Roy, Vincent et al. MicroPubl Biol

Gagne, Olivier, Hamiche, Karim, Narbonne, Patrick, Labbe, Jean-Claude, Roy, Vincent

[MicroPubl Biol, 2018]

PAR-4/LKB1 is maternally expressed and required for the asymmetrical distribution of early embryonic determinants and viability in C. elegans (Morton et al. 1992; Watts et al. 2000; Tenlen et al. 2008). It is also implicated in a variety of postembryonic processes, including germline stem cell quiescence (Narbonne and Roy 2006; Narbonne et al. 2017), neuronal growth and polarity (Kim et al. 2010; Teichmann and Shen 2011), cytoskeletal rearrangements (Narbonne et al. 2010; Chartier et al. 2011), and metabolism (Narbonne and Roy 2009). Its expression pattern and subcellular localization has been determined in fixed animals by antibody staining (Watts et al. 2000). Here, we used CRISPR/Cas9-mediated genome editing to fluorescently label the two longest endogenous PAR-4 isoforms, PAR-4A and PAR-4C, with monomeric Neon Green (mNG) (Shaner et al. 2013), using the self-excising cassette (SEC) system (Dickinson et al. 2015) (Fig. 1A). We found ubiquitous mNG::par-4a & c expression and cytoplasmic and cortical enrichment of mNG::PAR-4A & C proteins in the germ line and early embryos (Fig. 1B-H), as previously described (Watts et al. 2000). Interestingly, we find that mNG::PAR-4A & C cortical enrichment is transiently lost in the pachytene area of the germ line (Fig. 1F), although it remains unclear whether this is functionally relevant. The intermediate strain that still contains the SEC (Dickinson et al. 2015) is predicted to be null for par-4a & c, potentially leaving the shorter par-4b isoform functional. To evaluate the requirement for par-4a & c we examined the embryonic lethality (at 25C) of the generated SEC-containing and SEC-excised strains. We found that the self-progeny of homozygous SEC-containing (e.g. par-4a & c null) animals is viable (Fig. 1H). This suggests that par-4b alone is sufficient to establish embryonic polarity and sustain the essential function of par-4. Consistent with this, to our knowledge, all existing par-4 alleles that impair embryonic development disrupt par-4b (Morton et al. 1992; Watts et al. 2000).

Bertrand, Vincent et al. Worm Breeder's Gazette "Identification of deletion alleles by whole-genome sequencing"

Bertrand, Vincent, Hobert, Oliver

[Worm Breeder's Gazette]

Vincent AL et al. (1975) J Parasitol "The ultrastructure of adult Brugia malayi (Brug, 1927) (Nematoda: Filarioidea)."

Frommes SP, Ash LR, Vincent AL

[J Parasitol, 1975]

The ultrastruct of the adult subperiodic Brugia malayi (Brug, 1927) within pulmonary arteries of male jirds (Meriones unguiculatus) was studied by transmission electron microscopy. The cuticle consists of 10 sublayers (2 of which are prominently banded) and a typical outer unit membrane. Evidence is presented showing that the subcuticular region of the lateral chords comprises a functional complex of basal infoldings, multivesicular bodies, and associated mitochondria, which is probably engaged in the exchange of solutes across a permeable cuticle. Microbodies with paired, prominent cores, intracisternal A-particle viruslike bodies, nonstaining glycogen patches, and other structures are also present in the lateral chords. The platymyarian somatic musculature shares some coelomyarian characteristics, e.g., apparent neuromuscular connections and prominent glycogen deposits surrounded by mitochondria and other organelles. The alimentary tract has features typical of many nematodes. The luminal segments of the male and female reproductive tracts and their germinal products, excluding microfilariae, are described. Affinities with related species are discussed.

Vincent Hyenne et al. (2007) International Worm Meeting "Defining the role of the brat family in C.elegans embryonic polarity."

