Paul Maddox et al. (2007) International Worm Meeting "Functional genomics identifies a Myb domain-containing protein family required for assembly of CENP-A chromatin."

Joost Monen, Arshad Desai, Francie Hyndman, Karen Oegema, Paul Maddox

[International Worm Meeting, 2007]

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2-associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain-containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.

Peng, Felix et al. (2015) International Worm Meeting "Analysis of foci created by the telomere binding protein POT-1 in C. elegans."

Maddox, Paul, Ahmed, Shawn, Shtessel, Ludmila, Peng, Felix

[International Worm Meeting, 2015]

Telomeres are tracts of tandem DNA repeats that protect the ends of linear chromosomes from replicative erosion and from being identified and repaired as double-strand breaks. Shelterin is a complex of proteins found to directly associate with telomeres and functions to distinguish telomeres from double-strand breaks. The shelterin component POT1 binds to single stranded telomeric DNA, which is present at the displacement loop created by strand invasion of the 3' overhang into the telomeric duplex at the chromosome terminus. POT1 homologues have recently been identified and characterised in C. elegans. A transgene expressing pot-1::mCherry reveals strong punctate foci within the germline corresponding to discrete telomeric termini. Here, we examine the dynamics of POT1 foci in the germline and during early development within living embryos using time-lapse imaging. We interrogate the behaviour of POT1 foci in the context of fused chromosomal ends harbouring telomeric repeats of varying lengths. We also analyse POT1::mCherry foci in genetic backgrounds where telomerase activity is abolished, leading to shortened telomeres, and in mutants causing telomere elongation, as occurs with mutant pot-1. Our results suggest that POT1 creates large, discrete structures within the nucleus and we are testing the hypothesis that dynamics of POT1 foci during early development could be relevant to telomere biology.

Narbonne, Patrick et al. (2013) International Worm Meeting "Regulation of self-renewal and differentiation capacities of germline stem cells during ageing."

Labbe, Jean-Claude, Maddox, Paul S., Narbonne, Patrick

[International Worm Meeting, 2013]

Stem cells have the amazing capacity to proliferate and expand in numbers almost indefinitely in vitro. In vivo however, the efficiency of stem cell-dependent processes, such as regeneration/repair and reproduction, declines with age in most organisms. It is thus likely that stem cell capacities decay during ageing in vivo, but the mechanisms remain unclear. Yet, understanding this phenomenon could lead to the identification of genes or pathways that could be targeted by drugs to restore stem cell function in ageing humans. This may be useful to treat many degenerative disorders, but also to elucidate how cancer stem cells somehow escape from this regulation and acquire/maintain the ability to expand in numbers within an ageing organism. I use the C. elegans germline stem cells (GSCs) as a model to study stem cell activity in vivo during ageing. By staining the germ line of unmated hermaphrodites fixed at different ages with an M-phase marker (anti-phospho-H3ser10) and a differentiation marker (anti-HIM-3), I have determined their mitotic index until 10 days of adulthood at 25 deg C. The mitotic index of GSCs progressively decreases as worms age, leading to a decreased GSC pool size. At a population level, the mitotic index reaches a basal level (below 1%) after 4 days, and then remains more or less stable until day 10. GSC population size follows the same trend, reaching a basal size (~100) after 5 days and remaining stable until day 10. Similarly, mitosis-to-meiosis transition of the GSCs is slower in 4 day-old adults compared to 1 day-old. In continuously mated hermaphrodites, the mitotic index remains above 1% for a longer time, but eventually also declines in older animals, and a similar trend is observed in males. This suggests that GSC division rate is coupled to oocyte demand until a certain age, after which the capacity of GSCs to expand in numbers ceases to respond properly to the demand. This is consistent with stem cell capacities progressively deteriorating with age in vivo. I am currently generating mitotic index curves relative to age for a variety of aging, metabolic, epigenetic and developmental mutant animals to find out which genes and genetic pathways are involved in this process.

Narbonne, Patrick et al. (2015) International Worm Meeting "Localized insulin signaling inhibition couples germline stem cell activity to oocyte needs in aging C. elegans adults."

Narbonne, Patrick, Maddox, Paul S., Labbe, Jean-Claude

[International Worm Meeting, 2015]

The efficiency of stem cell-dependent processes declines with age, largely owing to a decline in stem cell capacities, but the mechanisms remain unclear. Using the C. elegans germline stem cells (GSCs) as model, we found that two general mechanisms underlie stem cell activity decline during aging: a passive decline that results from aging, and an active induction of quiescence to meet stem cell progeny needs. The adult C. elegans germline can be viewed as a biological assembly line, with stem cells as the raw material that is turned, in a stepwise manner, into mature oocytes. Under replete growth conditions, the two gonad arms produce about 300 oocytes over three days in self-fertilizing hermaphrodites, but can produce more if they mate with males. We found that the accumulation of unfertilized oocytes, which occurs after sperm depletion, triggers a feedback signal leading to a reversible complete germline arrest, including the induction of stem cell quiescence. Accumulation of intermediate differentiation products or sperm does not trigger the feedback, which once induced, is able to strongly suppress stem cell proliferation throughout the gonad. The active induction of stem cell quiescence in otherwise healthy and well-fed adults only occurs when there is an accumulation of terminally-differentiated stem cell progeny (oocytes) and requires a localized, daf-16-independent down-regulation of insulin signaling in the germline. This in turns locally activates the energy-sensing kinase AMPK, which can then phosphorylate regulators of the ERK/MAPK pathway, including MPK-1 itself, to inhibit MPK-1 activity and shut down GSC proliferation and differentiation. Our results demonstrate that stem cell assembly units (from the stem cells to their terminally-differentiated progeny) can function as self-regulated, independent entities within multi-cellular organisms. This allows different stem cell populations, across and within tissues, to differentially respond to systemic information according to the local needs.

