Hung M-S et al. (1995) International C. elegans Meeting "Sequence of vab-8, a gene involved in anterior-posterior guidance"

Garriga G, Way JC, Wolf FW, Hung M-S, Wightman BC

[International C. elegans Meeting, 1995]

The vab-8 gene is necessary for cell and axon migrations in the anterior-posterior direction. vab-8 mutant animals have a withered tail (the Wit phenotype), resulting from a failure of the CAN neuron migration, and are uncoordinated as a result of axon outgrowth/guidance defects. We have cloned the vab-8 gene and are determining its sequence. vab-8 lies on the cosmid C35G11 (Chr.V) adjacent to myo-3. The sequence of some incomplete cDNAs and the adjacent genomic sequence suggest that the vab-8 product might be secreted and processed: the predicted protein sequence contains many adjacent pairs of basic amino acids that could be targets of a Kex2- type protease (WBG v. 13, #5, p. 53). According to this idea, the vab-8 product might be a secreted signal directing anterior-posterior guidance events. This model needs to be confirmed (e.g. by mosaic analysis). a ~7kb DNA fragment is sufficient to rescue the Wit phenotype of vab-8 mutants, but a much larger fragment is required to rescue the Unc phenotype. Several transcripts derive from this region, which may encode distinct but related proteins that could function in axon guidance and cell migration (see abstract by Wolf et al.).

Hung M-S et al. (1996) East Coast Worm Meeting "The C. elegans vab-8 gene encodes two kinesin-related proteins necessary for axon outgrowth and cell migration"

Hung M-S, Garriga G, Wightman BC, Wolf FW, Way JC

[East Coast Worm Meeting, 1996]

The vab-8 gene of is necessary for posterior migrations of several cells and axons in C. elegans. One class of vab-8 mutants show defects in the posterior migration of the CAN neuron and in the posterior extension of the AVA, AVB, AVD, and AVK axons, while a second class of mutants are defective only in the extension of these axons (1). We have found that the sequence of genomic and cDNA clones predicts that vab-8 encodes two proteins of 1066 and 541 amino acids. The larger protein is distantly related to kinesin: it has a kinesin-like motor domain at its N-terminus, a central region with a predicted coiled-coil structure, and a globular C-terminus. The mRNA for the smaller protein is initiated at a distinct exon within an intron of the larger transcript. This smaller protein thus has a unique 29 amino acid N- terminus, but the remaining C-terminal 513 amino acids are identical between the two proteins. A DNA fragment encoding both proteins can rescue all the phenotypes of vab-8 mutants, and a smaller fragment encoding the 541 amino acid protein can rescue the migration defect of the CAN cell but not the axon guidance defect. As inferred from lacZ fusions, the vab-8 gene is expressed in the AVK and CAN neurons, and also in many other cell types whose function is not noticeably impaired in vab-8 mutants, including body, pharynx, and vulval muscles; the developing vulva and spermatheca-uterine valve; and in the excretory cell. Fusions corresponding to the small transcript are expressed in the CAN neuron, while fusions corresponding to the large product are expressed in AVK but not CAN; these results are consistent with cell- autonomous function. Analysis of genetic mosaics indicates that wild-type vab-8 product must be expressed in the CAN cells for them to migrate correctly. These results, together with previous phenotypic analyses, suggest that vab-8 encodes a novel motor protein that functions in orientation of cells as a prelude to cell or axon migration. Wightman et al. (1996). The C. elegans gene vab-8 guides posteriorly directed axon outgrowth and cell migration. Development 122, 671-682.

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. .

H Mimi Zhou et al. (2001) International C. elegans Meeting "Direct Visualization of the Movement of Motor Proteins and their Cargo within Neurons of Living C. elegans"

Ingrid Brust-Mascher, Jeff Eastham, H Mimi Zhou, Jonathan M Scholey

[International C. elegans Meeting, 2001]

