Jason R Kennerdell et al. (2004) West Coast Worm Meeting "INVESTIGATIONS INTO THE MECHANISMS OF SAX-3 MEDIATED AXON GUIDANCE"

Jason R Kennerdell, Cori Bargmann, Joe Hao

[West Coast Worm Meeting, 2004]

The transmembrane receptor SAX-3 is involved in the guidance decisions of many cells and axons of C. elegans. Like its fly ortholog, Roundabout (Robo), SAX-3 is involved in guidance at the ventral midline, where it prevents aberrant midline crossing of ventral cord axons. In addition, SAX-3 is involved in long-range dorsal-ventral guidance of the axon of the AVM neuron, in anterior-posterior placement of axons in the nerve ring, and in the posterior migration of the CAN neurons. One ligand for SAX-3 is the secreted molecule SLT-1, which directs the ventral guidance of the AVM neuron. SLT-1 is expressed in dorsal muscles, where it acts as a repellent for axons expressing SAX-3 to direct them ventrally. SLT-1 is also expressed in the head of the animal, where it could potentially play a role in nerve ring guidance. Interestingly, however, SAX-3 is more than just a receptor for responding to SLT-1, because mutations in sax-3 result in a number of phenotypes that are not present in animals with null mutations in slt-1. In sax-3 mutants there are axon guidance defects in the nerve ring, 80% embryonic lethality, and notched head phenotypes. These phenotypes are absent in slt-1 mutants. These slt-1 -independent phenotypes of sax-3 could be explained by a second ligand for SAX-3 or by ligand-independent functions of SAX-3. We're interested in understanding the sax-3 phenotypes that are absent in the slt-1 mutant. Previous genetic screens have failed to identify mutants other than sax-3 with the characteristic anterior nerve ring axon guidance defect, suggesting that there is not one discrete nerve ring ligand for SAX-3. Screens can often miss genes that function redundantly, and perhaps this is the case in nerve ring axon guidance. A second ligand functioning redundantly with SLT-1 might appear as a sax-3 -like phenotype in a slt-1 mutant background. We're using sensitized genetic screens in a slt-1 mutant background to find potential ligands for sax-3. The resulting mutants will be described.

Jason R Kennerdell et al. (2002) West Coast Worm Meeting "Investigations into the mechanisms of SAX-3 mediated axon guidance"

Cori I Bargmann, Jason R Kennerdell, Catherine Pawson, Joe Hao

[West Coast Worm Meeting, 2002]

The transmembrane receptor SAX-3 is involved in the guidance decisions of many cells and axons of C. elegans. Like its fly ortholog, Roundabout (Robo), SAX-3 is involved in guidance at the ventral midline, where it prevents aberrant midline crossing of ventral cord axons. In addition, SAX-3 is involved in long-range dorsal-ventral guidance of the axon of the AVM neuron, in anterior-posterior placement of axons in the nerve ring, and in the posterior migration of the CAN neurons. One ligand for SAX-3 is the secreted molecule SLT-1, which directs the ventral guidance of the AVM neuron. SLT-1 is expressed in dorsal muscles, where it acts as a repellent for axons expressing SAX-3 to direct them ventrally. SLT-1 is also expressed in the head of the animal, where it could potentially play a role in nerve ring guidance. Interestingly, however, SAX-3 is more than just a receptor for responding to SLT-1, because mutations in sax-3 result in a number of phenotypes that are not present in animals with null mutations in slt-1. In sax-3 mutants there are axon guidance defects in the nerve ring, lethality, and notched head phenotypes. These phenotypes are absent in slt-1 mutants. These slt-1-independent phenotypes of sax-3 could be explained by a second ligand for SAX-3 or by ligand-independent functions of SAX-3.

