Pacquelet A et al. (2008) Dev Biol "PAR-6 levels are regulated by NOS-3 in a CUL-2 dependent manner in Caenorhabditiselegans."

Pacquelet A, Zanin E, Gotta M, Ashiono C

[Dev Biol, 2008]

The PAR proteins have an essential and conserved function in establishing polarity in many cell types and organisms. However, their key upstream regulators remain to be identified. In C. elegans, regulators of the PAR proteins can be identified by their ability to suppress the lethality of par-2 mutant embryos. Here we show that a nos-3 loss of function mutant suppresses the lethality of par-2 mutants by regulating PAR-6 protein levels. The suppression requires the activity of the sex determination genes fem-1/2/3 and of the cullin cul-2. FEM-1 is a substrate-specific adaptor for a CUL-2-based ubiquitin ligase (CBC(FEM-1)). Interestingly, we find that CUL-2 is required for the regulation of PAR-6 levels and that PAR-6 physically interacts with FEM-1. Our data strongly suggest that PAR-6 levels are regulated by the CBC(FEM-1) ubiquitin ligase thereby uncovering a novel role for the FEM proteins and cullin-dependent degradation in regulating PAR proteins and polarity processes.

Pacquelet A (2017) Results Probl Cell Differ "Asymmetric Cell Division in the One-Cell C. elegans Embryo: Multiple Steps to Generate Cell Size Asymmetry."

Pacquelet A

[Results Probl Cell Differ, 2017]

The first division of the one-cell C. elegans embryo has been a fundamental model in deciphering the mechanisms underlying asymmetric cell division. Polarization of the one-cell zygote is induced by a signal from the sperm centrosome and results in the asymmetric distribution of PAR proteins. Multiple mechanisms then maintain PAR polarity until the end of the first division. Once asymmetrically localized, PAR proteins control several essential aspects of asymmetric division, including the position of the mitotic spindle along the polarity axis. Coordination of the spindle and cytokinetic furrow positions is the next essential step to ensure proper asymmetric division. In this chapter, I review the different mechanisms underlying these successive steps of asymmetric division. Work from the last 30years has revealed the existence of multiple and redundant regulatory pathways which ensure division robustness. Besides the essential role of PAR proteins, this work also emphasizes the importance of both microtubules and actomyosin throughout the different steps of asymmetric division.

Pacquelet A et al. (2015) J Cell Biol "PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division."

Tassan JP, Pacquelet A, Uhart P, Michaux G

[J Cell Biol, 2015]

During asymmetric cell division, the mitotic spindle and polarized myosin can both determine the position of the cytokinetic furrow. However, how cells coordinate signals from the spindle and myosin to ensure that cleavage occurs through the spindle midzone is unknown. Here, we identify a novel pathway that is essential to inhibit myosin and coordinate furrow and spindle positions during asymmetric division. In Caenorhabditis elegans one-cell embryos, myosin localizes at the anterior cortex whereas the mitotic spindle localizes toward the posterior. We find that PAR-4/LKB1 impinges on myosin via two pathways, an anillin-dependent pathway that also responds to the cullin CUL-5 and an anillin-independent pathway involving the kinase PIG-1/MELK. In the absence of both PIG-1/MELK and the anillin ANI-1, myosin accumulates at the anterior cortex and induces a strong displacement of the furrow toward the anterior, which can lead to DNA segregation defects. Regulation of asymmetrically localized myosin is thus critical to ensure that furrow and spindle midzone positions coincide throughout cytokinesis.

Pacquelet, Anne et al. (2011) International Worm Meeting "Role of the cullin CUL-5 in the regulation of polarity and asymmetric division."

Pacquelet, Anne, Michaux, Gregoire

[International Worm Meeting, 2011]

PAR proteins are essential regulators of polarity in a variety of cell types, including neurons, epithelial cells as well as asymmetrically dividing cells. We use the first division of the C. elegans embryo to study the mechanisms regulating PAR proteins and polarity in the context of asymmetric cell division. In the one-cell embryo, PAR-3, PAR-6 and PKC-3 define an anterior cortical domain while PAR-1 and PAR-2 define a posterior domain. We previously showed that an ubiquitin ligase formed by the cullin CUL-2, the substrate specific adaptor FEM-1 and the cofactors FEM-2 and FEM-3 contributes to polarity in the one-cell embryo by regulating PAR-6. Interestingly, we found that another cullin, CUL-5, also regulates polarity in the early embryo. In particular, loss of cul-5 enhances the lethality and polarity defects of par-2(ts) mutant embryos grown at semi-restrictive temperature. Preliminary results indicate that these defects are further enhanced when fem-3 is inactivated, suggesting a possible redundancy between the CUL-2/FEM-1,2,3 and CUL-5 based ubiquitin ligases. We are currently further characterizing the role of CUL-5 during polarity establishment and asymmetric cell division.