The neuron is a highly polarized cell that elaborates two processes, a dendrite that is specialized for neuronal signal reception and an axon that is specialized for signal conduction and transmission. The formation and function of these processes depends upon the microtubule-based transport of cellular components from their sites of synthesis in the neuronal cell body to their sites of utilization at the terminus. We have developed a time-lapse fluorescence microscopic assay that allows us to visualize the movement of MT-based motor proteins and their cargo along neurites within living C. elegans (Orozco et al., 1999, Nature, 398 (6729):674). We are using this assay to dissect the kinesin-II / IFT raft transport pathway that contributes to the formation and function of chemosensory neurons and sensory cilia (Signor et al., 1999, JCB 147:519), and the monomeric UNC-104 synaptic vesicle transport pathway that is required for proper synaptic transmission (Zhou et al., 2001, J. Neurosci., in press). Using time-lapse fluorescence microscopy we have visualized fluorescent kinesin-II motor (KAP-1) and raft proteins (OSM-1, OSM-6 and CHE-2) moving along dendrites and sensory cilia at 0.7 m m/s in the anterograde direction and 1.1 m m/s in the retrograde direction. Retrograde ciliary transport of these proteins is abolished in the absence of CHE-3 dynein, leading to a plausible model for bi-directional transport in chemosensory neurons. UNC-104 motor proteins move in both directions along neuronal processes, some of which were definitely identified as axons and others as dendrites, suggesting a role for this pathway in synaptic transmission at axo-dendritic and dendro-dendritic synapses. Using kymograph analysis, we observed that UNC-104::gfp particles moved bidirectionally at an average velocity of 1.0 m m/s, which is close to the in vitro velocity of microtubule gliding driven by purified monomeric kinesin at high motor density. These data are consistent with the hypothesis that multiple UNC-104 motors move synaptic vesicles along axons and dendrites at 1.0 m m/s, and that these motors are recycled by a retrograde transport system that coincidentally moves at the same velocity.

Wang, Juan et al. (2017) International Worm Meeting "Biogenesis and Function of Social Extracellular Vesicles (EVs)."

Barr, Maureen, Wang, Juan, Silva, Malan

[International Worm Meeting, 2017]

Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.

Wolf FW et al. (1996) West Coast Worm Meeting "VAB-8 IS A UNIQUE KINESIN-RELATED PROTEIN THAT FUNCTIONS CELL AUTONOMOUSLY IN POSTERIORLY MIGRATING CELLS AND GROWTH CONES"

Way JC, Garriga G, Wightman BC, Wolf FW, Hung M-S

[West Coast Worm Meeting, 1996]

vab-8 is required for most posteriorly directed cell and axon growth cone migrations (Wightman et al., Dev. 122:671(1996)). We cloned vab-8 and found that it encodes a highly divergent kinesin-related protein. The N-terminal 330 amino acids of VAB-8 contains kinesin motor domain signature sequences. Moreover, the putative VAB-8 motor domain is predicted to have a nearly identical secondary structure to that of the recently crystallized human kinesin motor domain (Kull et al. Nature 380:550(1996)). Additionally, both kinesins and VAB-8 have long divergent C-terminal sequences with regions that can form coiled-coil dimerization domains. These similarities suggest that vab-8 may function as a microtubule binding motor protein. However, VAB-8 is currently the most divergent kinesin-like molecule known. vab-8 encodes two activities that can be defined by the effects vab-8 mutations have on AVK axon outgrowth and CAN cell migration. Strong vab-8 alleles affect both AVK outgrowth and CAN migration, whereas Unc vab-8 alleles affect AVK outgrowth but not CAN migration. Molecularly vab-8 encodes at least two proteins: a larger protein (VAB-8L) that includes the putative motor domain and a smaller protein (VAB-8S) that shares C-terminal sequences with VAB-8L but lacks the motor domain. Sequencing of vab-8 alleles showed that strong alleles are frameshift and nonsense mutations that prematurely terminate both vab-8 proteins, and that the Unc allele ev411 is a splice donor site mutation that specifically disrupts VAB-8L. Furthermore, DNA clones encoding VAB-8S rescued the CAN migration but not AVK outgrowth defects of a strong vab-8 mutant. Taken together, these results show that VAB-8L, but not VAB-8S, is necessary for AVK axon outgrowth, and that VAB-8S is sufficient for CAN cell migration. By mosaic analysis we determined that vab-8 acts cell autonomously for CAN cell migration. Losses of an extrachromosomal array carrying the wild-type vab-8 gene in the CAN cell lineage but not other lineages in a strong vab-8 mutant background caused CAN cell migration defects. We propose that vab-8 functions in the interpretation of a cue that directs cells and growth cones posteriorly. Antibodies raised against the protein region common to VAB-8L and VAB-8S stained body wall muscle cells of wild-type but not vab-8 mutant animals. This staining was associated with dense body muscle attachment sites and was graded, with the strongest staining in the anterior of each cell. Because we were unable to detect vab-8 immunoreactivity in cells in which vab-8 is known to function, we expect that this muscle staining pattern represents a subset of the vab-8 expression pattern. We cannot detect any muscle defects in vab-8 mutants. Focal adhesions, which contain many of the molecular components found in dense bodies, play important roles in cell and axon growth cone migration. Thus the muscle expression pattern of vab-8 may serve as a model for understanding the role of vab-8 in posteriorly directed guidance.

