Vanneste, Christopher Allen et al. (2009) International Worm Meeting "Circumferential Actin and Elongation."

Mains, Paul, Vanneste, Christopher Allen

[International Worm Meeting, 2009]

Our work is focused on C. elegans embryonic elongation, which involves a smooth muscle like, actin-mediated contraction within the lateral epidermal (seam) cells. This contraction results in the embryo undergoing the four-fold lengthening into the vermiform worm shape without an increase in cell size or number. Our lab has previously shown that elongation involves the contractile cassette of let-502 (Rho-binding kinase), mel-11 (myosin phosphatase) and nmy-1/2 (non-muscle myosin heavy chains) (Piekny et al., 2003). MEL-11/myosin phosphatase inhibits elongation by dephosphorylating myosin while LET-502/Rho-binding kinase triggers elongation by inhibiting MEL-11 via sequestering it to the membrane. Priess and Hirsh (1986) showed that the cytoskeletal networks in the worm epidermis are organized differently in the lateral vs. the dorsal/ventral cells and work suggests that only the microfilaments in the lateral cells contract during elongation. However, the mechanism by which the highly organized actin networks are set up and how these differ in lateral vs. dorsal/ventral cells has yet to be elucidated. Here we describe FHOD- 1, a highly conserved Formin Homology Domain protein that may contribute to the spatial differences in the microfilament networks. Formins are actin nucleators, acting as progressive caps that form long unbranched actin filaments that are bundled into stress fibres. There are contradictory reports of human FHOD-1''s interaction with the small GTPase RAC and with Rho-binding Kinase (Gasteier et al., 2003. Westendorf et al., 1999 & 2001. Takeya et al. 2008). We found that fhod-1(RNAi) enhances the lethality of let-502 and suppresses that of mel-11, indicating that fhod-1(+) acts to induce elongation. We have generated antibodies to FHOD-1 and shown that it forms a filamentous pattern similar to that of actin. Significantly, FHOD-1 is only found in the lateral seam cells, consistent with the findings of Priess and Hirsh that the actin network is organized differently in the lateral cells compared to their neighbours. A deletion allele (tm2363) generated by the National Bioresource Project (which we believe is a complex rearrangement) shows defects in that actin network restricted to the seam cells. Currently we are trying to determine the fhod-1''s null phenotype and narrowing down FHOD-1''s timing of expression to see if it is the sole formin involved in organizing the seam cells actin fibres.

Allen AT et al. (2011) Genetics "Coexpressed D1- and D2-like dopamine receptors antagonistically modulate acetylcholine release in Caenorhabditis elegans."

Chase DL, Allen AT, Wani KA, Betts KE, Maher KN

[Genetics, 2011]

Dopamine acts through two classes of G protein-coupled receptor (D1-like and D2-like) to modulate neuron activity in the brain. While subtypes of D1- and D2-like receptors are coexpressed in many neurons of the mammalian brain, it is unclear how signaling by these coexpressed receptors interacts to modulate the activity of the neuron in which they are expressed. D1- and D2-like dopamine receptors are also coexpressed in the cholinergic ventral-cord motor neurons of Caenorhabditis elegans. To begin to understand how coexpressed dopamine receptors interact to modulate neuron activity, we performed a genetic screen in C. elegans and isolated mutants defective in dopamine response. These mutants were also defective in behaviors mediated by endogenous dopamine signaling, including basal slowing and swimming-induced paralysis. We used transgene rescue experiments to show that defects in these dopamine-specific behaviors were caused by abnormal signaling in the cholinergic motor neurons. To investigate the interaction between the D1- and D2-like receptors specifically in these cholinergic motor neurons, we measured the sensitivity of dopamine-signaling mutants and transgenic animals to the acetylcholinesterase inhibitor aldicarb. We found that D2 signaling inhibited acetylcholine release from the cholinergic motor neurons while D1 signaling stimulated release from these same cells. Thus, coexpressed D1- and D2-like dopamine receptors act antagonistically in vivo to modulate acetylcholine release from the cholinergic motor neurons of C. elegans.

Allen MA et al. (2011) Genome Res "A global analysis of C. elegans trans-splicing."

