Fielmich, Lars-Eric et al. (2017) International Worm Meeting "Spatiotemporal control of gene knock-out and protein-protein heterodimerization to study mitotic spindle positioning."

Van den Heuvel, Sander, Schmidt, Ruben, Fielmich, Lars-Eric, Akhmanova, Anna

[International Worm Meeting, 2017]

Tissue development and homeostasis depend on the correct axis and plane of cell division, which are determined by the position of the mitotic spindle. Studies of the C. elegans one-cell embryo have provided much insight in the regulation of spindle positioning during asymmetric cell division. Pulling forces that act from the cell cortex on astral microtubules were found to position the spindle. These pulling forces depend on a force-generating complex (FG) that anchors the dynein motor to the cortex. The FG is a conserved complex consisting of a membrane-bound Ga protein that, in the GDP-bound form, binds the GPR-1/2 linker protein, which interacts with the LIN-5 coiled-coil protein. The discovery of a non-canonical role of Ga as a membrane-anchor for the FG was surprising at the time, and still is only partly understood. The Guanine-nucleotide Exchange Factor (GEF) RIC-8 and GTPase-Activating Protein (GAP) RGS-7 regulate the GDP/GTP cycle of Ga and are also required for proper force generation. It remains unclear how this corresponds to Ga functioning as a membrane anchor. Hampering analysis, complete inactivation of ric-8 and rgs-7 appears difficult to achieve by RNAi and null mutants are not viable. We therefore developed an inducible, tissue-specific gene knock-out system based on Cre-Lox recombination. We introduced Lox sites in the endogenous ric-8 and rgs-7 loci to enable KO of the genes. To assure spatiotemporal control of the KO, we use a two-component recombinase system. Thus, gene KO can be induced in the tissue of choice, for example the germline, while maintaining a healthy strain. A complementary approach focuses on the individual functions of FG components. Hereto, we use the light-inducible heterodimerization of LOV and ePDZ to reconstitute cortical FG complexes that lack one or several components. Using a PH::LOV as membrane anchor and an ePDZ::dynein knock-in, we were able to recruit endogenous dynein directly to the membrane and bypass the FG. This was insufficient for pulling force generation and suggests that the FG is more than a dynein anchor. To test the role of Ga signaling, we generated and are currently analyzing an ePDZ::GPR-1 knock-in to reconstitute a cortical FG that lacks the Ga component. We will present our latest results at the meeting.

Updike, Dustin et al. (2011) International Worm Meeting "P granules extend the nuclear pore complex environment in the C. elegans germ line."

Strome, Susan, Updike, Dustin

[International Worm Meeting, 2011]

C. elegans germline development relies heavily upon post-transcriptional regulation in the germ plasm. Within the germ plasm, ribonucleoprotein aggregates called P granules overlie nuclear pore clusters and receive mRNAs as they are exported from the nucleus. A number of P-granule components, including the Vasa-related proteins GLH-1, GLH-2, and GLH-4, contain phenylalanine-glycine (FG) repeat domains, which are a common feature of nuclear pore complex (NPC) proteins. Within the NPC, FG-rich domains form a cohesive meshwork of filaments through hydrophobic interactions involving the phenylalanines in the FG motifs, creating a size-exclusion barrier that prevents diffusion of molecules larger than 45 kDa between the nucleus and the cytoplasm. We demonstrated that P granules, like NPCs, are held together by weak hydrophobic interactions and that they also establish a size-exclusion barrier similar to that of NPCs within the germ plasm. By expressing P-granule components in heterologous (intestinal) cells, we show that GLH-1 and its FG domain are not sufficient to form granules, but require factors like PGL-1 to nucleate the localized concentration of GLH proteins. GLH-1 is necessary but not sufficient to target intestinal PGL granules to the nuclear periphery. Our results provide insights into the roles of the PGL and GLH families of proteins and suggest that P granules extend the NPC environment in the germ line to create a specialized hydrophobic microenvironment that may facilitate post-transcriptional processing events while selectively excluding large protein complexes from gaining access to mRNAs and endogenous siRNAs.

