Houston, Jack et al. (2021) International Worm Meeting "Investigating the regulation of CDC-20 recruitment to kinetochores"

Lara-Gonzalez, Pablo, Houston, Jack, Kim, Taekyung, Desai, Arshad, Oegema, Karen

[International Worm Meeting, 2021]

The WD-40 repeat protein CDC-20 is the co-activator of the Anaphase Promoting Complex/Cyclosome (APC/C), the E3 ubiquitin ligase responsible for mitotic exit, as well as a core subunit of the mitotic checkpoint complex that restrains APC/C activity when chromosomes are not yet attached to the mitotic spindle. CDC-20 rapidly fluxes through the kinetochore region of chromosomes via association with a conserved binding motif, known as the ABBA motif, in BUB-1. CDC-20 flux through kinetochores is essential for mitotic checkpoint activation, which delays anaphase onset until all chromosomes attach to microtubules, and for promoting anaphase onset following kinetochore-microtubule attachment (Kim and Lara-Gonzalez et al, 2017, Genes and Development 31:1089-1094). Here, we investigate the regulation of BUB-1-dependent recruitment of CDC-20 to kinetochores. We found that mutating a conserved Polo-like Kinase 1 (PLK-1) docking site in BUB-1 eliminated CDC-20 kinetochore recruitment to the same extent as mutating the ABBA motif; in addition, the peak kinetochore recruitment of the mutant BUB-1 was reduced to ~50% of wildtype levels, likely due to a role for BUB-1-bound PLK-1 in promoting kinetochore recruitment of BUB-1. To address if the defect in CDC-20 kinetochore recruitment was a consequence of reduced BUB-1 kinetochore localization or was due to BUB-1-docked PLK-1 regulating the ABBA motif-CDC-20 interaction, we selectively mutated the BUB-1-associated protein BUB-3 to impair its recognition of the phosphorylated kinetochore scaffold KNL-1. The BUB-3 phospho-recognition mutant reduced peak BUB-1 kinetochore recruitment to ~30% of wildtype levels; however the consequences on anaphase onset and CDC-20 kinetochore localization were significantly less severe than observed for PLK-1-docking mutant BUB-1. These observations support a model in which BUB-1-associated PLK-1 is essential for CDC-20 kinetochore recruitment, either by direct phospho-regulation of the ABBA motif or by controlling access of the ABBA motif to CDC-20. Our current experiments are focused on distinguishing between these two potential mechanisms.

Richardson, Claire E. et al. (2009) International Worm Meeting "The IRE-1 Branch of the Unfolded Protein Response Functions in C. elegans Pathogen Resistance During Larval Development."

Kim, Dennis H., Kooistra, Tristan, Richardson, Claire E.

[International Worm Meeting, 2009]

The Unfolded Protein Response (UPR), a mechanism to sense and respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER), has an important role in development and stress resistance from C. elegans to mammals. We asked if the UPR functions in C. elegans immune defense against the bacterial pathogen Pseudomonas aeruginosa. We found that the conserved IRE-1-XBP-1 branch of the UPR is required specifically for immunity during larval development. IRE-1 is an endoribonuclease that activates the transcription factor XBP-1 by splicing a short exon from its mRNA. Using quantitative PCR, we show that the relative amount of IRE-1-spliced xbp-1 transcript increases after pathogen exposure. Furthermore, a fluorescent reporter for IRE-1 activation is induced after exposure to Pseudomonas specifically at the site of infection - the intestine. We analyzed the relationship between the IRE-1 branch of the UPR and the PMK-1 pathway, which is known to promote C. elegans immunity, and found that IRE-1 activation in response to Pseudomonas is downstream of the PMK-1 pathway. The IRE-1-XBP-1 branch of the UPR has been recently shown to be required for cellular defense against pore-forming toxins, also functioning downstream of PMK-1 (Bischoff et al, 2008). Our data point to a more general role for the UPR in innate immunity against infection. Since PMK-1 pathway activates transcription of putative pathogen resistance proteins, we suggest that the IRE-1 branch of the UPR promotes pathogen resistance by accommodating a PMK-1-driven influx of pathogen resistance proteins to the secretory pathway. Bischof, L.J., Kao, C.-Y., Los, F.C.O., Gonzalez, M.R., Shen, Z., Briggs, S.P., van der Goot, F.G., Aroian, R.V. (2008) Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo. PLOS pathogens, 4, 1-11.

Carmi I et al. (1998) West Coast Worm Meeting "FUNCTION OF THE NUCLEAR HORMONE RECEPTOR SEX-1, AN X-CHROMOSOME SIGNAL ELEMENT"

Meyer BJ, Carmi I

[West Coast Worm Meeting, 1998]