Jean-Claude Labbe, Marianne Desrosiers, Vincent Hyenne

[International Worm Meeting, 2007]

We are interested in understanding the molecular mechanisms controlling cell polarity and asymmetric cell division. Asymmetric cell division relies on the cells polarization prior to division and allows the generation of cell diversity. In C.elegans zygote, polarity is established along an antero-posterior axis and leads to the formation of two unequal cells with distinct fates after division. Polarization induces the segregation of cell fate determinants in the anterior or posterior part of the zygote as well as posterior displacement of the mitotic spindle during metaphase/anaphase. The evolutionary conserved PAR proteins (PAR-1 to -6 and PKC-3) are essential for these events: loss of any of them results in a loss of polarity and an abnormal symmetric division. However, the molecular mechanisms involving PAR protein functions remain elusive. Recently, several genes have been identified as new modulators of PAR-protein dependent cell polarity, as their disruption can suppress the embryonic lethality of par-2(it5ts) animals. Two of these genes, nos-3 and fbf-1/2, have Drosophila orthologs that were shown to form a complex with the tumor suppressor Brat and negatively regulate the translation of Hunchback mRNA during embryonic polarization. This suggested that orthologs of Brat could also regulate embryonic polarity in C.elegans. While there are 5 genes encoding predicted orthologs of Brat in C.elegans, we have found that 3 of them, namely ncl-1, nhl-1 and nhl-2 (together referred to as CeBrats), are able to suppress par-2(it5ts) lethality. This suggests that, like in Drosophila, they could function in C.elegans PAR-protein dependent polarity through translational repression. Phenotypic analysis demonstrated that ncl-1, nhl-1 and nhl-2 suppress most of the early polarity phenotypes associated with par-2 depletion. Although disrupting these CeBrats collectively did not result in significant embryonic lethality, embryos individually mutant for these 3 CeBrats displayed distinct polarity phenotypes: ncl-1 and nhl-1 mutants have a more posterior spindle localization whereas nhl-1 and nhl-2 have an enhanced asynchrony of division at the second cell stage. These results suggest that these CeBrats could function through at least partially independent pathways. We are currently establishing whether ncl-1, nhl-1 and nhl-2 function, together or independently, within a complex with nos-3 and fbf-1/2 to regulate translation, and we are searching for putative mRNA targets of such a complex, using a combination of biochemical, bioinformatic and genetic approaches. This study should shed light on a new molecular mechanism controlling cell polarity and asymmetric cell division in a PAR-protein dependent manner.

Vincent Menuz et al. (2005) International Worm Meeting "Identification of Genes Affecting Resistance to Anoxia in Caenorhabditis elegans"

Vincent Menuz, Monique Fornallaz, Michael O Hengartner, Jean-claude Martinou, Marie Gomez

[International Worm Meeting, 2005]

In obligatory aerobe organisms, molecular oxygen is necessary for the production of ATP through the electron transport chain in the mitochondria. Adult C. elegans has evolved the capacity to survive more than 24 hours in the complete absence of oxygen (anoxia). The goal of our study is to identify the mechanisms in C. elegans that confer its resistance to anoxia. During anoxia, C. elegans enters into a reversible state of suspended animation in which cell division, developmental progression and movement cease. Young adults worms (72 hours post L1 stage) were exposed to anoxia for different periods of time. A time course study indicated that 85%, 67%, 30% and 2% of the worms survived 48h, 72h, 96h and 168h of anoxia respectively. We chose to screen for mutants whose survival rate was either higher or lower than the survival rate of wild type worms after 72 h anoxia. A random screen of a mutant library allowed us to identify eight mutants which were unable to survive to anoxia. Among those mutants, the strongest phenotype was displayed by the srf-3 mutant whose survival did not exceed 2% at 72h of anoxia. SRF-3 codes for a nucleotide sugar transporter capable of translocating UDP-galactose and UDP-N-acetylglucosamine into the lumen of the Golgi apparatus. The sensitivity of this mutant to hyperoxia, heat shock, negative pressure, hypertonic and hypotonic shock was not different than that of wild type worms under the same conditions. We are currently investigating the mechanisms by which SRF-3 deficiency sensitizes C. elegans to anoxia.

Hulsey-Vincent H et al. (2023) MicroPubl Biol "A Fiji process for quantifying fluorescent puncta in linear cellular structures."

Alvinez N, Dahlberg C, Hulsey-Vincent H, Witus S, Kowalski JR

[MicroPubl Biol, 2023]

Understanding the cell biology of protein trafficking and homeostasis requires reproducible methods for identifying and quantifying proteins within cells or cellular structures. Imaging protocols for measuring punctate protein accumulation in linear structures, for example the neurites of <i>C. elegans,</i> have relied on proprietary software for a full range of analysis capabilities. Here we describe a set of macros written for the NIH-supported imaging software ImageJ or Fiji (Fiji is Just ImageJ) that reliably identify protein puncta so that they can be analyzed with respect to intensity, density, and width at half-maximum intensity (Full-Width, Half-Maximum, FWHM). We provide an explanation of the workflow, data outputs, and limitations of the Fiji macro. As part of this integration, we also provide two independent data sets with side-by-side analyses using the proprietary IgorPro software and the Fiji macro (Hulsey-Vincent, et al. A, B., 2023 submitted). The Fiji macro is an important new tool because it provides robust, reproducible data analysis in a free, open-source format.

