Ellenbecker, Mary et al. (2022) MicroPubl Biol

Ellenbecker, Mary, Voronina, Ekaterina

[MicroPubl Biol, 2022]

Dynein light chain (DLC-1) is a light chain component of the dynein motor complex, it functions as an allosteric regulator of multi-subunit protein complexes and promotes P granule integrity in the C. elegans embryo. P granules are RNA-protein complexes located in the C. elegans germline that are important for RNA regulation and fertility. To further study the role of DLC-1 during C. elegans embryogenesis we performed quantitative tandem mass tag mass spectrometry on embryos after dlc-1 knock down. The amount of core P granule components and nucleoporin proteins did not change after dlc-1(RNAi). These results show that DLC-1 does not help regulate P granule protein levels and support the model that DLC-1 facilitates phase separation of P granule components in vivo .

Wang, Xiaobo et al. (2015) International Worm Meeting "A role for dynein light chain in germline stem cell maintenance in C. elegans."

Ellenbecker, Mary, Voronina, Ekaterina, Wang, Xiaobo, Rasoloson, Dominique

[International Worm Meeting, 2015]

FBF-1 and FBF-2, two similar PUF family translational repressors, are required for maintenance of germline stem cell in C. elegans (Crittenden et al., 2002). Here we report identification of dynein light chain DLC-1 as a specific cofactor of FBF-2, but not FBF-1. In germline stem cells, FBF-2 localizes to perinuclear RNA granules called P granules (Voronina et al., 2012). This localization is important for FBF-2 binding to target mRNAs and its activity as a translational repressor. By contrast, FBF-1 does not localize to P granules, and its function does not depend on P granule integrity. The goal of this study is to investigate mechanisms of FBF-2 localization to P granules. DLC-1 is an LC8-type light chain, a cargo-binding component of dynein motor complex. Dynein traffics organelles, proteins, and mRNAs toward the minus ends of microtubules. Knock-down of dlc-1 by RNAi correlates with loss of FBF-2 perinuclear accumulation even when P granules are not affected and also causes a decrease in FBF-2 activity as a translational repressor. By contrast, both FBF-1 localization and its repressor function are not affected by dlc-1 knock-down. In vivo, FBF-2 is found in a complex with DLC-1. In the in vitro pulldown assay, DLC-1 interacts with FBF-2, but not FBF-1, suggesting that specificity of DLC-1 contribution to FBF-2 function is based on selectivity of protein-protein interactions. Interaction between DLC-1 and FBF-2 depends on the regions of sequence divergence between FBF-2 and FBF-1. Mutations disrupting the interaction of FBF-2 with DLC-1 do not interfere with the in vitro ability of FBF-2 to bind RNA, but disrupt FBF-2 localization to P granules in vivo.We conclude that DLC-1 binds to FBF-2 and promotes its localization to P granules, which is important for FBF-2-mediated translational regulation and germline stem cell maintenance. Dynein motor complex has been implicated in formation, transport, and dynamics of RNA granules in several cell types. Our findings suggest that LC8-type light chains such as DLC-1 may directly regulate recruitment of specific components to the RNA granules.

Day, Nicholas et al. (2021) International Worm Meeting "DLC-1 promotes germ granule integrity in C. elegans embryo"

Wang, Xiaobo, Day, Nicholas, Ellenbecker, Mary, Voronina, Ekaterina

[International Worm Meeting, 2021]