Tim Yu et al. (2001) International C. elegans Meeting "Roles of unc-34 and unc-40 in repellent guidance by sax-3"

Wendell Lim, Cori Bargmann, Tim Yu, Marc Tessier-Lavigne, Joe Hao

[International C. elegans Meeting, 2001]

Attractive and repulsive guidance receptors play key roles in the development of the nervous system by directing growing axons to their proper targets. The signaling mechanisms by which these receptors communicate with the growth cone cytoskeleton are not well understood. We are studying mechanisms of axon repulsion mediated by sax-3 , a member of the Robo family of guidance receptors. sax-3 mutants have defects in anterior and posterior cell migrations, in the formation of the nerve ring in the head, and in ventral axon guidance in the body. The slt-1 gene, a homolog of Drosophila and vertebrate Slit, encodes one probable ligand for sax-3 in several of these guidance decisions. Members of the Enabled/VASP family of proteins have been shown to be important modulators of actin dynamics and are potential downstream effectors of axon guidance receptors. We find that the C. elegans Enabled protein UNC-34 is a mediator of SAX-3 signaling in repulsive guidance from SLT-1. Genetic interactions with gain and loss of function mutations in the sax-3 pathway demonstrate that unc-34 works together with sax-3 in several guidance decisions. UNC-34 associates with the SAX-3 cytoplasmic domain in vitro , suggesting that these interactions are direct. These genetic and biochemical interactions are conserved across species, since Robo and Enabled also act together in Drosophila midline guidance (Bashaw et al, 2000). These results support a direct role for UNC-34 in repellent guidance. Unexpectedly, SAX-3 function is also potentiated by an UNC-6/Netrin-independent function of the Netrin receptor UNC-40. Genetic interactions between unc-40 and sax-3 are similar to interactions between unc-34 and sax-3 , and point to a role for UNC-40 in SAX-3 guidance. Such a role is further supported by the finding that UNC-40 and SAX-3 cytoplasmic domains directly interact. The interaction between UNC-40 and SAX-3 is also observed with their vertebrate counterparts, DCC and Robo, where it is implicated in a different guidance decision (Stein et al, 2001). Our results suggest that SAX-3 and UNC-40 may interact directly in guidance complexes that contribute to repulsive guidance. A combinatorial logic thus dictates alternative functions for UNC-40/DCC, which can act in attraction to UNC-6, repulsion from UNC-6 (with UNC-5), or repulsion from Slit (with SAX-3).

Motegi, Fumio et al. (2009) International Worm Meeting "PAR-2 is a microtubule-binding protein that links microtubules and cortical polarity in C. elegans zygotes."

Motegi, Fumio, Zonies, Seth, Seydoux, Geraldine

[International Worm Meeting, 2009]

The C. elegans zygote becomes polarized shortly after fertilization by the sperm-donated microtubule-organizing center (MTOC), which induces PAR polarity on the cortex. How the MTOC communicates with the cortex is not known. Possible signals include proteins that accumulate on the MTOC''s pericentriolar material (PCM) and the microtubules themselves (Motegi and Seydoux, 2007 for review). We have obtained evidence for a direct role for microtubules by studying PAR-2. PAR-2 is a RING finger domain protein that accumulates on the cortex nearest the MTOC during polarization (Boyd et al, 1996). PAR-2 also accumulates on the MTOC itself, most prominently during mitosis. We have found that PAR-2 has a strong affinity for microtubules in vitro (KDapp<0.2 mM). This affinity depends on three separate microtubule-binding domains, which overlap with the domain in PAR-2 required for localization to the cortex (Hao et al., 2006). Mutations in one of the domains reduce PAR-2 affinity for microtubules by 5 fold. When expressed in zygotes depleted of endogenous PAR-2, this PAR-2 mutant was defective in localizing to MTOCs, but was able to localize to the posterior cortex and rescue the par-2 embryonic lethality as efficiently as wild-type, suggesting that microtubule binding is not essential under normal conditions. The microtubule-binding defective mutant, however, was not able to localize to the cortex in zygotes that lack or delay PCM assembly or have reduced cortical actin contractility. In those zygotes, wild-type PAR-2 can still accumulate on the cortex nearest the MTOC, but PAR-2 defective in microtubule binding cannot and remains in the cytoplasm. We conclude that microtubule binding contributes to PAR-2 localization under conditions when the MTOC signal is compromised. Along with previous findings, these data suggest that polarity establishment in the zygote depends on two redundant mechanisms: a first mechanism dependent on efficient PCM assembly and actin contractility, and a second mechanism dependent on microtubules and PAR-2. Boyd, L., Guo, S., Levitan, D., Stinchcomb, D.T., Kemphues, K.J. (1996). Development 122, 3075-3084. Hao, Y., Boyd, L., Seydoux, G. (2006). Developmental Cell 10, 199-208. Motegi, F., Seydoux, G. (2007). JCB 179, 367-369.