Vincent Bertrand et al. (2007) International Worm Meeting "Acquisition of subtype specific features by a single interneuron in C. elegans."

Vincent Bertrand, Oliver Hobert

[International Worm Meeting, 2007]

One characteristic of the nervous system is its high diversity of neuronal subtypes. How this diversity is generated is the object of intense studies in several organisms. In C. elegans, the identity, connectivity and lineage of every neuron is known and thus it constitutes an excellent system to address this question. We are using the well-characterised AIY interneuron to analyse how pan-neuronal and neuronal subtype-specific features are acquired during embryonic development. A battery of at least 40 subtype-specific genes expressed in the AIY interneuron has recently been identified. These genes are directly activated in the post-mitotic AIY interneuron by two homeobox transcription factors TTX-3 and CEH-10 that cooperate to bind to their promoters. A key question to understand the acquisition of the specific identity of AIY is to determine how ttx-3 and ceh-10 are initially co-activated in the AIY interneuron during embryonic development. Via a combination of loss of function and promoter analysis experiments, we have shown that ttx-3 and ceh-10 are activated in the AIY lineage in a sequential manner: ttx-3 is first activated in the AIY precursor before its last cleavage (mid-embryogenesis), while ceh-10 is activated later in the postmitotic AIY interneuron in a ttx-3-dependent manner. We are now conducting a genetic screen in order to identify new genes involved in AIY identity specification. By using a worm sorting machine, we have screened 90 000 genomes so far and recovered 12 mutants affecting the AIY interneuron fate. We have cloned one of those mutants and shown that it affects a zinc finger transcription factor which directly activates ttx-3 expression in the AIY precursor. We are now extending the genetic screen and pursuing the analysis of the new mutants isolated. This study should lead to a better understanding, at a cellular and promoter resolution level, of neuronal subtype identity acquisition during embryonic development.

Cannataro, Vincent et al. (2009) International Worm Meeting "TMD-1 / Tropomodulin Regulates Intestinal Lumen Diameter in C. elegans."

Silva, Malan, Cannataro, Vincent, Gallagher, Thomas, Cox, Elisabeth

[International Worm Meeting, 2009]

Tubes are a central architectural feature of many human tissues including glands, the urogenital and gastrointestinal tracts, the respiratory tract and the vascular system. Despite their importance, much remains unknown about the molecular mechanisms involved in tube development. This work explores a new role for tropomodulins in regulation of endothelial tube formation. Tropomodulins are one of the few proteins known to regulate the slow growing ends of actin filaments. The C. elegans tmd-1 gene encodes two transcripts (tmd-1a and tmd-1b) that have similar domain structure to mammalian tropomodulins. We have been examining the role of TMD-1 in development of the C. elegans intestine, which is a relatively simple, transparent tube that forms by a cord-hollowing mechanism similar to that used by some capillaries and renal tubules. Antibody staining using a rabbit anti-TMD-1 antibody demonstrates that TMD-1 localizes to the luminal surface of the C. elegans intestine as it undergoes morphogenesis. Interestingly, embryos homozygous for the tmd-1(tm724) allele, which contains a large deletion that affects both transcripts1, exhibit areas of intestinal lumen diameter that are 65% larger than wild-type. tmd-1(sf20) homozygous mutants, which have a premature stop codon affecting both transcripts2, also exhibit luminal expansions (50% larger than wild-type). Currently, investigations are underway to determine the molecular mechanisms by which TMD-1 regulates intestinal lumen diameter. Possibilities include modulation of actomyosin contractility, cooperation with the actin-spectrin cytoskeleton to provide mechanical strength to the luminal surface, and/or regulation of vesicle trafficking. This work is supported by National Institutes of Health grant R15HD059952. 1 Yamashiro et al. (2008) J. Cell Sci. 121: 3867-77. 2 Stevensen et al. (2007) J. Mol. Bio. 374: 936-50.

Portegijs, Vincent et al. (2015) International Worm Meeting "Regulation of spindle positioning through phosphorylation of LIN-5 in asymmetric cell division."