P granules are RNA-protein complexes located in the germline of Caenorhabditis elegans that are important for RNA regulation, germ cell identity and fertility. P granules are present throughout the C. elegans germline lifecycle, during which they undergo several dynamic transitions. For example, P granules are perinuclear during most of germline development but become cytoplasmic during oocyte maturation and during embryogenesis P granules segregate asymmetrically with the P cell lineage that produces the primordial germ cells. Here we identify dynein light chain (DLC-1) as an important determinant of P granule formation and subcellular localization in the C. elegans embryo. DLC-1 is a bimolecular hub that interacts with a variety of cellular proteins and functions in diverse processes from dynein-dependent transport to allosteric regulation of ribonucleoprotein complexes. We used an in silico motif scanning approach to search for new DLC-1 binding partners, identifying several P granule components as a result. Direct interaction between DLC-1 and PGL-1, PGL-3, GLH-4 and MEG-4 was established using a biochemical binding assay in vitro. We confirmed that DLC-1 is in complex with P granule components PGL-1 and PGL-3 in the germline and embryo in vivo using proximity ligation assay. Loss of dlc-1 disrupts assembly of embryonic P granules as assessed by localization of multiple P granule components but does not impact the levels of PGL-1 or PGL-3 protein expression. Analysis of a temperature sensitive dynein heavy chain mutant C. elegans suggests that the dynein motor related function of DLC-1 is important for P granule segregation during asymmetric cell divisions and contributes to phase separation of core P granule components at select stages of embryonic development. DLC-1 is a member of the conserved LC8 protein family, whose members contribute to germ granule dynamics in other organisms such as zebrafish. Our findings may shed light on general mechanisms behind LC8 contribution to germ granule phase separation.

Novak, Preston et al. (2015) International Worm Meeting "Splicing machinery potentiates post-transcriptional regulation of germline development."

Ellenbecker, Mary, Feilzer, Sara, Wang, Xiaobo, Novak, Preston, Voronina, Ekaterina

[International Worm Meeting, 2015]

Germline stem cell proliferation in C. elegans is controlled in a large part by the FBF genes (fbf-1 and fbf-2; Crittenden et al., 2002). In the course of searching for FBF cofactors, we found that components of splicing machinery may contribute to FBF activity. Individual knockdowns of several splicing factors caused enhanced sterility of fbf-1 and fbf-2 single mutants compared to the wild type worms. This sterility was associated with masculinization of germline, a phenotype associated with disruption of FBF function. We further found that knockdown of these splicing factors caused misexpression of a transgenic reporter controlled by fog-1 3'UTR, which is normally repressed in stem cells by FBFs. Next, we tested whether splicing factors influence other post-transcriptional regulation in germline in addition to their contribution to FBF function or oogenesis. We find that splicing factor knockdown is also able to enhance defects in spermatogenesis caused by the temperature-sensitive mutation in fog-1, arguing that functional splicing cascade influences multiple post-transcriptional processes. Finally, we found that splicing factor knockdown does not worsen embryonic lethality caused by a mutation unrelated to post-transcriptional regulation (dhc-1ts). In all these assays, we think that knockdown of splicing factors decreased the efficiency of RNA processing, but did not completely eliminate it.We conclude that inefficient pre-mRNA splicing may interfere with multiple post-transcriptional regulatory events, which has to be taken into account when conducting genetic interaction screens.

Osterli, Emily et al. (2021) International Worm Meeting "COP9 signalosome component CSN-5 stabilizes stem cell regulators FBF-1 and FBF-2"

Voronina, Ekaterina, Terzo, Mikaya, Osterli, Emily, Ellenbecker, Mary, Wang, Xiaobo, Zeng, Baisen

[International Worm Meeting, 2021]

RNA-binding proteins FBF-1 and FBF-2 are required for stem cell maintenance in C. elegans, although the mechanisms by which FBF protein levels are regulated remain unknown. Using a yeast two-hybrid screen, we identified an interaction between both FBFs and CSN-5, a component of the COP9 (constitutive photomorphogenesis 9) signalosome. This highly conserved COP9 complex can affect protein stability through a range of mechanisms including deneddylation, deubiquitination, and phosphorylation (Wolf et al., 2003). We discovered that CSN-5 promotes the accumulation of FBF-1 and FBF-2 proteins in C. elegans stem and progenitor cells. Phenotypic analysis results were consistent with csn-5 contributing to FBF function since csn-5 germlines are masculinized (produce only sperm similar to fbf-1/2 loss of function) and show reduced numbers of stem cells. Similar phenotypes were observed in worms mutant for another COP9 holoenzyme component, csn-6. Curiously, phenotypes of the csn-2 mutant were clearly distinct, where oocytes were still forming and stem cell numbers were not as affected. Additionally, FBF protein levels were not as affected in the csn-2 mutant as observed in the csn-5 mutant. This suggests that csn-5's effect on FBFs might be independent of the COP9 holoenzyme. Mapping protein-protein interactions between FBFs and CSN-5 suggested that the MPN (Mpr1/Pad1 N-terminal) metalloprotease domain of CSN-5 interacts with the RNA-binding domain of FBFs at physiologically relevant (micromolar) concentrations. Furthermore, these conserved domains of the human homologs PUM1 and CSN5 interact as well, thus identifying a protein complex conserved in evolution. Investigating CSN-5 contribution to FBF protein activity and stem cell maintenance will have implications for human stem cell biology and improve our understanding of diseases like cancer.