Updike, Dustin et al. (2009) International Worm Meeting "P granules extend the nuclear pore environment in the C. elegans germ line."

Strome, Susan, Updike, Dustin

[International Worm Meeting, 2009]

Germ granules are large, non-membrane-bound, ribonucleoprotein (RNP) organelles found in the germline cytoplasm of most, if not all, animals[1]. Like germ granules across species, P granules in C. elegans are found at the nuclear periphery[2], and are closely associated with nuclear pores in the germ line[3]. The C. elegans VASA homologs, GLH-1, GLH-2, and GLH-4, which are constitutively associated with P granules, resemble nuclear pore (NUP) proteins in being rich in FG (PheGly) repeats[4]. We hypothesized that the association between P granules and nuclear pores is facilitated by hydrophobic interactions between the FG repeats of the GLHs and NUPs. Consistent with this, P granules are dispersed when hydrophobic interactions are disrupted by aliphatic alcohols. In nuclear pores, hydrophobic interactions between FG repeat domains create a size exclusion barrier. We have shown that P granules impose a similar size exclusion barrier. The integral connection between P granules and nuclear pores is supported by results from a genome-wide RNAi screen for components required for proper P granule assembly and localization, in which we identified several nuclear pore associated factors. We are currently using a sensitive assay to measure interactions between FG repeat domains from GLH-1, GLH-2, GLH-4 and nuclear pore components identified in our screen. We propose that P granules extend the nuclear pore environment and provide a unique cytoplamic domain for post-transcriptional regulation in the germ line. 1.Eddy, E.M., Germ plasm and the differentiation of the germ cell line. Int Rev Cytol, 1975. 43: p. 229-80. 2.Strome, S. and W.B. Wood, Immunofluorescence visualization of germ-line-specific cytoplasmic granules in embryos, larvae, and adults of Caenorhabditis elegans. Proc Natl Acad Sci U S A, 1982. 79(5): p. 1558-62. 3.Pitt, J.N., J.A. Schisa, and J.R. Priess, P granules in the germ cells of Caenorhabditis elegans adults are associated with clusters of nuclear pores and contain RNA. Dev Biol, 2000. 219(2): p. 315-33. 4.Gruidl, M.E., et al., Multiple potential germ-line helicases are components of the germ-line-specific P granules of Caenorhabditis elegans. Proc Natl Acad Sci U S A, 1996. 93(24): p. 13837-42.

Sheth, Ujwal et al. (2009) International Worm Meeting "P granule-nuclear pore complexes are principal sites of mRNA export in C. elegans germ cells."

Pitt, Jason, Sheth, Ujwal, Priess, James R

[International Worm Meeting, 2009]

Germline-specific P granules contain numerous RNA-binding proteins, but little detectable mRNA except under quiescent states. P granules are perinuclear during most of development, but are cytoplasmic in oocytes and early embryos. Although functions of cytoplasmic P granules have not been determined, loss of perinuclear P granules is associated with inappropriate expression of at least some "masked" maternal mRNAs and transformation of germ cells into somatic cells [1]. The perinuclear P granules are associated specifically with clusters of nuclear pore complexes (NPCs), representing about 75% of the total NPCs. Here, we wanted to determine whether the NPCs associated with P granules (Pg-NPCs) are active sites of mRNA export, and found that mRNA export factors such as NXF-1 are enriched at the base of the Pg-NPCs under normal growth conditions. We discovered that heat shock could be used to induce expression of transgenic mRNA in late pachytene nuclei, and used this assay to examine mRNAs containing several types of 3''UTRs. These include 3''UTRs from mRNAs that are (a) normally translated in the gonad, (b) not translated in the gonad, or (c) targeted for degradation by RNAi. None of these nascent mRNAs were retained in P granules, and instead each showed a similar, transient accumulation in P granules before entering the general cytoplasm. This transient accumulation depended on P granule proteins such as GLH-1, and was not seen in somatic cells. Thus, Pg-NPCs appear to contain a dynamic population of mRNAs. Although in time-lapse movies Pg-NPCs appear to be stable (>20 mins), we found in photobleaching experiments that P granule components such as PGL-1 are highly dynamic (<20 sec half-recovery times). We conclude that, under normal growth conditions, the Pg-NPCs do not appear to act as storage sites for masked mRNAs although it remains possible that cytoplasmic P granules have a role in storage. Instead, our results support the view that Pg-NPCs are sites of dynamic interactions between proteins and nascent mRNAs. We noted previously that the GLH family of P granule proteins contains numerous FG repeats [2], analogous to FG repeats in core nucleoporins that function in the export of mRNA. Interestingly, we found that a gonad-specific isoform of DDX-19 is highly enriched in FG repeats, and that DDX-19 and a novel C. elegans protein with FG repeats are enriched in P granules. Thus, we hypothesize that these repeats function in the extended export of mRNA/protein past the NPC and through P granules. [1] Ciosk et al., Science 311, 851(2006) [2] Schisa et al., Development 128, 1287 (2001).