Hodge D (1995) International C. elegans Meeting "INHIBITING THE FUNCTION OF CARNITINE PROVIDES CLUES ABOUT Caenorhabditis elegans SPERM MOTILITY."

Hodge D

[International C. elegans Meeting, 1995]

Carnitine, a molecule which transports long chain fatty acids across the inner mitochondrial membrane for beta-oxidation, is present in high concentration in marnmalian sperm and in collaboration with Dr. Joe Bahl we found that carnitine was also abundant in C. elegans sperm. I then tested to see if carnitine was necessary for C. elegans sperm motility. Activated sperm placed in tetradecyl acetic acid, a carnitine inhibitor solution, ceased motility within a given time, the inhibition time. These same sperm then regained motility when removed from this inhibitor solution. Furthermore, I discovered that the inhibition time increases significantly as sperm age increases. This data suggests that either the amount of carnitine increases with sperm age or that there is an alternative energy source, which does not require carnitine as a transporter, that increases with sperm age. Presently, I am trying to increase the amount of L-camitine in sperm by feeding it to virgin males and trying to determine if there is an alternative energy source that sperrn utilize for motility.

Catharine Rankin et al. (2003) International Worm Meeting "The effects of avr-14 on short- and long-term memory."

Nicholas Dube, Stephan Steidl, Catharine Rankin

[International Worm Meeting, 2003]

avr-14 encodes an -type subunit of a glutamate-gated chloride channel (GluCls) in Caenorhabditis elegans. avr-14 is expressed on the glutamatergic mechanosensory neurons of the tap-withdrawal response that synapse onto the command interneurons of the circuit. Because avr-14 is expressed on the touch cells it was hypothesized that it might play a role in experience-dependant plasticity in the response to tap. Worms with a mutation in avr-14 were tested for short-term and long-term habituation of the tap response. The mutation selectively affects short-term habituation for shorter interstimulus intervals (ISIs; 10s and 30s) while not affecting habituation for long ISIs (45s and 60s). These results provide support for the hypothesis that in C. elegans there are multiple ISI-dependant short-term memory systems. avr-14 worms show long-term memory for habituation training comparable to wild-type controls when given distributed training and testing with a 60s ISI. avr-14 worms, unlike wild-type controls, also show long-term memory when trained and tested with a 10s ISI. Furthermore, long-term memory for this training protocol is protein synthesis dependant, as administering heat shock in between blocks of training blocks the formation of long-term memory. This suggests that in wild-type worms the presence of avr-14 blocks the conversion from short-term to long-term memory following training with short ISIs.Thanks to Joe Dent for avr-14.

Ahmed Mohamed et al. (2005) International Worm Meeting "The VAB-1 Eph RTK is Required for Proper Axon Guidance and Neuronal Cell Positioning."