Hillier LW, Allen MA, Blumenthal T, Waterston RH

[Genome Res, 2011]

Trans-splicing of one of two short leader RNAs, SL1 or SL2, occurs at the 5' ends of pre-mRNAs of many C. elegans genes. We have exploited RNA-sequencing data from the modENCODE project to analyze the transcriptome of C. elegans for patterns of trans-splicing. Transcripts of 70% of genes are trans-spliced, similar to earlier estimates based on analysis of far fewer genes. The mRNAs of most trans-spliced genes are spliced to either SL1 or SL2, but most genes are not trans-spliced to both, indicating that SL1 and SL2 trans-splicing use different underlying mechanisms. SL2 trans-splicing occurs in order to separate the products of genes in operons genome wide. Shorter intercistronic distance is associated with greater use of SL2. Finally, increased use of SL1 trans-splicing to downstream operon genes can indicate the presence of an extra promoter in the intercistronic region, creating what has been termed a "hybrid" operon. Within hybrid operons the presence of the two promoters results in the use of the two SL classes: Transcription that originates at the promoter upstream of another gene creates a polycistronic pre-mRNA that receives SL2, whereas transcription that originates at the internal promoter creates transcripts that receive SL1. Overall, our data demonstrate that >17% of all C. elegans genes are in operons.

Eleanor Allen et al. (2004) East Coast Worm Meeting "Establishment of anteroposterior neuronal polarity"

Anastasia Bakoulis, Eleanor Allen, Scott Clark, Dave Hunt

[East Coast Worm Meeting, 2004]

Each neuron type in C. elegans projects axons with characteristic trajectories along the dorsoventral and/or anteroposterior body axes. Axons extend from the cell body at distinct orientations, revealing an intrinsic cellular polarity. Although evident in a mature neuron, when or how neuronal polarity is established is unclear. In addition, the processes that direct growth cone extensions along the anteroposterior body axis are poorly understood. To gain insight into the molecular mechanisms that determine neuronal polarity and anteroposterior axon guidance, we have begun screens for mutants with defects in the growth of the AVG axons. AVG is located in the retrovesicular ganglion and projects a very short anteriorly directed process and a long posteriorly directed process that extends to the tail. AVG pioneers the right ventral nerve bundle and is thought to provide cues that promote the proper assembly and organization of the ventral nerve cord. We isolated several mutations that alter AVG axonal outgrowth. zd101 caused the anteriorly directed AVG axon to over extend and enter the nerve ring; the posteriorly directed axon was unaffected. zd101 is an allele of klp-7 , which encodes a kinesin-related protein involved in the regulation of microtubules. We had found previously that klp-7 mutations cause neurons that normally extend only a single axon to project a second axon. Together these observations indicate that the regulation of microtubules can influence both the initiation and termination of axon extension. Several mutations were recovered that cause the apparent reversal of AVG anteroposterior polarity, as defined by the lengths of anterior and posterior axons. These mutants exhibited a range of three AVG axonal phenotypes: animals with a wildtype anterior axon and a posterior axon shorter than wild type, animals with anterior and posterior processes about equal in length and animals with a very long anterior axon and a very short or no posterior axon. When the axons were about equal in length, the anterior process terminated in the nerve ring and the posterior process stopped before the vulva. However, when the posterior axon was absent and the anterior axon was very long, the long axon typically entered the nerve ring and the looped back to the tail via the dorsal cord or lateral fascicle. These observations suggest that if the neuronal polarity of AVG is completely reversed, the long axon can be guided to the tail and that the AVG guidance cues are not restricted to the ventral nerve cord. Furthermore, these cues might only influence the direction of growth and not extension per se . We also found several genes that influence the anteroposterior polarity of other neurons, suggesting that multiple processes are involved in the establishment of anteroposterior neuronal polarity.

Allen E et al. (2015) Neuroscience "Sensory systems: their impact on C. elegans survival."

