Rie Sakamoto et al. (2001) International C. elegans Meeting "A C.elegans JSAP/JIP3 homolog, UNC-16, interacts with kinesin light chain, KLC-2"

Dana Byrd, Naoki Hisamoto, Yishi Jin, Kunihiro Matsumoto, Rie Sakamoto, Masato Kawasaki

[International C. elegans Meeting, 2001]

The c-Jun N-terminal kinase (JNK) of MAP kinase (MAPK) superfamily is involved in the various stress responses and apoptosis in mammal. The components of JNK MAPK cascade are well conserved from worm to human. The C. elegans JNK cascade is mediated by JNK-1 (MAPK) and JKK-1 (MAPKK), and functions in type D GABAergic motor neurons to modulate coordinated locomotion. In mammalian JNK MAPK pathways, JIP3/JSAP acts as a scaffolding protein. C. elegans has a JIP3/JSAP homolog encoded by the unc-16 gene. To identify additional components involved in the C. elegans JNK pathway, we screened for UNC-16-binding proteins by using a yeast two-hybrid system. One of the isolated genes is klc-2 encoding kinesin right chain. Co-immunoprecipitation experiments reveal that KLC-2 associates with UNC-16 and UNC-116 kinesin heavy chain when they are co-expressed in mammalian cells. To investigate the klc-2 function in C. elegans , we isolated a klc-2 mutant by TMP/UV method. The klc-2(km11) mutation produces a truncated form of the KLC-2 protein lacking its C-terminal portion. The mutant exhibits Unc and weak Egl phenotypes. We will discuss the connection between conventional kinesin, scaffolding protein and JNK signaling in the neuronal system.

Dana Byrd et al. (2002) West Coast Worm Meeting "UNC-16, the C. elegans JIP3, plays kinesin-1-dependent and independent roles in directing vesicular traffic"

Masato Kawasaki, Dana Byrd, Rie Sakamoto, Naoki Hisamoto, Yishi Jin, Kunihiro Matsumoto

[West Coast Worm Meeting, 2002]

The proper localization of synaptic components is a regulated process that requires the pairing of multiple cargo vesicles with different motor proteins. Despite the identification of many motors, the specific interactions of cargoes and motors are poorly understood. We have previously shown that loss-of-function mutations in unc-16, the C. elegans homolog of JSAP1/JIP3/dSYD, cause mislocalization of synaptic vesicle and glutamate receptor markers (Neuron 32: 787-800). Moreover, mutations in unc-16partially suppress the synaptic vesicle retention defect in unc-104 KIF1A mutants. UNC-16 physically interacts with the KLC-2/UNC-116 kinesin-1 complex via KLC-2. Mutations in unc-116 KHC/KIF5 and klc-2 share a similar synaptic vesicle mislocalization phenotype with unc-16 mutants, and mislocalize a functional UNC-16::GFP. Conversely, UNC-116::GFP is mislocalized in unc-16 and klc-2 mutants. These results are consistent with a direct interaction of UNC-16 with the kinesin-1 motor complex and support an evolutionarily conserved function of UNC-16/JIP3 in mediating kinesin-1 transport. However, the function of UNC-16 may be more complex than simply serving to regulate kinesin-1-cargo interactions because UNC-16 and kinesin-1 have separable functions. For example, mutations in unc-116do not suppress the unc-104synaptic vesicle retention defect; unc-116 mutants, but not unc-16, have axon outgrowth defects; and GLR-1::GFP is mislocalized in unc-16 mutants, but not in unc-116, jnk-1, or unc-104 mutants. Therefore, we propose that UNC-16 likely has kinesin-1-independent functions.