Pacquelet, A. et al. (2015) International Worm Meeting "PAR-4/LKB1 and anillin prevent myosin from uncoupling cytokinetic furrow and mitotic spindle positions during asymmetric division."

Uhart, P., Pacquelet, A., Michaux, G.

[International Worm Meeting, 2015]

It is well established that the mitotic spindle regulates cytokinetic furrowing, thereby ensuring that cleavage occurs through the central spindle and properly segregates DNA. However, asymmetrically localized myosin has also been found to have the ability to induce cytokinesis, notably in asymmetrically dividing drosophila and C. elegans neuroblasts. How cells coordinate signals from the spindle and myosin to ensure that cleavage occurs through the central spindle and correctly segregates DNA is unknown. This is particularly intriguing in cells, such as in the C. elegans one-cell embryo, where myosin localization does not correlate with spindle position and may trigger a tug-of-war between the two mechanisms to properly position the furrow.We identified a novel pathway which regulates myosin and is thereby essential to coordinate furrow and spindle positions during asymmetric cell division. In the C. elegans one-cell embryo, myosin localizes at the anterior cortex while the mitotic spindle localizes towards the posterior pole of the embryo. We find that the kinase PAR-4/LKB1 regulates anillins (ANI-2 and ANI-1) together with the cullin CUL-5, as well as an anillin independent pathway involving the kinase PIG-1/MELK. In the absence of both ANI-1 and PIG-1/MELK, the spindle is still localized towards the posterior of the embryo but myosin strongly accumulates at the anterior cortex and induces a drastic displacement of the furrow towards the anterior: this severely uncouples spindle and furrow positions and can lead to DNA segregation defects. Altogether our results show that regulation of asymmetrically localized myosin by PAR-4/LKB1, PIG-1/MELK and ANI-1 is critical to ensure that furrow and central spindle positions coincide throughout cytokinesis.

Anne Pacquelet et al. (2006) Development & Evolution Meeting "A Novel Role For NOS-3 In PAR Protein-Dependent Cell Polarity"

Jean-Claude Labbe, Thomas Marty, Esther Zanin, Anne Pacquelet, Monica Gotta

[Development & Evolution Meeting, 2006]

Asymmetric cell division is a fundamental process that generates cell diversity during development. An essential step for this process is the establishment of a polarity axis in the mother cell prior to division. We are interested in understanding the molecular mechanisms involved in polarity establishment, using the early embryo as a model system. In the one-cell embryo, PAR proteins are responsible for the establishment and maintenance of cell polarity. PAR-3, PAR-6 and PKC-3 are members of a protein complex (referred to as the PAR-3/6/3 complex) localized at the anterior cortex of the embryo while PAR-2 and PAR-1 are present at the posterior cortex. Mutations that disrupt PAR-2 function result in embryos in which the PAR-3/6/3 complex is mislocalized, leading to polarity defects and embryonic lethality. It has been previously shown that the lethality of par-2 mutants can be suppressed by reducing PAR-6 levels or by depleting CDC-42, an activator of the PAR-3/6/3 complex. This indicates that the lethality due to the absence of PAR-2 can be suppressed by conditions that affect the levels, the localization or the activity of the PAR-3/6/3 complex. To identify new genes that regulate the PAR-3/6/3 complex, a genome-wide RNAi screen looking for suppressors of par-2 lethality was performed. One of the identified suppressors is NOS-3, the homologue of Drosophila Nanos. nos-3 suppresses most of the phenotypes associated with loss of par-2 function, in particular early cell division defects. nos-3 also suppresses the lethality of a null allele of par-2, indicating that NOS-3 impinges on the PAR pathway independently of the PAR-2 protein. Consistently, we found that nos-3 regulates PAR-6 levels in the embryo. We also identified other par-2 suppressors which are likely to be involved in regulating PAR-6 levels together with nos-3. We are currently characterizing the molecular mechanisms by which nos-3 and these additional suppressors control PAR-6 levels.

Pacquelet A et al. (2019) Curr Biol "Simultaneous Regulation of Cytokinetic Furrow and Nucleus Positions by Cortical Tension Contributes to Proper DNA Segregation during Late Mitosis."

