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[
MicroPubl Biol,
2021]
The AID system has emerged as a powerful tool to conditionally deplete proteins in a wide-range of organisms and cell types (Nishimura et al. 2009; Holland et al. 2012; Zhang et al. 2015; Natsume et al. 2016; Trost et al. 2016; Brown et al. 2017; Daniel et al. 2018; Chen et al. 2018; Camlin and Evans 2019). The system is comprised of two components. A plant F-box protein Transport Inhibitor Response 1 (TIR1) is expressed and forms a complex with endogenous Skp1 and Cul1 proteins to form a functional SCF ubiquitin ligase (Nishimura et al. 2009; Natsume and Kanemaki 2017). TIR1 can either be expressed constitutively or in a tissue-specific manner depending on promoter choice. A degron sequence from the IAA17 protein is fused to the protein of interest (Nishimura et al. 2009; Natsume and Kanemaki 2017). Commonly used auxin-inducible degrons include 44 amino acid (AID*) and 68 amino acid (mAID) fragments of IAA17 (Morawska and Ulrich 2013; Li et al. 2019). Addition of the plant hormone auxin bridges an interaction between TIR1 and the degron and the SCF ligase ubiquitylates the degron-fused protein leading to proteasomal degradation.
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[
MicroPubl Biol,
2021]
Gamete fusion is a pivotal step during fertilization to create an organism of the next generation. In C. elegans, since oocytes have no thick egg coat like the zona pellucida, perhaps spermatozoa directly bind to and fuse with the oocyte plasma membrane (PM). Thus, C. elegans is an excellent model to investigate how a spermatozoon and an oocyte fuse together.
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[
Development,
2009]
A diverse group of developmental biologists who study cell polarity gathered in late March 2009 at the RIKEN Center for Developmental Biology in Kobe, Japan, for a symposium entitled ;Shape and Polarity''. The organizers, Masatoshi Takeichi, Fumio Matsuzaki, Hitoshi Sawa [RIKEN Center for Developmental Biology (CDB), Kobe, Japan] and Carl-Philipp Heisenberg (Max Planck Institute, Dresden, Germany), put together an engaging program that highlighted recent progress towards understanding the mechanisms of cell polarization during development, and the functions of cell polarity in shaping development.
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[
MicroPubl Biol,
2020]
The Auxin-Inducible Degradation (AID) system is a powerful technique in the C. elegans toolkit that enables conditional and reversible protein depletion with high temporal and spatial specificity (Zhang et al. 2015; Martinez et al. 2020; Ashley et al. 2020; Martinez and Matus 2020). This system relies on tagging a gene of interest with a short AID degron sequence and transgenic expression of TIR1, an inducible E3 ubiquitin ligase normally found only in plants (Nishimura et al. 2009; Zhang et al. 2015). Upon exposure to the plant-derived hormone Auxin, TIR1 is activated and targets AID-tagged proteins for proteasomal degradation (Nishimura et al. 2009; Zhang et al. 2015) (Figure 1A). While there are qualitative reports that Auxin does not overtly affect the morphology or behavior of wild-type C. elegans (Zhang et al. 2015), this has not been quantitatively assessed. Determining whether Auxin significantly affects C. elegans morphology and behavior, even in subtle ways, is important given the C. elegans communitys rapid uptake of the AID system (Kasimatis et al. 2018; Nance and Frkjr-Jensen 2019; Ashley et al. 2020; McDiarmid et al. 2020). Here, we use our high-throughput machine vision tracking system, the Multi-Worm Tracker (MWT) (Swierczek et al. 2011), to investigate whether exposure to Auxin affects a suite of morphological, locomotor, mechanosensory, and short-term habituation learning phenotypes in our labs derivate of Bristol N2 wild-type worms and the CGC wild-type reference strain, PD1074 (Yoshimura et al. 2019) (Figure 1B).