Zhang Y, Allen E, Alcedo J, Ren J

[Neuroscience, 2015]

An animal's survival strongly depends on a nervous system that can rapidly process and integrate the changing quality of its environment and promote the most appropriate physiological responses. This is amply demonstrated in the nematode worm Caenorhabditis elegans, where its sensory system has been shown to impact multiple physiological traits that range from behavior and developmental plasticity to longevity. Because of the accessibility of its nervous system and the number of tools available to study and manipulate its neural circuitry, C. elegans has thus become an important model organism in dissecting the mechanisms through which the nervous system promotes survival. Here we review our current understanding of how the C. elegans sensory system affects diverse physiological traits, whose coordination would be essential for survival under fluctuating environments. The knowledge we derive from the C. elegans studies should provide testable hypotheses in discovering similar mechanisms in higher animals.

T Allen et al. (1999) International C. elegans Meeting "Biological Roles of Troponin T"

T Allen, A Burkeen, L Hong, EA Bucher, J Ward

[International C. elegans Meeting, 1999]

The in vivo functions and interactions of TnT in muscle contraction are poorly understood. Thus, we are studying the four C. elegans TnT genes, which collectively give rise to at least eight distinct transcripts, by comparisons of their encoded proteins, their expression patterns, the individual and combined effects of TnT gene RNAi loss of function, and consequences of site-directed mutagenesis. The mup-2/TnT-1 gene codes for a single isoform expressed in embryonic/larval body wall muscles and in the hermaphrodite oviduct. RNAi of TnT-1 phenocopies the mup-2 null embryonic phenotype. The TnT-2 gene encodes the major larval and adult body wall isoform. The TnT-3 gene, which is expressed in early embryonic body wall muscle and in pharyngeal muscle and other minor muscle groups, generates at least four distinct transcripts. The TnT-4 gene, which is active in pharyngeal muscle, gives rise to two isoforms differing in the N-terminal region. RNAi of TnT-4 causes L1 arrest, consistent with a likely defect in pharyngeal function (starvation). Comparisons of C. elegans and vertebrate TnTs reveals two highly conserved regions. One is a cluster of charged residues located in the N-terminal region of TnT believed to interact in a calcium-independent manner with tropomyosin. Mutations near this charged cluster in human cardiac TnT are linked with familial hypertrophic cardiomyopathy. There is also a highly conserved heptad repeat of leucines in the C-terminal region, which is thought to allow interaction with troponin I and to be essential for TnT's role in regulating muscle contraction. Surprisingly, one of the TnT-3 isoforms replaces, through alternative RNA splicing, the heptad repeat with a sequence of about 70 amino acids having some similarity to filaggrin, which binds intermediate filaments, as well as to the Wolf-Hirschhorn Syndrome Critical Region 1 protein (WHSC1), which is expressed in rapidly growing embryonic tissues. The effects in vivo of site-directed mutations within the conserved charged cluster and the heptad repeat of TnT-1 are being examined, and our current data support that mutation of these domains causes dominant hypercontracted phenotypes, consistent with an essential role of these regions in inhibition of tension development.

Allen TS et al. (2000) East Coast Worm Meeting "In vivo Studies of Troponin T Functions."

Ward J, Burkeen A, Hong L, Allen TS, Bucher EA

[East Coast Worm Meeting, 2000]

The in vivo functions and interactions of TnT in muscle contraction are poorly understood. Thus, we are studying the four C. elegans TnT genes, which give rise to at least eight distinct transcripts, by comparing their encoded proteins, their expression patterns, their individual and combined RNAi phenotypes, and the effects of site-directed mutagenesis. The mup-2/TnT-1 gene encodes a single isoform expressed in embryonic and larval body wall muscles, and in the hermaphrodite oviduct. RNAi of TnT-1 phenocopies the mup-2 null embryonic Mup phenotype. The TnT-2 gene encodes the major larval and adult body wall isoform. RNAi of TnT-2 causes an uncoordinated phenotype and larval arrest. The TnT-3 gene, which is expressed in early embryonic body wall muscle and in pharyngeal muscle and other minor muscle groups, generates at least four distinct transcripts. No phenotypes have yet been associated with RNAi of TnT-3. The TnT-4 gene, which is expressed in pharyngeal muscle, gives rise to two isoforms differing in the N-terminal region. RNAi of TnT-4 causes L1 arrest, consistent with a likely defect in pharyngeal function (starvation). We are examining the TnT-2 and TnT-4 phenotypes further, and undertaking RNAi of TnT loci in combination. Comparisons of C. elegans and vertebrate TnTs reveal a highly conserved cluster of charged residues located in the N-terminal region of TnT believed to interact in a calcium-independent manner with tropomyosin. Mutations in human cardiac TnT near this cluster are linked with familial hypertrophic cardiomyopathy. There is also a highly conserved heptad repeat of leucines in the C-terminal region, which is thought to allow interaction with troponin I and to be essential for TnTs role in regulating muscle contraction. One of the TnT-3 isoforms replaces, the heptad repeat with a sequence of about 70 amino acids having some similarity to filaggrin, which binds intermediate filaments, as well as to the Wolf-Hirschhorn Syndrome Critical Region 1 protein (WHSC1), which is expressed in rapidly growing embryonic tissues. The effects in vivo of site-directed mutations of these domains appear to cause dominant hypercontracted phenotypes suggesting essential roles in acto-myosin inhibition.