Dana Byrd et al. (2001) International C. elegans Meeting "UNC-16, A JNK signaling scaffold, regulates vesicle transport in C. elegans"

Yishi Jin, Masato Kawasaki, Naoki Hisamoto, Kunihiro Matsumoto, Mercy Walcoff, Dana Byrd

[International C. elegans Meeting, 2001]

Localization of synaptic components must be both precise and dynamic to allow proper information flow between neurons and their targets. In first larval stage (L1) animals, the GABAergic DD motor neurons form synapses with ventral body wall muscle, and the localization of SNB-1::GFP, a synaptic vesicle marker, is restricted to the ventral processes. Loss-of-function mutations in the unc-16 gene result in the mislocalization of SNB-1::GFP to dorsal dendritic processes of L1 stage DD neurons without a detectable change in a postsynaptic GABA receptor marker in the muscle. The localization of presynaptic markers in many neurons is similarly altered in unc-16 mutants. SNB-1::GFP is mislocalized to the dorsal dendritic processes of the VD motor neurons and out along the dendritic endings of several sensory neurons in the head. Intriguingly, GLR-1::GFP, a postsynaptic marker, is also mislocalized along the ventral cord and in the anterior endings of some interneurons. unc-16 encodes a protein homologous to mouse JSAP1/JIP3 and Drosophila Sunday Driver (dSYD). Like its mammalian homologs, UNC-16 physically interacts with JNK signaling components. Deletion mutants of KNK signaling genes result in slight mislocalization of synaptic vesicle markers in L1 DD motor neurons and exacerbate the phenotype of weak unc-16 alleles. UNC-16 and JNK signaling molecules are expressed in neurons and function cell autonomously in DD neurons. Our results suggest that UNC-16 may regulate synaptic vesicle transport through its combined interactions with both JNK signaling molecules and motor proteins. In support of a role for UNC-16 in regulating vesicle transport, unc-16 mutations partially suppress mutations in unc-104 , a synaptic vesicle specific kinesin. The suppression is not allele specific, suggesting that the interaction of unc-16 and unc-104 may not be direct, but that both UNC-104 and UNC-16 may contribute to a similar process. Mutations in unc-16 do not suppress unc-116 kinesin heavy chain mutations. Instead, a weak unc-116 mutation results in SNB-1::GFP mislocalization in L1 DD motor neurons similar to that seen in unc-16 mutants. We propose that UNC-16 functions by both forming a linkage between vesicular cargo and motor proteins and serving as a scaffold for signaling molecules that direct cargo selection, motor activity, or motor direction.

Rie Sakamoto et al. (2002) Japanese Worm Meeting "A JNK/UNC-16 signaling complex regulates synaptic vesicle localization through a conventional kinesin in C. elegans"

Rie Sakamoto, Yishi Jin, Naoki Hisamoto, Dana Thyra Byrd, Kunihiro Matsumoto, Masato Kawasaki

[Japanese Worm Meeting, 2002]

The c-Jun N-terminal kinase (JNK) of the MAP kinase (MAPK) superfamily is involved in various stress responses and apoptosis in mammals. The C. elegans JNK cascade is composed of JNK-1 (MAPK) and JKK-1 (MAPKK) and functions in type-D GABAergic motor neurons. The C. elegans unc-16 gene encodes a protein homologous to mammalian JSAP1/JIP3, which acts as a scaffold protein in the JNK pathway by binding with MLK (MAPKKK), MKK7 (MAPKK) and JNK3 (MAPK). Like JSAP1/JIP3, UNC-16 physically interacts with JNK-1 and JKK-1, forming a JNK signaling module. unc-16(ju79), jnk-1(gk7), and jkk-1(km2) mutant animals exhibit mislocalization of a synaptic vesicle marker, SNB-1::GFP, in L1 DD motor neurons. This suggests that the JNK-1 pathway containing UNC-16 regulates synaptic vesicle localization. To understand the mechanism regulated by UNC-16, we screened for UNC-16-binding proteins using yeast two-hybrid system. One of the isolated genes is klc-2 encoding a kinesin light chain. Co-immunoprecipitation experiments reveal that KLC-2 associates with UNC-16 and UNC-116 kinesin heavy chain when they are co-expressed in mammalian cells. We constructed the klc-2(km11) mutation that produces a truncated form of the KLC-2 protein lacking its C-terminal portion. Similar to unc-16(ju79), jnk-1(gk7), and jkk-1(km2) mutants, the SNB-1::GFP marker is mislocalized along the dorsal DD processes in the klc-2(km11) L1 larvae. These results raise the possibility that the UNC-16-JNK-1 pathway regulates synaptic vesicle localization through the KLC-2-UNC-116 conventional kinesin in C. elegans.