Fielmich, Lars-Eric, Van den Heuvel, Sander, Portegijs, Vincent, Galli, Matilde

[International Worm Meeting, 2015]

Asymmetric cell division is required throughout development to generate cells of unequal size, composition and fate. The correct distribution of cell fate determinants depends on the plane of cell cleavage, which is determined by the position of the mitotic spindle. Spindle positioning is mediated by a conserved Galpha-GPR-1/2&shy;-LIN-5 complex at the cortex, which interacts through dynein with astral microtubules and thereby promotes the generation of pulling forces. We aim to understand how the developmental regulation of cortical pulling forces determines the plane of cell cleavage and contributes to asymmetric cell division.Previous studies of our lab identified four amino acids in the C-terminal domain of LIN-5 that are phosphorylated by the polarity kinase PKC-3. This phosphorylation leads to reduced pulling forces in the anterior, but additional mechanisms contribute to asymmetric spindle positioning. In recent studies, we identified additional phosphorylations of LIN-5 with critical functions in vivo. Moreover, we defined the LIN-5 C-terminal domain that mediates interaction with GPR-1. Hereto, we used reverse yeast two-hybrid screening to identify single amino acid changes in LIN-5 that prevent GPR-1 binding. All of the identified mutations are confined to a 30 amino-acid domain located upstream of the PKC-3 phosphorylated residues of LIN-5. This GPR-1 binding region contains two additional in vivo phosphorylated LIN-5 residues. We used Cas9/CRISPR-mediated genome editing to substitute these amino acids for non-phosphorylatable alanine or phosphomimetic glutamic acid / aspartic acid. Combined with UV-laser spindle severing experiments, we found that phosphorylation of these residues contributes to GPR-1 binding and cortical pulling forces. In a parallel study, we identified residues in the N-terminus of LIN-5 that are phosphorylated by CDK-1. Again, examination of Cas9/CRISPR-mediated knock-in alleles revealed that these N-terminal residues are critical for LIN-5 function. Taken together, our data show that phosphorylation of LIN-5 contributes to the spatiotemporal regulation of the cortical pulling forces that position the mitotic spindle.

Mancuso, Vincent P. et al. (2011) International Worm Meeting "Extracellular leucine-rich repeat proteins and maintenance of epithelial integrity."

Poggioli, Corey, Hall, David, Storer, Luke, Parry, Jean, Sundaram, Meera, Mancuso, Vincent P.

[International Worm Meeting, 2011]

Elron (extracellular Leucine-Rich Repeat only) proteins are known to have roles in synapse formation, axon guidance, and cell-cell adhesion. Here we describe distinct roles for the two transmembrane Elron proteins K07A12.2 and LET-4/SYM-5/C44H4.2 in C. elegans epithelial cells. Both K07A12.2 and let-4 single mutants have lethal defects in embryonic elongation and excretory system function. In the low-penetrance embryonic phenotype, epidermal cell morphology appears normal until embryonic elongation, at which point epidermal lesions form. This suggests a role for K07A12.2 and LET-4 in the maintenance of epidermal cell integrity during the stress of elongation. Another transmembrane Elron, SYM-1, acts redundantly with LET-4 during elongation (Davies et al., 1999). Both K07A12.2 and LET-4 GFP fusion reporters localize to the apical face of epidermal cells during elongation, suggesting an interaction with the embryonic sheath. Individuals that do not have the embryonic elongation defect develop a lethal excretory system defect. In the excretory system defect phenotype, both K07A12.2 and let-4 single mutants accumulate fluid in the excretory system and die as fluid-filled L1s. The excretory system includes three tandem unicellular epithelial tubes: the excretory canal cell, the duct cell, and the pore cell. Although both K07A12.2 and LET-4 are required for a functioning excretory system, each functions in a different aspect of the excretory system. K07A12.2 mutants lack a pore cell autojunction, though it is currently unknown whether it fails to form or is lost during development. This phenotype suggests a role for K07A12.2 in initial formation of the pore cell, or in maintenance of the tube shape. A failure of either process could explain the excretory system defect. In let-4 mutants, the pore autojunction is unaffected, but the duct cell lumen becomes distorted and discontinuous just before hatch, indicating a role for LET-4 in lumen integrity. LET-4::GFP is localized to the luminal (apical) side of the duct cell, and may interact with the extracellular matrix inside the lumen of the duct and pore cells. This work suggests roles for Elron proteins in mediating interactions between epithelia and the apical extracellular matrix to help maintain cell shape and integrity. Davies AG, et al. (1999) Genetics 153, 117-134.