Allen JE et al. (1998) Parasite Immunol "Profound suppression of cellular proliferation mediated by the secretions of nematodes."

MacDonald AS, Allen JE

[Parasite Immunol, 1998]

Loss of T lymphocyte proliferation and the emergence of a host response that is dominated by a Th2-type profile are well-established features of human filarial infection. Down-regulation and modulation of host T cell responses during lymphatic filariasis has been investigated by implantation of parasite stages into inbred mice. Adherent peritoneal exudate cells (PEC) from mice transplanted with adult or larval Brugia malayi parasites are profoundly anti-proliferative but do not prevent antigen-specific cytokine production by T cells. We demonstrate here that the excretory/secretory (E/S) products of the adult parasite are sufficient to induce PEC that block proliferation if injected daily into mice. We have previously shown that in vivo production of host IL-4 is required for the generation of suppressive cells. Because the induction of host IL-4 is characteristic of infection with nematodes, we asked whether E/S from two other nematode parasites, Nippostrongylus braziliensis and Toxocara canis were also capable of generating a suppressor cell population. Injection of E/S from these two parasites also led to a reduction in T cell proliferation suggesting that this mechanism of down-regulating host responses is a feature common to nematode parasites.

Allen TS et al. (1995) International C. elegans Meeting "Analyses of Troponin T Mutations in C. elegans."

Myers CD, Goh P-Y, Bogaert T, Bucher EA, Allen TS

[International C. elegans Meeting, 1995]

To elaborate the mechanisms by which troponin T (TnT) functions in the assembly and contraction of developing and mature muscle, we are studying a TnT homolog in C. elegans that is encoded by the mup-2 gene locus. mup-2 is defined by a heat sensitive allele, designated e2346ts, and by an unconditional allele, up1, believed null. The e2346ts mutation creates a premature stop at codon 342, eliminating 64 residues. The up1 mutation creates a premature stop at codon 94, preventing expression of the regions homologous to the functional domains of vertebrate TnT. Both alleles cause muscle positioning defects (Mup) during embryogenesis and result in kinked, dead larvae. Because mutants for these alleles can show muscular contraction prior to death, the embryonic phenotype is distinct from the paralysed, arrested elongation at two-fold (Pat) phenotype shown by worms with mutations in genes identified tentatively as encoding troponin C and tropomyosin (Williams and Waterston. J. Cell Biol. 124:475, 1994), two proteins that interact with TnT. Our working hypothesis is that the putative tropomyosin and troponin C mutations prevent activation (disinhibition) of thin filaments and thus block muscular contraction. Conversely, the mutations in TnT impair inhibition (thin filaments are "unregulated") and thus allow aberrant force production, causing tearing of the dorsal muscle quadrants ventrally. mup-2 also has larval and adult functions. When e2346ts mutants are shifted from 15 C to 25 C after embryogenesis, the muscle quadrants remain properly positioned, but the worms tremble when placed in liquid solution. The trembling suggests dysfunctional contraction/relaxation cycles, in line with our working hypothesis. Additionally, the temperature-shifted hermaphrodites are sterile: oocytes accumulate in the oviduct, presumably because contraction of the oviductal myogenic sheath is defective. Our analysis also shows defects in migration of the gonadal sheath cells to the distal gonadal arms. A function of TnT in cell migration and in nonstriated tissue contraction is surprising, because vertebrate TnT is found exclusively in striated muscle; however, in situ hybridization studies support that mup-2 is expressed in these cells. Genetic interaction studies, site-directed mutagenesis, and physiological assays that we are developing will contribute towards defining the in vivo functions of TnT.