(2019) Development "The people behind the papers - Julia Brandt, Mary Rossillo and Niels Ringstad."

[Development, 2019]

A fundamental aim in developmental biology is to understand how the various cell types of the body are specified by differential gene regulation. <i>Caenorhabditis</i><i>elegans</i> nervous system development provides a powerful system for studying this, as exemplified by a new Development paper reporting on how the BAG neurons that help the worm sense oxygen and carbon dioxide are specified. We caught up with first authors Julia Brandt and Mary Rossillo and their supervisor Niels Ringstad (Associate Professor at the Skirball Institute of Biomolecular Medicine and Department of Cell Biology at New York University) to find out more about the story.

Yang L et al. (1998) West Coast Worm Meeting "Two new genes required for positioning QR descendants along the anterior-posterior body axis"

Kenyon CJ, Yang L

[West Coast Worm Meeting, 1998]

The QL and QR neuroblasts, which are born as bilaterally symmetric left/right homologs in the posterior of the worm, generate identical cell lineages. During the first larval stage, the descendants of QR migrate anteriorly whereas those of QL migrate posteriorly. One gene required for the anterior migration of the QR lineage which gives rise to SDQR and AVM, but not for the posterior migration of the QL lineage which gives rise to SDQL and PVM, is mig-13. This gene was cloned and characterized in our lab by Mary Sym (unpublished). mig-13 encodes a transmembrane protein that acts outside of the Q cells to promote anterior cell migrations. To further understand the regulation of anterior cell migration, we are studying two mutants with altered QR descendant migrations, mu232 and mu290. These mutants were isolated in two recent screens for Q cell migration mutants conducted in our lab by Queelim Ch'ng and Mary Sym. In these two mutants, as in mig-13 mutants, SDQR and AVM (which are normally found in the anterior body region) are found just anterior to the birthplace of QR whereas the final positions of SDQL and PVM are unaffected. In addition, like mig-13 mutants, the QR descendant AQR (which is normally found in the head) can be found anywhere along the anterior-posterior axis, suggesting that these genes are not required for motility but are required for positioning these cells. Thus, these new genes are candidates for components of a signalling pathway involving mig-13. We have mapped mu232 and mu290 to chromosomes V and III, respectively. Further mapping and additional phenotypic analysis are in progress.

Hodgkin JA et al. (1993) Worm Breeder's Gazette "Caenorhabditis Genetic Map and Nomenclature E-Mailing List"

Hodgkin JA, O'Callaghan M

[Worm Breeder's Gazette, 1993]

Most of the points covered in the accompanying 'Nomenclature Guidelines -- New Recommendations' contribution were circulated by e-mail before the June 1993 meeting, to principal investigators with e-mail addresses, for their comments and reactions. This procedure appeared to be efficient and helpful. We wish to expand our e-mailing list for nomenclature issues, and for recommended methods of data submission. For this reason, we sent a message in August 1993 inviting other potentially interested parties to join this list, using the e-mail addresses from the 1993 WBG Subscriber Directory. We repeat this message below, for the benefit of readers who have e-mail access but did not receive our invitation. If you are interested, send us an e-mail message asking to be added to the list. From cgc@mrc-lmb.cam.ac.uk Dear WBG subscriber At present, CGC e-mail messages about methods of data submission, nomenclatorial issues and so on, are sent to all investigators with assigned lab codes (strain and allele designations) and known e-mail addresses. We would like to extend this mailing list to other interested parties who can be reached by e-mail. If you wish to be included on this expanded e-mailing list, please reply to this message, and you will be added to the list. Investigators already on the list need take no action. Regardless of whether you join the list, you will still be able to take note of any significant new recommendations, because these will be duly announced in issues of the Worm Yours sincerely, Jonathan Hodgkin Mary O'Callaghan