van Weeghel R et al. (1993) International C. elegans Meeting "A cdc-42 homolog in C. elegans."

Culotti JG, Hung M-S, van Weeghel R, Way JC

[International C. elegans Meeting, 1993]

Pinan-Lucarre, Berangere et al. (2009) International Worm Meeting "Transgenic C. elegans expressing the fungal prion protein HET-s as a model for the study of amyloid infectivity and toxicity."

Qiao, Yujie, Saupe, Sven, Pinan-Lucarre, Berangere, Driscoll, Monica

[International Worm Meeting, 2009]

Amyloids are protein fibers rich in beta sheet structure that are associated with numerous neurodegenerative diseases, including Alzheimer disease. Amyloids have two major biological properties. First, they can be infectious--meaning that they can spread the altered amyloid conformation to the same protein or even among proteins of distinct primary sequence (the latter phenomenon is known as cross-polymerization). Infectious amyloids are called prions. Second, amyloids can be toxic, whether they are infectious or not. Both properties have tremendous fundamental and biomedical impact. [Het-s] is a prion of the fungus Podospora anserina involved in a defense cell suicide mechanism named heterokaryon incompatibility, which disables mixing of different strains by somatic fusion, an ubiquitous feature in filamentous fungi. The HET-s protein exists in a soluble state or in an aggregated, prion state named [Het-s]. The [Het-s] prion is not toxic by itself in P. anserina or in yeast. However cell death is rapidly triggered by the lethal interaction between 2 alleles of the het-s locus: het-S (het-"large S") and het-s (het-"small s") when the HET-s protein adopts the prion form. This prion has been extensively characterized and it is the only prion for which tridimensional structure is available to date. The critical point is that in the [Het-s] system, transgenic expression of het-s allows propagating a non-toxic prion while co-expression of het-s and het-S generates toxicity. We constructed strains expressing het-s or the prion domain only het-s(218-289) (the 218-289 fragment of the protein) as GFP fusions under the control of the ubiquitous promoter dpy-30. The fluorescence of pdpy-30het-s::gfp appears to be mainly diffuse in the cytoplasm while it shows strong aggregation in pdpy-30het-s(218-289)::gfp strains. We constructed strains expressing het-s(218-289)::gfp in the touch neurons using mec-4 promoter and observed that the fusion protein is mostly aggregated into a large fiber spanning the cell body and the proximal part of the axon. These aggregates remain to be shown as infectious [Het-s] amyloids. In future experiments, we will aim to generate neuronal toxicity through the co-expression of het-s(218-289) and het-S. This C. elegans model for prion studies, filling a gap between simple fungal systems and highly complex mammalian systems, might allow a better understanding of the molecular and cellular basis of amyloid infectivity and toxicity.

Dustin L Updike et al. (2005) International Worm Meeting "The TIP60/SWR1 Chromatin Remodeling Complex Functions with the Selector Gene PHA-4 to Endow Cells with Pharyngeal Competence"

Dustin L Updike, Susan E Mango

[International Worm Meeting, 2005]

