King DC et al. (2000) European Worm Meeting "Genetic analysis of polyglutamine-mediated cellular dysfunction in C. elegans"

Hart AC, King DC, Faber PW

[European Worm Meeting, 2000]

Huntington's Disease (HD) is one of 8 distinct human neurodegenerative disorders caused by expansion of an uninterrupted polyglutamine (polyQ) segment in the corresponding protein. The fact that the expanded polyQ segment is the only shared feature between these 8 proteins directly implicates the polyQ segment in neurotoxicity. In general, more than 40 consecutive glutamine residues in these proteins lead to the disease phenotype whereas fewer than 40 will not cause neurodegeneration. All 8 diseases are inherited in a genetically dominant manner and show an age of onset that is dependent on the length of the polyQ segment. In addition, each disease leads to a progressive deterioration towards death over 10-20 years. Despite the common features of these neurodegenerative disorders the precise molecular nature of polyQ toxicity is unknown. We are using the well characterized C. elegans nervous system to gain insight in the cellular mechanism of polyQ neurotoxicity. Previously we described a C. elegansmodel for polyQ-mediated cellular dysfunction utilizing small N-terminal fragments of the HD protein huntingtin (Htn) expressed in the ASH sensory neuron (PNAS 96, 179-184, 1999). These fragments contain polyQ stretches of 2, ", 95 or 150 residues. Expression of Htn-Q150, but not Htn-Q2, Htn-Q23 or Htn-Q95, caused age dependent ASH degeneration without ASH cell death in 8-day-old animals (as measured in dye-filling assays and immunohistochemical experiments). Despite the absence of cell death this degeneration was partly dependent on ced-3 caspase function suggesting the involvement of the apoptotic cell death pathway in Htn-Q150 mediated neurodegeneration. To identify genes that normally function to counteract polyQ toxicity we performed an F2 EMS screen for mutations that lead to enhanced polyQ toxicity in the ASH neuron without compromising ASH survival/function in the absence of expanded polyQ segments. We anticipate that such mutations will define genes involved in turn-over and stability of Htn-Q150 or form part of the unknown cellular pathway(s) perturbed by expanded polyQ segments. We retained mutant strains that showed a >30% Htn-Q150 mediated ASH degeneration in 3 day old animals (as compared to <1% degeneration in the parental strain in 3 day old animals). After screening 30,000 mutagenized animals 7 independent mutations have been identified (rt13, rt58, rt61, rt62, rt64, rt65 and rt67). In rt13 mutant animals expressing Htn-Q150 about 80% of the ASH neurons were absent in 3 day old animals. In the absence of Htn-Q150 expression no ASH cell death was observed in these animals even at 8 days of age. In addition, rt13 mutant animals are wild-type in two ASH mediated behavioral assays (nose-touch and osmotic avoidance). These results indicate that rt13 does not generally impair ASH survival and function. Importantly, neither expression of Htn-Q2 nor a toxic control protein devoid of polyglutamines induced ASH cell death in 3 or 8 day old rt13 animals. The Htn-Q150 mediated cell death in these animals is partly dependent on ced-3 caspase function, again implicating the apoptotic cell death pathway in this process. rt13 has been mapped to a small chromosomal interval that lacks obvious candidate genes. Phenotypic and complementation analysis of the 6 remaining mutations is under way. Identification of the molecular nature of rt13 and related mutations might provide important insights in the molecular nature of polyQ pathogenesis and lead to novel therapeutic approaches for these neurodegenerative disorders.

RC Chan et al. (1999) International C. elegans Meeting "Immunoaffinity purification of the C. elegans dosage compensation complex"

RC Chan, M Albrecht, C Tsai, BJ Meyer

[International C. elegans Meeting, 1999]