Ian D Chin-Sang, Ahmed Mohamed

[International Worm Meeting, 2005]

The vertebrate Eph receptor tyrosine kinases (RTKs) and their ligands, the Ephrins, have been found to have diverse developmental roles including early aspects of embryogenesis, segmental boundary formation, cell migration, neurogenesis, axon guidance, dendritic spine morphogenesis and tumorigenesis. Mutations that affect the only C. elegans Eph RTK, VAB-1, cause defects in neuroblast migration and epidermal morphogenesis. Recent work has also revealed the presence of some weak neuronal defects that include aberrant axon crossing in the ventral midline, disruption in the ventral guidance of amphid axons, anterior axon outgrowth in the nerve ring, and ipsilateral outgrowth of pharyngeal neurons (Bulow and Hobert, Neuron 2004 41:723-36; Hao et al., Nueron 2001 32:25-38; Morck et al., Developmental Biology 2003 260:158-75; Zallen et al., Development 1999 126:3679-92). To further understand the role of VAB-1 in neuronal development, we characterized the neuronal and behavioral phenotypes of both loss-of-function and gain-of-function VAB-1 Eph signaling in neurons. We specifically analyzed the PLM mechanosensory neuron, the canal-associated neurons (CANs) and the DVB GABAergic neuron. We found that vab-1 mutants had weak neuronal defects where axons overextended beyond their usual target region. To investigate the effects of a VAB-1 gain-of-function, we created a hyper-active form of VAB-1 by targeting the intracellular portion of VAB-1 to the plasma membrane via N-terminal myristoylation (MYR). The MYR-VAB-1 resulted in a constitutively active tyrsoine kinase and induced a penetrant phenotype where axons terminated prematurely before reaching their target, a phenotype opposite of the vab-1 loss-of-function mutants. Our results suggest that the VAB-1 Eph RTK is required for targeting or limiting axons and neuronal cells to a specific region perhaps by transducing a repellent or stop cue.

Gitai Z et al. (1998) West Coast Worm Meeting "UNC-6 CAN REDIRECT AXONS IN VIVO"

Bargmann CI, Tessier-Lavigne M, Gitai Z

[West Coast Worm Meeting, 1998]

The UNC-6 netrin is thought to be involved in both the attraction and repulsion of circumferentially migrating cells and axons. Consistent with that idea, such migrations are disrupted in unc-6 mutants. It has also been shown that in vitro, the UNC-6 vertebrate homolog, Netrin-1, is sufficient to elicit both axonal outgrowth and turning. However, it has yet to be clearly demonstrated whether UNC-6 can play an instructive role in directing axon migrations in vivo. To address that question we have focused on the HSN motor neuron. In wild type animals UNC-6 is expressed ventrally, and the HSNUs initial axon projection is directed ventrally. The disruption of this ventral migration is one of the most penetrant defects associated with unc-6 mutants. We used the muscle-specific fragment of the UNC-129 promoter (kindly provided by Joe Culotti) to misexpress UNC-6 in the dorsal muscle. In an unc-6 mutant background, where the only source of UNC-6 is the ectopic dorsal source, the HSN axons can be misdirected dorsally. This shows that axons can be redirected to grow toward an ectopic source of UNC-6 in vivo. The attractive migration towards UNC-6 requires the UNC-40 receptor. Little is known, however, about the mechanism by which UNC-6Us interaction with UNC-40 signals to the cyctoskeleton to elicit directed migration. Axon growth cones are normally exposed to many different signals directing their growth. In worms with ectopic dorsal UNC-6, we appear to have migrations directed solely by UNC-6. This strain can now be used to identify and study molecules required for presentation, reception, and transduction of the UNC-6 / UNC-40 signal.

Pandey, Amita et al. (2009) International Worm Meeting "UNC-53 antagonizes VAB-8 function in posterior migrations of neuronal cell bodies and growth cones."