A critical question in developmental biology is how complex programs of gene expression are orchestrated by a class of regulators known as selector genes. Selector genes code for transcription factors that autonomously govern the fates of groups of cells related to each other by virtue of their cell type, position or affiliation to an organ (1). For example, the FoxA transcription factor PHA-4 dictates the identity of cells within the C. elegans pharynx. Embryos that lack pha-4 fail to generate pharyngeal cells, and these cells acquire an alternative ectodermal fate instead (2,3). PHA-4 functions in combination with additional transcription factors to modulate genes at distinct stages and in different cell types during pharyngeal development (4,5). Here we explore the role of PHA-4 during transcriptional regulation. We identified components of the TIP60/SWR1 complex in a screen for loci that interact genetically with PHA-4. The predicted histone acetyltransferase mys-1 and chromatin remodeling factor ssl-1 each enhanced partial loss of pha-4 activity. In other organisms, the SSL-1 complex exchanges nucleosomes with histone H2A for those containing the variant H2Az (6). Nucleosomes containing H2Az appear less stable than those carrying H2A, which may facilitate the subsequent removal of nucleosomes for activation or repression (7). Consistent with this idea, C. elegans H2Az enhances the pharyngeal phenotype of pha-4 alleles, similar to ssl-1. Furthermore, we observe H2Az associated with PHA-4 target promoters in nascent pharyngeal cells. We propose that the MYS-1/SSL-1 chromatin remodeling complex functions with PHA-4 to endow cells with pharyngeal competence by restructuring the chromatin environment around PHA-4 target genes. Chromatin restructuring may render these promoters susceptible to binding by additional transcription factors that modulate pharyngeal gene expression in conjunction with PHA-4.1) R. S. Mann, S. B. Carroll, Curr. Opin. Genet. Dev. 12, 592 (2002). 2) S. E. Mango, E. J. Lambie, J. Kimble, Development 120, 3019 (1994). 3) M. A. Horner et al., Genes Dev. 12, 1947 (1998). 4) J. Gaudet, S. Muttumu, M. Horner, S. E. Mango, PLoS Biol. 2, e352 (2004). 5) W. Ao, J. Gaudet, W. J. Kent, S. Muttumu, S. E. Mango, Science 305, 1743 (2004). 6) G. Mizuguchi, X. Shen, J. Landry, W. H. Wu, S. Sen, C. Wu, Science 303, 343 (2004). 7) D. W. Abbott, V. S. Ivanova, X Wang, W. M. Bonner, J. Ausio, J. Biol. Chem. 276, 945 (2001).

J Liu et al. (1999) International C. elegans Meeting "Hox factors and other components in patterning of embryonic and postembryonic myogenic lineages"

J Liu, AZ Fire

[International C. elegans Meeting, 1999]

Bodywall muscles are derived from four of the embryonic founder cells as well as the postembryonic M blast cell. We are studying patterning of both the embryonic and postembryonic muscles in order to understand how myogenic cell fates are specified during development. As an entry point to study muscle patterning during embryonic development, we characterized enhancer elements from the hlh-1 regulatory region that are able to drive reporter expression in muscle precursors of the MS, D and C lineages. Analyses of these elements suggested regulatory roles of the Hox genes and the hlh-1 gene itself. These analyses also suggested regulatory mechanisms involving unknown bZIP, bHLH and novel transcription factors. We are currently in the process of genetically identifying these factors. The postembryonic M lineage provides an alternative model to study myogenic fate specification. The M lineage gives rise to 14 bodywall muscles, 2 coelomocytes and 16 sex muscles. A number of genes had previously been identified to function in patterning the M lineage, including the Hox gene mab-5 and the bHLH transcription factor twist (1, 2, 3). The role of the Hox factors in patterning the M lineage has been something of a mystery: mab-5 is expressed during the entire M lineage, but mab-5 mutants cause only limited lineage transformation. We will describe a series of experiments indicating a more central role for the Hox genes in activating twist and specifying the M lineage. We found that lin-39 mab-5 double mutants fail to activate twist and completely lack products of the M lineage. Expression (either ectopic or in a normal pattern) of either Hox gene is sufficient to activate twist expression. However, twist activation is not sufficient for specification of the M lineage. Current efforts are directed towards identification of other factors involved in specifying the M lineage. 1. Kenyon, C. (1986), Cell 46: 477-487 2. Harfe B. D., Vaz Gomes A., Kenyon C., Liu J., Krause M., Fire A. (1998) Genes & Dev. 12: 2623-2635 3. A. Corsi, S. Kostas, E. Jorgensen, A. Fire, and M. Krause (poster)