Kato, Mihoko et al. (2015) International Worm Meeting "LINKIN, a cell adhesion molecule required during collective migration of the male gonad."

Yu, Collin Z., Kato, Mihoko, Chou, Tsui-Fen, Sternberg, Paul W., DeModena, John

[International Worm Meeting, 2015]

During epithelial collective migration, partial tissue organization is maintained through cell adhesion. The developing C. elegans male gonad undergoes a collective cell migration consisting of the linker cell, which acts as a leader migratory cell, and a chain of interconnected epithelial-like follower cells. This organization differs from the hermaphrodite gonad, in which the leader cells are immediately followed by germ cells. We have identified a conserved protein, LNKN-1, required for maintaining cell adhesion during male gonadal migration. LNKN-1 defines the LINKIN proteins, a family of previously uncharacterized single-pass transmembrane proteins conserved from unicellular eukaryotes to humans. We identified seven atypical FG-GAP domains in the extracellular domain, which potentially fold into a beta-propeller structure resembling the ligand-binding domain of alpha-integrins. By antibody staining and YFP-translational fusion, we observe expression of LNKN-1 in the plasma membrane of all gonadal cells with apical and lateral enrichment. Through rescue experiments, we showed that both the extracellular and intracellular domains are required for LNKN-1 function. We identified RUVB-1/RUVBL1, RUVB-2/RUVBL2 and TBA-2/alpha-tubulin as interactors of LINKIN by mass spectrometry using SILAC-treated HEK 293T cells and testing candidates for lnkn-1-like phenotypes in C. elegans male gonad migration. We propose that LINKIN promotes tissue cohesion during migration by adhering through a beta-propeller extracellular domain and regulating microtubule dynamics through RUVBL proteins at its intracellular domain. We are currently building on the LINKIN pathway by investigating whether LINKIN binds homophilically or has a heterophilic binding partner. Additionally, we are examining other lnkn-1 mutants, including a point mutation of a highly conserved residue and a new LINKIN deletion mutant.

Chen, Wenjun et al. (2019) International Worm Meeting "Germline RNA helicases drive the phase separation and perinuclear anchoring of germ granules to promote piRNA-mediated genome surveillance."

Zhang, Donglei, Lee, Heng-Chi, Tu, Shikui, Weng, Zhiping, Chen, Wenjun, Munro, Ed, Lang, Charles, Bennett, Karen, Brown, Jordan