Miho Tanaka-Hino et al. (2001) International C. elegans Meeting "C. elegans SEK-1 MAPK cascade regulates neuronal asymmetric development mediated by Ca2+ signalling"

Kunihiro Matsumoto, Cornelia I Bargmann, Shunji Nakano, Masato Kawasaki, Miho Tanaka-Hino, Alvaro Sagasti, Naoki Hisamoto, Jun Ninomiya-Tsuji

[International C. elegans Meeting, 2001]

Lateral asymmetric patterns of gene expression are key to the development in many systems. Within the bilaterally symmetric pair of olfactory AWC neurons in Caenorhabditis elegans , the candidate odorant receptor STR-2 is asymmetrically expressed in either the left or the right neuron, but never both. The asymmetric AWC cell fate is regulated by axon contact during development and by Ca2+ signalling. The mitogen-activated kinase (MAPK) pathway is a highly conserved signalling cascade that converts extracellular signals into various outputs. We identified a C. elegans MAPK kinase (MAPKK), SEK-1, that can activate p38 MAPK. Here we show that SEK-1 is required for asymmetric expression in AWC neurons. Genetic and biochemical analyses reveal that SEK-1 functions within the same pathway downstream of UNC-43, the type II Ca2+- and calmodulin-dependent protein kinase (CaMKII), and NSY-1 MAPKK kinase (MAPKKK). We conclude that the NSY-1-SEK-1 MAPK pathway establishes asymmetric cell fate decision during neuronal development. This MAPK cascade is activated by Ca2+ signalling through CaMKII.

Aoki, Scott Takeo et al. (2013) International Worm Meeting "Structural characterization of the P-granule protein scaffold."

Aoki, Scott Takeo, Kimble, Judith

[International Worm Meeting, 2013]

C. elegans P-granules are essential for both the development and maintenance of the germline tissue. Disruption of P-granule formation interferes with proper germ cell proliferation and differentiation, resulting in sterility (1, 2). P-granule assembly requires structural scaffold proteins PGL-1 and PGL-3 (3, 4). These nematode-specific paralogs are sufficient to form granules in cells and multimerize through self-association (5, 6). We aim to understand the structural organization of the P-granule scaffold to better understand how the organelle regulates mRNA trafficking and turnover. We are able to express and purify recombinant PGL-1 and PGL-3. Full-length recombinant protein self-assembles into large soluble aggregates. Protease digestion analyses identify a single domain that dimerizes in solution. We are currently trying to obtain a high-resolution crystal structure of the protease-protected fragment, as well as determine the role of the N- and C- terminal regions in scaffold oligomerization. Several different types of RNA granules are required in eukaryotes for cell homeostasis, differentiation, and response to stress. The fundamental mechanisms involved in P-granule organization will undoubtedly shed light on other granules involved in RNA regulation. References: 1. Updike, D., Strome, S. (2010) J Androl 31: 53-60. 2. Voronina, E., Paix, A., Seydoux, G. (2012) Development 139: 3732-3740. 3. Kawasaki, I., et al. (1998) Cell 94: 635-645. 4. Kawasaki, I., et al. (2004) Genetics 167: 645-661. 5. Updike, D.L., Hachey, S.J., Kreher, J., Strome, S. (2011) J Cell Biol 192: 939-948. 6. Hanazawa, M., Yonetani, M., Sugimoto, A. (2011) J Cell Biol 192: 929-937..