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Yamanaka, MIkiko, Nishimura, Hitoshi, Shiraki, Riona, Shimada, Yoshihiro, Hashimoto, Masaharu, Kaneko, Mayumi, Ohkura, Kouhei, Omote, Masaaki, Hashiba, Youki, Karuo, Yukiko
[
International Worm Meeting,
2021]
In Caenorhabditis elegans, spermiogenesis undergoes two pivotal events; sperm formation and activation. Pseudopods extend from round spermatids to form motile spermatozoa, whereas membranous organelles (MOs) in spermatids fuse with the plasma membrane (PM) to activate spermatozoa. During the latter process, MOs release their contents extracellularly, and some proteins that are essential for fertilization relocate from the MO membrane onto the sperm surface, resulting in the acquisition of sperm fertility. These cytological features of MO fusion are similar to those of the acrosome reaction in mouse spermatozoa, representing one event for sperm activation. Thus, we hypothesized that C. elegans and the mouse might share a common mechanism for sperm activation. To explore this, we first screened a chemical library to obtain compounds that trigger C. elegans spermiogenesis. Of 480 entries contained in the library, we got several compounds as C. elegans spermiogenesis activators and chose one of the positive agents, named DDI-4, for further analyses. Intriguingly, 100 microM DDI-4 could induce the acrosome reaction in ~85% of mouse cauda epididymal spermatozoa, while ~70% became acrosome-reacted with 10 microM the calcium ionophore A23187. Moreover, DDI-4 promoted tyrosine phosphorylation of mouse sperm proteins, a typical capacitation signature, at least in vitro. These results indicate that DDI-4 can activate both C. elegans and mouse spermatozoa in vitro. In other words, these two species presumably possess targets of DDI-4 that function in sperm activation. To obtain clues regarding the DDI-4 targets or DDI-4-related factors, we screened mutants whose spermatids were resistant to DDI-4 in ethyl methanesulfonate-treated worms. Since two mutant strains were eventually isolated, we are currently investigating those strains by next-generation sequencing to identify mutated genes by which spermatids will become incapable of being activated with DDI-4. Information of such genes might contribute to elucidating the common mechanism for sperm activation in C. elegans and the mouse.
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[
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.
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[
MicroPubl Biol,
2021]
During the process of cell differentiation, specific cytoskeletal proteins can sequentially assemble into a wide variety of diverse molecular superstructures. Nematode spermatogenesis provides a powerful system for studying these transitions since sperm-specific transcription ceases prior to the meiotic divisions and translation ceases shortly thereafter (Chu and Shakes, 2013). Therefore, structural transitions that follow the meiotic divisions must be carried out by the remodeling of already synthesized proteins. The Major Sperm Protein (MSP) is a nematode-specific cytoskeletal element whose polymerization dynamics drive the pseudopod-based motility of the activated sperm (Roberts, 2005). In C. elegans, MSP additionally functions as the extracellular signaling molecule for triggering both ovulation and oocyte maturation (Miller et al., 2003). MSP is highly abundant in sperm, where it reaches 10-15% of total and 40% of soluble cellular protein (Roberts 2005). Within developing spermatocytes, MSP is packaged into fibrous bodymembranous organelle (FB-MO) complexes (Fig. 1A, Roberts et al., 1986). By assembling into paracrystalline FBs, MSP is both sequestered away from the critical meiotic processes of chromosome segregation and cytokinesis while also being packaged for efficient segregation into spermatids during the post-meiotic partitioning process (Chu and Shakes 2013, Nishimura and LHernault, 2010, Price et al., 2021). Following the meiotic divisions and sperm individualization, FBs disassemble, and MSP disperses as dimers throughout the spermatid cytoplasm (Fig. 1A). When sperm activate to form motile spermatozoa, MSP polymerization within the pseudopod drives the motility of the crawling sperm (Chu and Shakes, 2013). Thus, MSP exists in at least three distinct molecular states: 1) in highly organized paracrystalline FBs within developing spermatocytes 2) as unpolymerized dimers within spermatids, and 3) in dynamically polymerizing filaments and fibers within crawling spermatozoa.
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[
International Worm Meeting,
2013]
Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disease with an unknown pathogenesis and limited therapeutic treatments. A major limitation hindering the development of therapeutic treatments is a lack of understanding of the disease's molecular pathways. In humans, a P56S point mutation in the VAPB/ALS8 MSP domain is associated with ALS and late-onset spinal muscular atrophy (SMA) (Funke et al., 2010; Millecamps et al., 2010; Nishimura et al., 2004). The N-terminal MSP domain is cleaved from the C-terminus of the VAPB protein, and is secreted in a cell-type specific manner (Tsuda et al., 2008). However, the P56S mutation inhibits secretion of the MSP domain. Genetic and biochemical evidence support the hypothesis that the MSP domain interacts with the VAB-1 Eph receptor, ROBO/SAX-3 receptor, and CLR-1 Lar-like protein tyrosine phosphatase receptor, which are collectively called growth cone guidance receptors (Miller et al, 2001; Miller et al., 2003; Tsuda et al., 2008; Han et al., 2012). In C. elegans, secreted vMSP acts on CLR-1 and ROBO/SAX-3 receptors expressed in striated muscle, promoting Arp2/3-dependent actin remodeling. This remodeling is critical for proper placement of mitochondria to actin-rich myofilament I-bands (Han et al., 2012). We hypothesize that the vMSP receptors form heteromeric complexes to promote signaling critical for actin remodeling and correct mitochondria placement. To begin testing this hypothesis, I am expressing combinations of VAB-1, SAX-3, and CLR-1 in cultured cells and investigating putative complex formation via co-immunoprecipitation. My preliminary data suggest that SAX-3 complexes with both VAB-1 and CLR-1. Data will also be presented on the role of C. elegans VAPB/VPR-1 in regulating mitochondria in motor neurons. The results could provide insight into growth cone guidance receptor interactions and pathways involved in ALS.