Gerhold, Abigail et al. (2015) International Worm Meeting "Investigating the impact of organismal physiology and tissue architecture on mitosis by in situ live imaging of adult germline stem and progenitor cells."

Gerhold, Abigail, Larouche, Myreille, Labbe, Jean-Claude, Maddox, Paul

[International Worm Meeting, 2015]

The study of dynamic mitotic events, such as spindle assembly, chromosome segregation and cytokinesis, has been largely confined to tissue culture and embryonic model systems, which are readily amenable to live imaging. How these fundamental mitotic processes occur in adult stem and progenitor cells, which divide within the complex environment of a mature tissue and organism, remains largely unknown. We are using in situ live imaging of C. elegans germ line stem and progenitor cells to investigate various aspects of mitosis in cells dividing within their native environment in an intact animal. We have shown that, unlike in the early embryo, regulation of anaphase onset in germ cells by the spindle assembly checkpoint resembles that which has been described for classical tissue culture models. In addition the timing of anaphase entry is highly sensitive to dietary restriction, attenuated insulin signaling, germ line function and developmental stage. These results provide in vivo validation of models for spindle assembly checkpoint regulation developed in tissue culture systems and demonstrate that mitotic progression in an adult stem and progenitor cell population is highly sensitive to changes in organismal physiology. Germ cell mitosis also occurs within a complex tissue architecture - germ cells are partially syncytial and are arranged in a circumferential monolayer with syncytial openings facing a common cytoplasmic core. We are exploring how this tissue organization is maintained during cell division and how it influences ring closure during cytokinesis.

Lister-Shimauchi, Evan et al. (2021) International Worm Meeting "Temporary loss of the shelterin proteins POT-1 or POT-2 alters telomeric protein localization for multiple generations"

Lister-Shimauchi, Evan, Maddox, Paul, Dinh, Michael, Ahmed, Shawn

[International Worm Meeting, 2021]

Telomere proteins protect the ends of linear chromosomes from shortening during cell replication. Deficiency for telomerase results in loss of telomeric sequence across multiple generations. However, there is no described role for telomeres in transgenerational epigenetic inheritance. The C. elegans Pot1 homologues POT-1 and POT-2, members of the shelterin complex, form foci at telomeres in germ cells. These foci disappear by fertilization and gradually accumulate during early development. We find that mutation of either Pot1 gene in sperm or oocytes alters levels of telomeric foci for multiple generations, despite progeny possessing wild-type Pot1 genes. pot-2 mutant gametes gave rise to progeny with abundant POT-1::mCherry and mNeonGreen::POT-2 foci during early development, an effect which persisted for six generations. In contrast, pot-1 mutant gametes gave rise to progeny with fewer foci for several generations. We investigated a range of genes involved in epigenetic inheritance. While Pot1 foci were not affected by loss of genes involved in sRNA pathways, they were greatly diminished for at least two generations by the loss of MET-2, SET-25, or SET-32, three methyltransferases that promote heterochromatin formation. We propose that POT proteins may interact with H3K9 methyltransferases during gametogenesis to induce a persistent form of transgenerational epigenetic inheritance that influences telomeric heterochromatin.

Zellag Mohamed, Reda et al. (2019) International Worm Meeting "Physiological control of C. elegans germline stem cell mitosis."

Maddox, Paul, Gerhold, Abigail, Zellag Mohamed, Reda, Labbe, Jean-Claude

[International Worm Meeting, 2019]

Accurate segregation of the replicated genome is an essential step in the production of genetically identical daughter cells during mitosis. The fidelity of chromosome segregation relies upon robust spindle assembly and the activity of a surveillance system called the spindle assembly checkpoint. These mitotic processes have been studied extensively in tissue culture and embryonic model systems, which are readily amenable to live imaging, but which generally lack physiological complexity. As a result, how mitosis is adapted to accommodate in vivo physiological variability remains largely unknown. We are using in situ live imaging of C. elegans germ line stem cells (GSCs) to investigate various aspects of mitosis in cells dividing within the physiologically complex environment of an intact animal. We have shown that modulating dietary intake, Insulin signaling or reproductive activity influences mitotic progression in GSCs and may affect mitotic fidelity. In addition, we have found that GSCs have a stronger spindle assembly checkpoint than cells in the early embryo, a trait which may be linked to their germ cell identity. Here we will present an optimized method for in situ live imaging of GSCs and our results indicating that GSC mitosis is highly sensitive to changes in organismal physiology. We suggest that several features of mitotic progression are affected by the physiological context in which cell division occurs, which may help to explain why certain conditions predispose cells to the development of aneuploidy.