Caroline Spike et al. (2006) Development & Evolution Meeting "A Novel P-granule Protein Required for glh-1 and rde-4 mRNA Accumulation in the Germline"

Valerie Reinke, Caroline Spike, Susan Strome

[Development & Evolution Meeting, 2006]

P granules are germ-cell-specific structures containing RNA and protein and required for proper germ cell development. We are interested in understanding how P granules are assembled or stabilized and whether they regulate mRNA translation or stability in germ cells. Mutations in glh-1, a gene encoding a VASA-like RNA helicase, fail to localize PGL-1 to P granules (Kawasaki et al. Cell 94, 635-645). Using EMS mutagenesis, we identified four mutations in a second gene (Y65B4BL.2) that reduce the accumulation of PGL-1 on P granules. We provisionally refer to this gene as dip-1 (diffuse PGL-1). Like pgl-1 and glh-1, dip-1 mutations cause germline underproliferation and temperature-sensitive maternal-effect sterility. Furthermore, glh-1 mRNA and protein levels are dramatically reduced in dip-1 mutants, suggesting that DIP-1 may be required to produce or stabilize glh-1 mRNAs. DIP-1 is a P-granule-associated protein, raising the exciting possibility that DIP-1 may link P granules to mRNA stability. We investigated this possibility by comparing the genome-wide expression profiles of isolated N2 and dip-1 germlines using microarrays. Although relatively few messages have altered abundances (either increased or decreased) in dip-1 mutant germlines, one gene that shows reduced mRNA accumlation is rde-4. Intriguingly, dip-1 mutants are resistant to pos-1(RNAi) and skn-1(RNAi) by feeding and we speculate that this is caused by a deficiency in RDE-4 protein accumulation in the germline.

Frank Nunes et al. (2006) European Worm Meeting "Evaluating the Role of the Neuronal MAP Kinase JNK-1 in Orientation Behavior and Thermotaxis"

Frank Nunes, Arne Henkel, Melanie Voss, Rudiger J. Paul, Marc Wolf

[European Worm Meeting, 2006]

The C.elegans MAP kinase JNK1 is a homolog of human stress activated protein kinases (SAPK). jnk1 deletion mutants are shortlived and more susceptible to heavy metal stress. On the other hand, If JNK1 was overexpressed, worms showed an increased resistance to oxidative stress and a prolonged lifespan (Oh et al., 2005, Villanueva et al., 2001). As shown by GFP reporter gene assays, JNK-1 is exclusively expressed in the worms nervous system (Kawasaki et al., 1999). By western blot analyses we could demonstrate an increasing activation of JNK-1 with rising but moderate temperatures, which should not be a harmful stress to C. elegans. Therefore we started to investigate the role of JNK-1 in thermotaxis behavior. Indeed jnk-1 deletion mutants [VC8 (gk7)] have an altered temperature preference compared to the wildtype (Bristol N2) when placed on a linear temperature gradient. The wildtype prefers temperatures between 15C and 20C whereas jnk-1 deletion mutants are predominantly found between 15C and 24C, thus having a broader temperature preference range. C. elegans explores its environment by modulating the frequency of occasional turns and reversals. When the environmental conditions are unfavorable reversals or sharp turns and consequently reorientation become more frequent (Gray et al., 2005). We have found that jnk-1-deletion mutants show significantly lower rates of orientation movements at 20C and 25C, which could be the reason for the observed altered temperature preference. Thus it is possible that cellular stress response as well as stress avoidance behavior are both activated in a coordinated manner through JNK-1.

Darryl Conte Jr et al. (2004) East Coast Worm Meeting "The germ granule protein PGL-1 is required for efficient RNA interference"

Darryl Conte Jr, Craig C Mello, Yingdee Unhavathaya

[East Coast Worm Meeting, 2004]