Allen TS et al. (1996) East Coast Worm Meeting "TROPONIN T DIVERSITY AND FUNCTION IN C. ELEGANS"

Myers CD, McArdle K, Bucher EA, Allen TS

[East Coast Worm Meeting, 1996]

Troponin T (TnT), in combination with tropomyosin, troponin I, and troponin C, confers calcium-sensitivity upon force production in striated muscle. The pathway by which TnT relays the calcium-signal to actin and myosin is unknown; however, the role of TnT in the relay is believed pivotal for several reasons. First, vertebrate striated muscle cells can express more than 100 isoforms of TnT through alternative pre- mRNA splicing. Second, the particular isoforms expressed in vertebrate muscle cells change during development and disease. Third, mutations in TnT are linked to muscle dysgenesis in Drosophila and to cardiomyopathy and death in humans. To understand the biological roles of TnT in developing and mature striated muscle, we are studying the diversity of TnT isoforms in C. elegans, as well as the physiological importance of individual isoforms. Three loci code for TnT in C. elegans, one defined genetically (mup-2, which codes for TnT-1), and two uncovered by the genome sequencing project (TnT-2, on cosmid F53A9, and TnT-3, on C14F5). The predicted products of all three genes have the hallmarks of TnT: extremely high conservation of residues both at the amino-terminus (8 aa, encoded by the first exon in all three genes) and within a leucine- zipper motif and its flanking region. Unlike vertebrate TnT, C. elegans TnT isoforms have a long (100-150 aa) carboxyl-terminal tail after the leucine zipper motif and its flanking region. Differences among products of the three genes are evident. TnT-3 mRNA is found in pharyngeal muscle, and TnT-1 and TnT-2 mRNAs are detected in body wall muscle. The TnT-3 transcript, unlike that of TnT-1 and TnT-2, can be alternatively spliced to generate multiple mRNAs, and three splicing patterns for TnT-3 mRNA have been detected by PCR. The alternative splicing occurs among exons coding for the carboxyl-terminal tail. TnT- 1 is distinguishable from TnT-2 primarily through the reduced length of TnT-1 (405 vs 428 residues; 12 of the 23 "deletions" occur in the carboxyl-terminal tail, and the other 11 occur adjacent to the conserved amino-terminus). Given the differences in the carboxyl-terminal tail regions of the TnT isoforms, it is interesting that the first recovered mutant allele of mup-2, e2346ts, arose from a premature stop at codon 341, eliminating the final 64 residues of the tail. The e2346ts mutation at 25oC produces a phenotype (muscle positioning defects during embryogenesis and late-embryonic/early-larval lethality) identical to that of a second allele, up1, which is a premature stop at codon 94 and is believed null. Our working hypothesis is that truncation of the carboxyl-terminal tail, as well as elimination of the entire protein, prevents the tropomyosin- troponin complex from stopping force production in a timely manner following a calcium-induced contraction. Thus, the tearing of the dorsal muscle quadrants toward the ventral surface in mutant worms is envisaged as a result of prolonged, aberrent muscle constraction. To test the working hypothesis, we examined whether myo-3 (st386) and pat-5 (st556) are epistatic to mup-2 (e2346ts). myo-3 (st386) eliminates myosin-containing thick filaments in body wall muscle. pat-5 (st556) is proposed to be a mutation in a voltage-gated calcium-channel of muscle (R. Lee and L. Avery, 1995 C. elegans Meeting Abstracts, p. 72); this mutation renders embryonic body wall muscle paralyzed, presumably by eliminating or diminishing calcium-influx needed for triggering contraction. Both mutations are epistatic to mup-2 (e2346ts), suggesting that manifestation of the mup phenotype in e2346ts worms requires muscle contraction, in accord with the working hypothesis.