wide-field and confocal microscopy shows extracellular MSP to be punctate with large (<0.5 microns) MSP-staining puncta near spermatids or spermatozoa. Confocal microscopy shows that MSP localizes to apparent membrane blebs at the surface of the sperm cell body, suggesting that the free puncta might originate from the membrane blebs. In the spermatheca, extracellular MSP appears to be more finely punctate suggesting that the large puncta may lose their integrity in this region to form a diffusible signal. Neither a pseudopod nor motility is required for MSP release because MSP puncta are located near wild-type or spe-8 mutant spermatids. However, MSP puncta produced by spermatids appears to provide a longer acting more local signal. Using high-pressure freezing and freeze substitution to prepare samples for transmission electron microscopy, we have identified unusual free 150-300 nm double-layered vesicles located near spermatozoa in extracellular spaces of the spermatheca and uterus. We are currently testing if MSP is localized within these structures using immuno-EM. Since, MSP release appears not to occur from spermatids within or dissected from males, we reasoned a cue from the hermaphrodite must initiate release. To test this hypothesis we devised an in vitro release assay. Spermatids treated in vitro with a female extract rapidly (20s to 2 min) form MSP-containing blebs at their cell surface and MSP appears to be lost from the cell upon further incubation. This activity is distinct from activation during spermiogenesis during which the sperm forms a pseudopod. Scanning electron microscopy also shows a difference in the cell morphology of spermatids treated with the extract. Activity from this extract is abundant, soluble, heat stable, yet abolished by boiling. MSP puncta are found within the uterus of mated germline deficient glp-4(bn2 ts ) animals, suggesting the proposed cue may originate from the soma. Based on these results we purpose a model in which spermatids and spermatozoa receive a signal from the hermaphrodite soma and shed vesicles containing MSP in response to trigger meiotic maturation. In this model, MSP-containing vesicles released by spermatozoa in the spermatheca are unstable, forming a diffusible MSP signal that correlates with meiotic maturation rates and MAP kinase activation. Biochemical and cell biological tests of this model are underway.