Dosage compensation (DC) in C. elegans equalizes X expression between males (XO) and hermaphrodites (XX) by reducing transcription from both hermaphrodite X chromosomes. A large protein complex specifically localizes to the X chromosomes of XX animals to implement DC. Members of this DC complex include the SMC (structural maintenance of chromosome) proteins DPY-27 and MIX-1, and a non-SMC protein DPY-26. The latter two proteins also function in chromosome segregation during mitosis or meiosis, demonstrating the recruitment of chromosome segregation proteins to the regulation of gene expression. A second class of genes ( sdc ) that coordinately controls sex-determination and DC is required for the X localization of the DC complex. To gain an in-depth view of the interactions among these various DC proteins and to identify other DC proteins, we developed an immunoaffinity purification scheme to enrich for the DC proteins localized on DNA. To characterize the DC complex, peptide antibodies were raised against the carboxyl terminal (c-ter) sequences of DPY-26, MIX-1 and DPY-27, and were used to precipitate the complex from crude embryonic extracts. The benefit of this approach is that the precipitate can be eluted with c-ter peptides under non-denaturing conditions to allow further fractionation and biochemical analysis. Our analysis provided the following findings: (1) The product of the genetically characterized DC gene dpy-28 was shown to be a member of the complex. Besides DC, DPY-28 also functions in meiosis (see Chan et al.), further reinforcing the model that DC evolved by recruiting factors essential to chromosome segregation. The addition of DPY-28 to the DC complex revealed a striking overall similarity between the DC complex and the Xenopus 13S condensin, required for mitotic chromosome condensation in vitro . This result suggests that DC may be mechanistically similar to mitotic chromosome compaction. (2) The SDC-2 and SDC-3 proteins were also present in the precipitate, suggesting that SDC proteins directly recruit the DC complex to X (see Dawes et al.) (3) Two other proteins of 165- and 80-kD were found. Work is in progress to identify these two proteins and to further purify the DC complex for biochemical analysis.

Tiraihi A et al. (2007) Biosystems "Early onset of regionalization in EMS lineage of C. elegans embryo: a quantitative study."

Tiraihi A, Tiraihi T

[Biosystems, 2007]

Early localization of C. elegans founder cell descendents in certain regions of embryo has been documented. The purpose of this investigation is to evaluate the onset of ABp and EMS descendent cell regionalization in the embryo using the random motility coefficient as a quantitative parameter. The forward migration index (FMI) was also calculated in order to evaluate the chemotatic biases of ABp-dc and EMS-dc during regionalization. The results showed that the random motility coefficient declined as the cells tended to regionalize. The mean squared displacement (MSD) versus time plot showed a non-linear model which indicated non-random cell movement. FMI showed progressive increase as the cells tended to regionalized, and it was significantly higher in EMS-dc than ABp-dc, moreover the chemotatic biases were higher in EMS-dc than ABp-dc. The circular plots showed that the statistical differences between the two lineages were significant, while ABp-dc showed significant differences in xy, xz and yz planes; EMS derived cells showed no significant differences except in yz planes. The conclusion of this study is that the onset of early regionalization occurs in EMS-dc sooner than in ABp-dc.

Zhong F et al. (2011) Genes Dev "Disruption of telomerase trafficking by TCAB1 mutation causes dyskeratosis congenita."

Alter BP, Artandi SE, Shkreli M, Jessop L, Zhong F, Giri N, Savage SA, Chen R, Myers T

[Genes Dev, 2011]

Dyskeratosis congenita (DC) is a genetic disorder of defective tissue maintenance and cancer predisposition caused by short telomeres and impaired stem cell function. Telomerase mutations are thought to precipitate DC by reducing either the catalytic activity or the overall levels of the telomerase complex. However, the underlying genetic mutations and the mechanisms of telomere shortening remain unknown for as many as 50% of DC patients, who lack mutations in genes controlling telomere homeostasis. Here, we show that disruption of telomerase trafficking accounts for unknown cases of DC. We identify DC patients with missense mutations in TCAB1, a telomerase holoenzyme protein that facilitates trafficking of telomerase to Cajal bodies. Compound heterozygous mutations in TCAB1 disrupt telomerase localization to Cajal bodies, resulting in misdirection of telomerase RNA to nucleoli, which prevents telomerase from elongating telomeres. Our findings establish telomerase mislocalization as a novel cause of DC, and suggest that telomerase trafficking defects may contribute more broadly to the pathogenesis of telomere-related disease.

Sharma R et al. (2014) Genes Dev "Differential spatial and structural organization of the X chromosome underlies dosage compensation in C. elegans."