SEX-1 is a nuclear hormone receptor (NHR) homolog that participates in the X-chromosome counting process that specifies sex in C. elegans. sex-1 is one of several counted X-signal elements that communicate the dose of X chromosomes to the target switch gene, xol-1. In XX animals, two doses of signal elements repress xol-1, permitting hermaphrodite development and activation of dosage compensation. In contrast, in XO animals, a single dose of signal elements is insufficient to repress xol-1, resulting in high xol-1 levels and male development. While sex-1 regulates xol-1 at the transcriptional level, other signal elements regulate xol-1 at a post-transcriptional level (M. Nicoll, this meeting). Extensive genetic analysis has shown that sex-1 is a dose-sensitive regulator of xol-1. Reducing the dose of sex-1 results in derepression of xol-1, promoting the male fate and killing hermaphrodites. Conversely, increasing sex-1 dose leads to xol-1 repression, promoting the hermaphrodite fate and killing males. The sex-specific lethality is due to dosage compensation defects. In addition, sex-1 exhibits synergistic interactions with other signal elements. The homology of SEX-1 to members of the nuclear hormone receptor superfamily and its highly conserved DNA binding domain are consistent with its role as a transcriptional repressor of xol-1. To further understand how changes in the dose of sex-1 affect xol-1, we initiated a molecular/biochemical characterization of the gene. We raised polyclonal antibodies against SEX-1 and examined the protein localization pattern. We found that the protein is ubiquitously expressed in nuclei of young embryos. SEX-1 expression peaks at the beginning of gastrulation (28-100 cell stage), then gradually diminishes, disappearing by the end of embryonic cell proliferation (~500 cell stage). The timing of expression is consistent with a direct role in xol-1 regulation: xol-1 must be repressed during gastrulation to allow hermaphrodite development. To determine whether SEX-1 directly regulates xol-1 transcription we are examining whether SEX-1 can bind xol-1 promoter sequences in vivo. We have constructed extra-chromosomal arrays containing xol-1 promoter sequences, as well as lacO sequences and lacI::gfp. LacI::GFP binds lacO sequences and thus marks the position of the arrays in the nucleus (Gonzalez-Serricchio and Sternberg, WBG 14(5):18). Using antibody staining, we are asking whether SEX-1 co-localizes with GFP in embryos bearing these arrays. Preliminary results suggest that SEX-1 and GFP co-localize more consistently in embryos bearing xol-1 arrays than in embryos containing control DNA, suggesting that SEX-1 may directly regulate xol-1.

Karla M Knobel et al. (2001) International C. elegans Meeting "Structural and topological organization of the C. elegans polycystin LOV-1"

Maureen M Barr, Karla M Knobel

[International C. elegans Meeting, 2001]

lov-1 and pkd-2 are required for C. elegans male mating behavior. These genes act in the same pathway and are required for response and location of vulva (Lov) aspects of mating 1,2 . LOV-1 and PKD-2 are both found exclusively in the male chemosensory neurons (CEMs, rays, hook) where they localize to sensory cilia, suggesting a role in sensory signal transduction. lov-1 and pkd-2 are similar to the human polycystic kidney disease (PKD) genes PKD1 and PKD2, respectively. Mutations in either PKD1 or PKD2 account for 95% of all autosomal dominant PKD cases (ADPKD). ADPKD is an extremely prevalent genetic disease, occurring in roughly 1 of every 800 persons. LOV-1, like polycystin-1 (encoded by PKD1), is a large and complex protein. LOV-1 is composed of 3178 amino acids and is predicted to contain a variety of functional domains. The extracellular amino terminus contains a mucin-like serine/threonine rich region, a number of glycosylation sites, and a G-protein coupled receptor proteolysis site (GPS) near the first putative transmembrane domain (TM1). Just after TM1 is a region of conserved homology with the lipoxygenase and alpha-toxin proteins called the PLAT/LH2 domain. Typically these domains regulate the interaction between the enzyme and plasma membrane associated proteins and lipids 3 . Following the LOV-1 PLAT/LH2 domain are 10 hydrophobic regions. The last six of these comprise a region of homology conserved in all polycystins. These six putative TM domains are related to those found in a variety of channel proteins. Polycystin-2 is comprised primarily of these 6 conserved TM domains and appears to be a calcium permeable channel 4,5 . A physical interaction between the carboxy termini of polycystin-1 and polycystin-2 is required to produce a functional channel 6 . The current model for polycystin function is that polycystin-1 is an extracellular sensor that mediates calcium flux through the polycystin-2 channel 7,8 . Likewise, we speculate that LOV-1 acts as a receptor that regulates PKD-2. Our main objective is to use in vivo experimental approaches to test if these structural and functional predictions for the polycystins are true. My first goal is to determine the overall membrane topology of LOV-1. That is, I will determine where the amino and carboxyl tail of LOV-1 is found relative to the plasma membrane. I will also determine which of the 11 hydrophobic regions found in the LOV-1 sequence are functional transmembrane spanning domains. My second goal is to determine whether the LOV-1 protein is cleaved at the GPS and to test whether the PLAT/LH2 domain is required for LOV-1 membrane localizaton and protein function. Ultimately, I will characterize calcium channel activity in vivo . By using C. elegans as a model system for ADPKD, we may begin to understand the physiological function(s) of the polycystins. 1. Barr, M. M., Demodena, J., Hall, D. H., Braun, D. & Sternberg, P. W. Nat Genet submitted . 2. Barr, M. M. & Sternberg, P. W. Nature 401 , 386-9. (1999). 3. Miled, N. et al. Biochimie 82 , 973-86. (2000). 4. Gonzalez-Perrett, S. et al. Proc Natl Acad Sci U S A 98 , 1182-1187 (2001). 5. Vassilev, P. M. et al. Biochem Biophys Res Commun 282 , 341-50. (2001). 6. Hanaoka, K. et al. Nature 408 , 990-4. (2000). 7. Calvet, J. P. & Grantham, J. J. Semin Nephrol 21 , 107-123. (2001). 8. Grantham, J. J. & Calvet, J. P. Proc Natl Acad Sci U S A 98 , 790-792. (2001).