Consortium Wormbase (2000) Worm Breeder's Gazette "Update on the C. elegans database WormBase"

Consortium Wormbase

[Worm Breeder's Gazette, 2000]

WormBase (www.wormbase.org) is an international consortium of biologists and computer scientists dedicated to providing the research community with accurate, current, accessible information concerning the genetics, genomics and biology of C. elegans and some related nematodes. WormBase builds upon the existing ACeDB database of the C. elegans genome by providing curation from the literature, an expanded range of content and a user friendly web interface. The team that developed and maintained ACeDB (Richard Durbin, Jean Thierry-Mieg) remains an important part of WormBase. Lincoln Stein and colleagues at Cold Spring Harbor are leading the effort to develop the user interface, including visualization tools for the genome and genetic map. Teams at Sanger Centre (led by Richard Durbin) and the Genome Sequencing Center at Washington University, St. Louis (led by John Spieth) continue to curate the genomic sequence. Jean and Danielle Thierry-Mieg at NCBI spearhead importation of large-scale data sets from other projects. Paul Sternberg and colleagues at Caltech will curate new data including cell function in development, behavior and physiology, gene expression at a cellular level; and gene interactions. Paul Sternberg assumes overall responsibility for WormBase, and is delighted to hear feedback of any sort. WormBase has recently received major funding from the National Human Genome Research Institute at the US National Institutes of Health, and also receives support from the National Library of Medicine/NCBI and the British Medical Research Council. WormBase is an expansion of existing efforts, and as such continues to need you help and feedback. Even with the increased scope and funding, all past contributors to ACeDB remain involved. The Caenorhabditis Genetics Center (Jonathan Hodgkin and Sylvia Martinelli) collaborate with WormBase to curate the genetic map and related topics. Ian Hope and colleagues continue to supply expression data to WormBase. Leon Avery will continue his superb website and serves as one advisor to WormBase. While the major means of access to WormBase is via the world wide web, downloadable versions of WormBase as well as the acedb software engine will continue to be available.

Rachel McMullan et al. (2006) European Worm Meeting "Rho is a Pre-Synaptic Activator of Neurotransmitter Release at Pre-Existing Synapses in C.elegans"

Rachel McMullan, Emma Hiley, Paul Morrison, Stephen J. Nurrish

[European Worm Meeting, 2006]

Rachel McMullan, Emma Hiley, Paul Morrison and Stephen J. Nurrish. Rho GTPases have important roles in neuronal development but their function in adult neurons is less well understood. We demonstrate that pre-synaptic changes in Rho activity at C.elegans neuromuscular junctions can radically change animal behaviour via modulation of two separate pathways. In one, pre-synaptic Rho increases acetylcholine (ACh) release by stimulating the accumulation of diacylglycerol (DAG) and the DAG-binding protein UNC-13 at sites of neurotransmitter release; this pathway requires binding of Rho to the DAG kinase DGK-1. A second DGK-1-independent mechanism is revealed by the ability of a Rho inhibitor (C3 transferase) to decrease levels of release even in the absence of DGK-1; this pathway is independent of UNC-13 accumulation at release sites. We do not detect any Rho induced changes in neuronal morphology or synapse number, thus Rho facilitates synaptic transmission by a novel mechanism. Surprisingly, many commonly available human RhoA constructs contain an uncharacterised mutation that severely reduces binding of RhoA to DAG kinase. Thus a role for RhoA in controlling DAG levels has not been previously appreciated.

Minniti AN et al. (1995) International C. elegans Meeting "THE HERMAPHRODITE-INITIATED SPERMIOGENESIS PATHWAY."

Minniti AN, Nance JF, Sadler C, Ward S

[International C. elegans Meeting, 1995]

During spermiogenesis, round nonmotile spermatids are rapidly transformed into asymmetrical crawling spermatozoa. In addition to spe-8 and spe-12, we found two new genes, spe-27 and spe-29, that when mutated, disrupt spermiogenesis in an identical manner: mutant hermaphrodites are self-sterile, while mutant males are fertile. Sperm from both sexes activate abnormally in vitro, suggesting that these gene products are expressed in both sperm, but only hermaphrodites require them for activation. The hermaphrodite's spermatids, however, can be activated by mating. This phenotype has been explained by a model that has two distinct pathways of activation, one for males and one for hermaphrodites (Shakes and Ward, 1989). The four genes are needed only for the hermaphrodite pathway. spe-27 and spe-12 have been cloned. Their mRNA is found exclusively in the germline during spermatogenesis. Their predicted protein sequences show no similarities to known proteins. We are currently cloning spe-29. To further understand how these genes act during spermiogenesis we are determining the subcellular localization of their gene products. In addition, suppressor analysis is under way (see abstract by Paul Muhlrad and Samuel Ward) to look for other genes in this pathway and for interactions between these genes.