TM Tam et al. (1999) International C. elegans Meeting "Effects of UNC-2, a a-1 Calcium Channel Subunit, on Neuronal Migration and Development"

TM Tam, WR Schafer

[International C. elegans Meeting, 1999]

Our lab is interested in the roles of voltage gated calcium channels in nervous system function and development. We have been studying the product of the gene unc -2, which encodes the homolgue of the vertebrate a -1 subunit of a non-L-type calcium channel. Typical unc -2 alleles display three main phenotypes: sluggishness, kinky uncoordinated movement and constitutive egg-laying (Egl-c). Mosaic analysis and RNA in situ hybridization have correlated the sluggish behavior to motorneurons and muscles, the kinky uncoordinated movement to motorneurons and Egl-c to egg-laying neurons. Recently, we have analyzed deletion and insertion alleles of unc -2, some of which are putative nulls. In these alleles, we have observed an additional phenotype: the final positions of the neurons descended from QR are abnormal. In particular, the dorsal/ventral positioning of the nuclei is often disrupted, suggesting that unc -2 is specifically required for the final dorsal/ventral migration of these cells. These defects are approximately 50% penetrance in the most severe deletion mutants (Mary Simms in Cynthia Kenyon's lab first noticed a very low penetrance defect in the EMS-generated allele unc -2(e55)). The QL migration as well as the ALM's appear normal. To determine the role of the calcium channel, we are testing expression pattern with GFP, and the cell autonomy of the migration defect by mosaic analysis. These experiments may indicate whether the UNC-2 calcium channel mediates a calcium influx directing the dorsal/ventral positioned Q cell migration. a unc a

Tam TM et al. (1998) West Coast Worm Meeting "GENETICS AND IMMUNOHISTOCHEMISTRY OF NEMATODE CALCIUM CHANNEL PROTEINS"

Wolf H, Schafer WR, Sanchez BM, Tam TM

[West Coast Worm Meeting, 1998]

Our lab is interested in the roles of voltage gated calcium channels in nervous sytem function and development. We focus on unc-2 which encodes the homologue of the vertebrate a-1 subunit of a non-L-type calcium channel. Typical unc-2 alleles display three main phenotypes: sluggishness, kinky uncoordinated movement and constitutive egg-laying (Egl-c). Mosaic analysis and RNA in situ hybridization correlates the sluggish behavior to motorneurons and muscles, the kinky uncoordinated movement to motorneurons and Egl-c to egg-laying neurons. We recently begun characterizing a set of stronger recessive alleles, which we think may be nulls. These alleles display a more dramatic movement deficit as well as two developmental defects. A drastic reduction in brood size is seen in three alleles, one of which maintains the low count even when mated to wild type males suggesting maternal effect. This suggests that unc-2 calcium channels are involved in early development and/or germ cell function. A number of unc-2 mutants also show defect in the positioning of neurons descended from migratory neuroblasts (a phenotype first identified by Mary Sym in Cynthia Kenyon's lab). Additional analysis of this phenotype will be presented. We are also interested in determining the expression pattern and intracellular localization of UNC-2 as well as UNC-36, which encodes the homologue of the vertebrate a-2 subunit. We raised antibodies against UNC-2 and UNC-36 and have preliminary data which seems to localize both subunits to body wall muscle. This data also shows lack of UNC-2 staining in a unc-36 point mutant which may suggest that the a-2 subunit is necessary for the localization of the a-1.