Wolf, Fred W., Pandey, Amita, Garriga, Gian

[International Worm Meeting, 2009]

Nervous system development involves the migrations of neuronal cell bodies and their axons along the dorsal-ventral and anterior-posterior axes of C. elegans in response to various navigational cues. The kinesin-like protein VAB-8 is both necessary and sufficient for the posterior migrations of cells and neuronal growth cones. VAB-8 appears to act through the Rac GEF activity of UNC-73 to promote guidance receptor activity that guides these migrations toward the tail 1,2. While VAB-8 and UNC-73 promote the function of these receptors, we have identified a molecule, UNC-53, that appears to inhibit their function. UNC-53 is a conserved protein that has an actin binding domain, a SH3 binding domain, a putative microtubule binding domain and an AAA domain. UNC-53 antagonizes VAB-8 function in the ALM and CAN neurons. Ectopic expression of VAB-8 reverses the polarity of the ALM, causing it to extend its anterior process toward the posterior. A reduction in unc-53 function enhances the frequency of the ALM polarity reversals. Similarly, UNC-53 also suppresses the CAN migration defects of vab-8 partial loss-of-function but not null mutants. We found that the mutation identified originally as enu-1(ev419) (enhancer of Unc) by Joe Culotti is an allele of unc-53. Unlike other unc-53 alleles, the ev419 mutation did not cause an Unc phenotype. But like the other alleles, ev419 enhanced the ALM reversal phenotype and suppressed the CAN migration defects of vab-8 mutants, suggesting that this allele defines an UNC-53 function that specifically antagonizes VAB-8. The ev419 mutation affects RNA splicing of the unc-53 transcript and appears to generate a protein that is lacking 23 amino acids. unc-53 was identified in a genome-wide RNAi screen for genes involved in endocytosis of yolk protein by oocytes3. We also found that ev419 and other unc-53 mutants are defective in oocyte endocytosis, suggesting that UNC-53''s function in promoting endocytosis antagonizes VAB-8. 1Strumpf, N.C and Culotti, J.G. (2007) Nature Neurosci. 10: 161-168. 2Watari-Goshima, N et al. (2007) Nature Neurosci. 10: 169-176. 3Balklava, Z. et al. (2007) Nature Cell Biology 9: 1066-1073. A.

Boxem, Mike (2009) International Worm Meeting "Mapping the protein interaction network that controls cell polarity."

Boxem, Mike

[International Worm Meeting, 2009]

Interactions between proteins are a key component of most or all biological processes. A key challenge in biology is to generate comprehensive and accurate maps (interactomes) of all possible protein interactions in an organism. This will require iterative rounds of interaction mapping using complementary technologies, as well as technological improvements to the approaches used. For example, we recently developed a novel yeast two-hybrid approach that adds a new level of detail to interaction maps by defining interaction domains(1). Currently, I am working to generate an interaction map of proteins involved in controlling cell polarity in C. elegans to improve our understanding of the molecular mechanisms that establish and maintain cell polarity in multicellular organisms. I will combine two fundamentally different interaction mapping techniques: the yeast two-hybrid system (Y2H) and affinity purification/mass spectrometry (AP/MS). This will provide more detail by identifying both direct interactions between pairs of proteins by Y2H, and the composition of protein complexes by AP/MS. Moreover, interactions missed by one technology may be detected by the other, leading to a more complete interaction map. I will integrate the physical interactions with phenotypic characterizations. To this end I will systematically characterize the interaction network in vivo using two distinct models of polarity: asymmetric division of the one-cell embryo, and stem-cell-like divisions of a multicellular epithelium (in collaboration with M. Wildwater and S. van den Heuvel). M. Boxem, Z. Maliga, N. Klitgord, N. Li, I. Lemmens, M. Mana, L. de Lichtervelde, J. D. Mul, D. van de Peut, M. Devos, N. Simonis, M. A. Yildirim, M. Cokol, H. L. Kao, A. S. de Smet, H. Wang, A. L. Schlaitz, T. Hao, S. Milstein, C. Fan, M. Tipsword, K. Drew, M. Galli, K. Rhrissorrakrai, D. Drechsel, D. Koller, F. P. Roth, L. M. Iakoucheva, A. K. Dunker, R. Bonneau, K. C. Gunsalus, D. E. Hill, F. Piano, J. Tavernier, S. van den Heuvel, A. A. Hyman, and M. Vidal, A protein domain-based interactome network for C. elegans early embryogenesis. Cell, 2008. 134(3): p. 534-545. .