Etienne J, Pacquelet A, Michaux G, Jousseaume M

[Curr Biol, 2019]

Coordinating mitotic spindle and cytokinetic furrow positioning is essential to ensure proper DNA segregation. Here, we present a novel mechanism, which corrects DNA segregation defects due to cytokinetic furrow mispositioning during the first division of C.elegans embryos. Correction of DNA segregation defects due to an abnormally anterior cytokinetic furrow relies on the concomitant and opposite displacements of the furrow and of the anterior nucleus toward the posterior and anterior poles of the embryo, respectively. It also coincides with cortical blebbing and an anteriorly directed cytoplasmic flow. Although microtubules contribute to nuclear displacement, relaxation of an excessive tension atthe anterior cortex plays a central role in the correction process and simultaneously regulates cytoplasmic flow as well as nuclear and furrow displacements. This work thus reveals the existence of a so-far uncharacterized correction mechanism, which is critical to correct DNA segregation defects due to cytokinetic furrow mispositioning.

Pacquelet, Anne et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The RNA-binding protein LARP-1 promotes oogenesis in a fem-3 dependent manner"

Pacquelet, Anne, Gotta, Monica, Zanin, Esther

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

In C. elegans hermaphrodites, a complex regulatory network regulates the switch from sperm to oocyte production. A key player in this regulation is CBC FEM-1 , an ubiquitin ligase formed by the cullin CUL-2, the substrate specific adaptor FEM-1 and the cofactors FEM-2 and FEM-3. CBC FEM-1 allows the degradation of TRA-1, a GLI-family transcription factor that promotes oogenesis. We identified the RNA-binding protein LARP-1 (LA-related protein 1) as a new regulator of sex determination. We found that simultaneous depletion of larp-1 and nos-3 results in germline masculinization. This phenotype is accompanied by a strong reduction in TRA-1 levels. Both the masculinization phenotype and the reduction in TRA-1 levels observed in nos-3;larp-1 mutants require fem-3 activity, suggesting that nos-3 and larp-1 regulate the sperm/oocyte switch by inhibiting fem-3 . Previous data indicated that NOS-3 represses fem-3 translation. Interestingly, we found that fem-3 mRNA levels were reduced in l arp-1 mutants but not in nos-3 mutants, showing that LARP-1 and NOS-3 regulate fem-3 by distinct mechanisms. We are currently investigating whether LARP-1 directly regulates fem-3 .

Demouchy F et al. (2023) Development "PAR-4/LKB1 prevents intestinal hyperplasia by restricting endoderm specification in C. elegans embryos."

Demouchy F, Michaux G, Nicolle O, Pacquelet A

[Development, 2023]

The kinase PAR-4/LKB1 appears as a major regulator of intestinal homeostasis, which prevents polyposis in humans. Moreover, its ectopic activation is sufficient to induce polarization and formation of microvilli-like structures in intestinal cell lines. Here, we use C. elegans to further examine the role of PAR-4 during intestinal development in vivo. We show that it is not required to establish enterocyte polarity and plays only a minor role in brush border formation. By contrast, par-4 mutants display severe deformations of the intestinal lumen as well as supernumerary intestinal cells, thereby revealing a novel function of PAR-4 in preventing intestinal hyperplasia. The presence of supernumerary enterocytes in par-4 mutants is not due to excessive cell proliferation but to the abnormal expression of the intestinal cell fate factors end-1 and elt-2 outside the E lineage. Notably, par-4 mutants also display a reduced expression of end-1 and elt-2 inside the E lineage. Our work thereby unveils an essential and dual role of PAR-4, which on one hand restricts intestinal specification to the E lineage and on the other hand ensures its robust differentiation.

Zanin E et al. (2010) J Cell Sci "LARP-1 promotes oogenesis by repressing fem-3 in the C. elegans germline."

Pacquelet A, Scheckel C, Zanin E, Gotta M, Ciosk R

[J Cell Sci, 2010]

LA-related protein 1 (LARP-1) belongs to an RNA-binding protein family containing a LA motif. Here, we identify LARP-1 as a regulator of sex determination. In C. elegans hermaphrodites, a complex regulatory network regulates the switch from sperm to oocyte production. We find that simultaneous depletion of larp-1 and the Nanos homologue nos-3 results in germline masculinization. This phenotype is accompanied by a strong reduction of the levels of TRA-1, a GLI-family transcription factor that promotes oogenesis. TRA-1 levels are regulated by CBC(FEM-1), a ubiquitin ligase consisting of the FEM proteins, FEM-1, FEM-2 and FEM-3 and the cullin CUL-2. We show that both the masculinization phenotype and the reduction of TRA-1 levels observed in nos-3;larp-1 mutants require fem-3 activity, suggesting that nos-3 and larp-1 regulate the sperm-oocyte switch by inhibiting the fem genes. Consistently, fem-3 mRNA levels are increased in larp-1 mutants. By contrast, levels of fem-3 mRNA are not affected in nos-3 mutants. Therefore, our data indicate that LARP-1 and NOS-3 promote oogenesis by regulating fem-3 expression through distinct mechanisms.