[International Worm Meeting, 2019]

piRNAs function as a guardian of the genome through the silencing of foreign or selfish nucleic acids. However, it remains unknown how piRNAs effectively identify foreign/selfish RNAs among many endogenous mRNAs. Intriguingly, many piRNA pathway factors are enriched in germ granules, the major sites of RNA export in germ cells, implying their localization may facilitate the detection and silencing of foreign nucleic acids. Here we showed that two germline helicases, GLH-1 and GLH-4, play a redundant role in germ granule formation. In glh-1;glh-4 double mutants, many germ factors, including PGL-1 and PRG-1, become completely dispersed into the cytoplasm. To investigate the roles of germline helicases in controlling germ granule formation, we created gene-edited worms that express various mutated GLH-1 helicases. We found that GLH-1 controls the formation and disassembly of germ granules through their binding and release of RNAs, respectively, which are coupled to distinct steps of its ATP hydrolysis cycle. Microscopic and proteomic analyses support a model that RNA binding by GLH-1 allows for the assembly of germ granule factors on RNAs, which promote the phase separation of germ granules. In addition, the FG repeats of GLH-1 is required for the anchoring of germ granules to perinuclear foci. Importantly, piRNA reporter analyses and small RNA cloning data suggest that normal germ granule dynamics and proper nuclear membrane association are both critical for the production of secondary small RNAs and gene silencing. Furthermore, we showed that these glh-1 mutants with abnormal germ granules also exhibit defects in transgenerational gene silencing. Together, our research suggests that the localization and dynamics of germ granules are both controlled by germline helicases and their RNA-bound complexes drive the phase separation of germ granules. In addition, our data support a model that germ granules function as a checkpoint, where small RNAs survey mRNA transcripts as they exit nuclei to identify RNA targets for gene silencing.

Adam Lunceford et al. (2007) International Worm Meeting "Functional analysis of Caenorhabditis elegans COQ-3: An O-methyltransferase required for synthesis of coenzyme Q."

Adam Lunceford, Catherine Clarke, Fernando Gomez, Ryoichi Saiki

[International Worm Meeting, 2007]

coq-3(qm188) homozygous mutant C. elegans derived from heterozygous mothers develop more slowly and are smaller in size than wild-type animals. The majority of these homozygous mutants are completely sterile with only a few laying 5-10 eggs. These second generation homozygous mutants arrest as L1 larvae. Based on amino acid sequence identity with yeast and human Coq3 polypeptides, the C. elegans coq-3 gene has been identified as a candidate O-methyltransferase in the biosynthesis of coenzyme Q (ubiquinone or Q). Q functions as an essential factor of the respiratory chain and as a lipid soluble antioxidant that prevents peroxidation of membrane lipids. The Q biosynthetic pathway consists of at least nine polypeptides encoded by coq genes designated coq-1 through coq-9. The Q biosynthetic pathway in eukaryotes includes two O-methylation steps catalyzed by Coq3, one C-methylation by Coq5, and at least two hydroxylases, encoded by the coq-6 and clk-1/coq7 genes. It is intriguing that the C. elegans coq-3 mutants exhibit a much more drastic phenotype than the C. elegans clk-1/coq7 mutants, which exhibit slow development, sluggish adult behaviors, and an extended life span. C. elegans clk-1/coq-7 mutants fail to produce Q, accumulate demethoxyubiquinone (DMQ), and are dependent on a dietary supply of Q for growth and fertility. However, dietary Q fails to rescue the fertility of the C. elegans coq-3 mutant. We have recovered a C. elegans coq-3 cDNA and have expressed it in the Saccharoymyces cerevisiae coq3 null strain. The rescued yeast strain is able to grow on glycerol, a non-fermentable carbon source. Q₆ biosynthesis is restored in this rescued strain, although not to wild-type levels. Expression of the C. elegans coq-3 cDNA in the Escherichia coli mutant GD1, which lacks the coq-3 ortholog ubiG, restores growth on succinate and enables the bacteria to produce Q₈. These data indicate that the COQ-3 polypeptide acts as an O-methyltransferase in the C. elegans Q biosynthetic pathway. Using the yeast and bacterial expression systems we hope to further characterize the functional roles of the C. elegans COQ-3 polypeptide. This work was supported by NIA Grant AG19777 and NIH Grant GM45952, and by NIH Cell and Molecular Training Grant GM07185 to AL and FG.