In C. elegans , very small quantities of dsRNA are sufficient to generate RNA interference not only in the animal that is exposed to dsRNA but also in subsequent generations (1, 2). These findings indicate that amplification and inheritance of a silencing agent(s) (such as siRNA) are important to the mechanism of RNAi. P-granules, the worm equivalent of germ granules, are expressed in the maternal germline, deposited into early embryos and are implicated in translational regulation of maternal mRNAs. Therefore, we hypothesized that the germline P-granules of C. elegans may be important for the transmission of a silencing factor and hence for RNAi. An examination of mutations in known P-granule components revealed that the PGL-1 protein is required for RNAi. siRNAs failed to accumulate in pgl-1 mutant worms exposed to dsRNA targeting a maternal mRNA, while siRNAs appeared to accumulate in the embryos of wild-type worms. Remarkably, animals challenged with dsRNA targeting zygotic mRNAs expressed in somatic tissues produced progeny with a reduced penetrance of RNAi phenotypes. These data are consistent with the hypothesis that PGL-1 may be important for efficient transmission of a silencing agent to the progeny of animals exposed to dsRNA. PGL-1 encodes a protein with an RGG-box found in RNA-binding proteins and is required for fertility in C. elegans (3). An attractive scenario is that PGL-1 interacts with and transmits siRNA to the progeny of animals exposed to dsRNA. Alternatively, PGL-1 may regulate the activity of the RNAi pathway by modulating the accessibility, localization or expression of RNAi pathway components or intermediates. We will present our recent efforts to elucidate the function of PGL-1 in RNAi and the relationship to the role of PGL-1 in development. 1) Fire et al. (1998) Nature 391:806-811 2) Grishok, Tabara and Mello (2000) Science 287:2494-2497 3) Kawasaki et al. (1998) Cell 94:635-645 This work is funded by National Institutes of Health grants GM058800 (to C.C.M.) and GM063348 (to D.C.)

Alberto Villanueva et al. (2001) International C. elegans Meeting "jkk-1 and mek-1 regulate body movement coordination and response to heavy metals through jnk-1 in Caenorhabditis elegans"

Xinzhu Deng, Xinhua Lin, Jose Lozano, Richard N Kolesnick, Alberto Villanueva, Michael O Hengartner, Albert Morales

[International C. elegans Meeting, 2001]

Although in vitro evidence suggests two c-Jun N-terminal kinase (JNK) kinases, MKK4 and MKK7, transactivate JNK, in vivo evidence confirming this paradigm is incomplete. In fact, data from Drosophila and murine knockouts indicate JNK deficiency may differ from the composite deficiency of MKK4 and MKK7. Recently, the Caenorhabditis elegans homologs of human JNK, jnk-1 (1) , and two MKK-7s, mek-1 (2) and jkk-1 (1), were cloned. Here we characterize jnk-1 , which encodes two isoforms JNK-1 a and JNK-1 b . A null allele, jnk-1(gk7), resulted in worms with defective body movement coordination and modest mechanosensory deficits. Similar to jkk-1 mutants (1), elimination of GABAergic signals suppressed the jnk-1(gk7) locomotion defect. jnk-1(gk7), like mek-1 nulls (2) , also showed copper and cadmium hypersensitivity. Conditional expression of JNK-1 a or JNK-1 b rescued both phenotypes, suggesting they are not due to developmental errors. While jkk-1 or mek-1 inactivation by RNA-mediated interference mimicked jnk-1(gk7) locomotion and heavy metal stress defects, respectively, mkk-4 / sek-1 inactivation did not, but rather yielded defective egg-laying . jnk-1(gk7);jkk-1(km2) and jnk-1(gk7);mek-1(ks54) double mutants displayed non-additivity regarding amplitude and heavy metal sensitivity, respectively, consistent with these MKK-7s being on separate JNK-1 pathways. Interestingly, both double mutants continued to manifest the modest mechanosensory deficits detected in jnk-1(gk7) mutant worms, suggesting the existence of other JNK-1 regulators. Our results delineate at least two different JNK pathways through jkk-1 and mek-1 in C. elegans . Further, three distinct genetic models, C. elegans , Drosophila and mice, now define interaction between MKK7, but not MKK4, and JNK. 1. Kawasaki, K. et al (1999) EMBO J. 18, 3604-3615. 2. Koga, M. et al (2000) EMBO J. 19, 5148-5156.