Kind J, Vaillant C, Meister P, Jost D, Askjaer P, Sharma R, Gomez-Saldivar G, van Steensel B

[Genes Dev, 2014]

The adjustment of X-linked gene expression to the X chromosome copy number (dosage compensation [DC]) has been widely studied as a model of chromosome-wide gene regulation. In Caenorhabditis elegans, DC is achieved by twofold down-regulation of gene expression from both Xs in hermaphrodites. We show that in males, the single X chromosome interacts with nuclear pore proteins, while in hermaphrodites, the DC complex (DCC) impairs this interaction and alters X localization. Our results put forward a structural model of DC in which X-specific sequences locate the X chromosome in transcriptionally active domains in males, while the DCC prevents this in hermaphrodites.

Rezai P et al. (2011) Biomicrofluidics "Effect of pulse direct current signals on electrotactic movement of nematodes Caenorhabditis elegans and Caenorhabditis briggsae."

Selvaganapathy PR, Gupta BP, Salam S, Rezai P

[Biomicrofluidics, 2011]

The nematodes (worms) Caenorhabditiselegans and Caenorhabditisbriggsae are well-known model organisms to study the basis of animal development and behaviour. Their sinusoidal pattern of movement is highly stereotypic and serves as a tool to monitor defects in neurons and muscles that control movement. Until recently, a simple yet robust method to initiate movement response on-demand did not exist. We have found that the electrical stimulation in a microfluidic channel, using constant DC electric field, induces movement (termed electrotaxis) that is instantaneous, precise, sensitive, and fully penetrant. We have further characterized this behaviour and, in this paper, demonstrate that electrotaxis can also be induced using a pulse DC electric signal. Worms responded to pulse DC signals with as low as 30% duty cycle by moving towards the negative electrode at the same speed as constant DC fields (average speed of C. elegans=296+/-43m/s and C. briggsae=356+/-20m/s, for both constant and pulse DC electric fields with various frequencies). C. briggsae was found to be more sensitive to electric signals compared to C. elegans. We also investigated the turning response of worms to a change in the direction of constant and pulse DC signals. The response for constant DC signal was found to be instantaneous and similar for most worms. However, in the case of pulse DC signal, alterations in duty cycle affected the turning response time as well as the number of responding worms. Our findings show that pulse DC method allows quantitative measurement of response behaviour of worms and suggest that it could be used as a tool to study the neuronal basis of such a behaviour that is not observed under constant DC conditions.

Semnani RT et al. (2008) J Immunol "Induction of TRAIL- and TNF-alpha-dependent apoptosis in human monocyte-derived dendritic cells by microfilariae of Brugia malayi."

Skinner JA, Venugopal PG, Chien D, Mahapatra L, Chaussabel D, Meylan F, Dorward DW, Nutman TB, Semnani RT, Siegel RM

[J Immunol, 2008]

Dysregulation of professional APC has been postulated as a major mechanism underlying Ag-specific T cell hyporesponsiveness in patients with patent filarial infection. To address the nature of this dysregulation, dendritic cells (DC) and macrophages generated from elutriated monocytes were exposed to live microfilariae (mf), the parasite stage that circulates in blood and is responsible for most immune dysregulation in filarial infections. DC exposed to mf for 24-96 h showed a marked increase in cell death and caspase-positive cells compared with unexposed DC, whereas mf exposure did not induce apoptosis in macrophages. Interestingly, 48-h exposure of DC to mf induced mRNA expression of the proapoptotic gene TRAIL and both mRNA and protein expression of TNF-alpha. mAb to TRAIL-R2, TNF-R1, or TNF-alpha partially reversed mf-induced cell death in DC, as did knocking down the receptor for TRAIL-R2 using small interfering RNA. The mf also induced gene expression of BH3-interacting domain death agonist and protein expression of cytochrome c in DC; mf-induced cleavage of BH3-interacting domain death agonist could be shown to induce release of cytochrome c, leading to activation of caspase 9. Our data suggest that mf induce DC apoptosis in a TRAIL- and TNF-alpha-dependent fashion.

Galimov ER et al. (2018) Cell Rep "Coupling of Rigor Mortis and Intestinal Necrosis during C.elegans Organismal Death."