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[
International Worm Meeting,
2011]
Sperm motility and oocyte maturation in C. elegans are dependent on the Major Sperm Protein (MSP). MSP is secreted from sperm and binds to the Eph receptor VAB-1 and other unknown receptors expressed on oocytes and sheath cells. The MSP domain is an evolutionarily conserved motif found in a number of proteins. A point mutation (P56S) in the MSP domain of human VAP/ALS8 has been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and late-onset spinal muscular atrophy (Nishimura et al., 2004). ALS patients and ALS mouse models exhibit reduced VAPB expression, suggesting that VAPB plays a widespread role in pathogenesis. Previous work has shown that the VAPB MSP domain is cleaved, secreted, and acts as a ligand for Eph receptors. The P56S mutation causes protein aggregation and failed secretion (Tsuda et al., 2008). VPR-1 is the worm VAPB homologue. Loss of this protein in the worm recapitulates many of the pathologies observed in ALS patients, including mitochondrial dysfunction and lipid metabolism defects. Further,
vpr-1 null mutant animals are sterile due to germ cells failing to proliferate and differentiate. Pan neuronal expression of VPR-1 in null mutant animals partially rescues the metabolic defects, and partially rescues the germ-line defect in some animals while causing tumors in others. Examination of the sterile phenotype has shown the distal tip cells of VPR-1 null mutant animals are enlarged and have shorter processes compared to those of wild-type animals. The morphology of sheath cells in null mutants is also severely disrupted. Using apoptosis markers, including a sheath cell driven CED-1::GFP transgene, we have observed an increase in germ line apoptosis in
vpr-1 mutant hermaphrodites. Silencing of selected genes involved in the physiological and DNA-damaged induced apoptosis pathways suggests that the increase in apoptosis is of a physiological basis rather than from DNA damage. Given the sheath cell defect, we are currently examining if these cells are capable of clearing apoptotic cells. My current goal is to delineate the basis of the apoptosis defect and to identify genes that modify the apoptosis phenotype.
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[
International Worm Meeting,
2009]
The Major sperm protein (MSP) of C. elegans has an important role in sperm motility and oocyte maturation and ovulation. MSP secreted from sperm binds to the VAB-1 Eph receptor and other receptors on the surface of oocytes and sheath cells. The MSP domain structure is highly conserved throughout evolution. The human gene VAPB contains an MSP domain and a single point mutation (P56S) within this domain causes an inherited form of the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS) (Nishimura et al., 2004). VAPB is found at reduced levels in sporadic ALS patients and a mouse ALS model. Prior work has shown that VAPB MSP domains are cleaved, secreted, and act as ligands for Eph receptors (Tsuda et al., 2008). However, the P56S MSP domain in flies fails to be secreted, suggesting that this protein has a signaling function involved in ALS. VPR-1 is the C. elegans homologue of human VAPB. Loss of VPR-1 in the worm causes multiple metabolic defects that are reminiscent of defects seen in some ALS patients. Further,
vpr-1 null mutants are sterile due to germline proliferation and differentiation defects. Neuronal-specific overexpression of VPR-1 in a null mutant background leads to partial rescue of the metabolic defects. In addition, neuronal overexpression partially rescues the germline proliferation and differentiation defects in some animals, while causing germline tumors in others. Binding studies suggest that MSP domain receptors are absent or at very low levels in proliferating germ cells, but expressed in the distal sheath and possibly the distal tip cell. The molecular basis of the VPR-1 germline development defects is unknown. My objective is to understand the biological function of VPR-1 in germline development. First, I am characterizing the germline defects of
vpr-1 null mutants using molecular markers. Second, I am generating transgenic lines that express VPR-1 in the germ line to test for rescue of the sterility phenotype. Finally, I am performing an RNAi screen to identify suppressors of the VPR-1 sterile phenotype, as well as modifiers of the tumor phenotype. My preliminary results will be presented.