Pincus Z, Gems D, Poole SE, Galimov ER, Benedetto A, Pryor RE

[Cell Rep, 2018]

Organismal death is a process of systemic collapse whose mechanisms are less well understood than those of cell death. We previously reported that death in C.elegans is accompanied by a calcium-propagated wave of intestinal necrosis, marked by a wave of blue autofluorescence (death fluorescence). Here, we describe another feature of organismal death, a wave of body wall muscle contraction, or death contraction (DC). This phenomenon is accompanied by a wave of intramuscular Ca²⁺release and, subsequently, of intestinal necrosis. Correlation of directions of the DC and intestinal necrosis waves implies coupling of these death processes. Long-lived insulin/IGF-1-signaling mutants show reduced DC and delayed intestinal necrosis, suggesting possible resistance to organismal death. DC resembles mammalian rigor mortis, a postmortem necrosis-related process in which Ca²⁺influx promotes muscle hyper-contraction. In contrast to mammals, DC is an early rather than a late event in C.elegans organismal death. VIDEO ABSTRACT.

Kim J et al. (2022) Elife "Condensin DC loads and spreads from recruitment sites to create loop-anchored TADs in C. elegans."

Morao AK, Street LA, Kim J, Winterkorn LH, Albritton SE, Ercan S, Ragipani B, Zhang B, Jimenez DS, Kramer M

[Elife, 2022]

Condensins are molecular motors that compact DNA via linear translocation. In Caenorhabditis elegans, the X-chromosome harbors a specialized condensin that participates in dosage compensation (DC). Condensin DC is recruited to and spreads from a small number of recruitment elements on the X-chromosome (rex) and is required for the formation of topologically associating domains (TADs). We take advantage of autosomes that are largely devoid of condensin DC and TADs to address how rex sites and condensin DC give rise to the formation of TADs. When an autosome and X-chromosome are physically fused, despite the spreading of condensin DC into the autosome, no TAD was created. Insertion of a strong rex on the X-chromosome results in the TAD boundary formation regardless of sequence orientation. When the same rex is inserted on an autosome, despite condensin DC recruitment, there was no spreading or features of a TAD. On the other hand, when a 'super rex' composed of six rex sites or three separate rex sites are inserted on an autosome, recruitment and spreading of condensin DC led to the formation of TADs. Therefore, recruitment to and spreading from rex sites are necessary and sufficient for recapitulating loop-anchored TADs observed on the X-chromosome. Together our data suggest a model in which rex sites are both loading sites and bidirectional barriers for condensin DC, a one-sided loop-extruder with movable inactive anchor.

Semnani RT et al. (2003) J Immunol "Brugia malayi microfilariae induce cell death in human dendritic cells, inhibit their ability to make IL-12 and IL-10, and reduce their capacity to activate CD4+ T cells."

Sabzevari H, Kubofcik J, Gilden JK, Zhou J, Nutman TB, Semnani RT, Liu AY

[J Immunol, 2003]

Parasite Ag-specific T cell unresponsiveness and diminished IFN-gamma production are immunologic hallmarks of patent infection with lymph-dwelling filarial nematodes. Although this diminished responsiveness is directed primarily against the intravascular microfilarial (MF) parasite stage and mediated in part by reduced APC function, the mechanisms involved are not fully understood. In this report, we demonstrate that human dendritic cells (DC) exposed to live MF up-regulate both the cell surface and gene expression of CD54 (ICAM-1). Moreover, live MF result in a 3-fold increase in DC death compared with MF-unexposed DC, primarily due to apoptosis. Notably, microarray and real-time RT-PCR data indicate that live MF concurrently up-regulate mRNA expression of proinflammatory molecules such as IL-8, RANTES, IL-1alpha, TNF-alpha, and IL-beta in DC, the presence of which is also detected at the protein level, while inhibiting the production of IL-12 (p40 and p70) and IL-10. Soluble excretory-secretory products from live MF diminished IL-12 and IL-10 production and induced DC death, although to a lesser degree. Moreover, exposure of DC to live MF resulted in a decrease in the ability of DC to promote CD4(+) T cell production of IFN-gamma and IL-5. Our findings clearly suggest that the interaction between live MF and DC is complex but contributes to the hyporesponsiveness and parasite persistence associated with the MF(+) state in the infected human. These data further suggest that MF induce an orchestrated response in APC that leads to a diminished capacity to function appropriately, which in turn has significant consequences for CD4(+) T cells.