Kuwabara, Patricia E. (2012) Worm Breeder's Gazette "Notable recent papers in biochemistry"

Kuwabara, Patricia E.

[Worm Breeder's Gazette, 2012]

Kuwabara, Patricia E et al. (2012) Worm Breeder's Gazette "A universal Cbr-unc-119::FRT*::galk::FRT* template for recombineering and biolistics"

Qiao, Mengmeng, Kuwabara, Patricia E

[Worm Breeder's Gazette, 2012]

Kuwabara, Patricia et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The C. elegans Patched-related protein PTR-2 is required for somatic cytokinesis"

Soloviev, Alexander, Kuwabara, Patricia, Zugasti, Olivier

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

The Hedgehog (Hh)/Patched (Ptc) signalling pathway promotes cell proliferation and plays a central role in establishing embryonic polarity and organ patterning in Drosophila and vertebrates. In humans, dysregulation this pathway can cause cancer and developmental abnormalities. C. elegans encodes a large number of Ptc and Hh-related proteins, but it remains unclear whether these proteins participate in cell-cell signalling (1,2). We previously showed that ptc-1 is essential for germ line cytokinesis (3). To determine whether PTC proteins play more global roles in somatic cytokinesis, we performed an RNAi screen. To this end, we identified the ptr-2 (ptc-related) gene. ptr-2(RNAi) embryos arrest with multinucleated cells, which we attribute to a defec t in cytokinesis (1). Given the multitude of ptc and ptr genes in the worm, we obtained a ptr-2 mutant to show that the disruption of cytokinesis by ptr-2(RNAi) is not due to off target effects. We find that ptr-2 mutants do not display a phenotype identical to that of ptr-2(RNAi) animals; however, cytokinesis defects are indeed present in the progeny of ptr-2 (m- z+) mothers. Time-lapse recordings of ptr-2(RNAi) embryos reveal defects in chromosome condensation/segregation, spindle positioning and cleavage furrow formation. We have generated PTR-2 antibodies and have examined the intracellular localisation of PTR-2. To gain a better understanding of PTR-2 dynamics, we have generated a pie-1::ptr-2::mRFP reporter, which is capable of rescuing a ptr-2 mutant. We will show that PTR-2 is dynamically distributed in vesicles throughout the cell-cycle and discuss the role of the cytoskeleton in PTR-2 localisation. 1. Zugasti et al., 2005 2. Burglin and Kuwabara, 2006 3. Kuwabar a et al., 2000

Kuwabara PE (1994) Worm Breeder's Gazette "Evidence For Interactions Between the Sex Determination and Dosage Compensation Pathways in the Germline"

Kuwabara PE

[Worm Breeder's Gazette, 1994]

Evidence for Interactions Between the Sex Determillation and Dosage Compensation Pathways in the Germline Patricia E. Kuwabara MRC-LMB Hills Road Cambridge CB2 2QH England

Patricia E Kuwabara et al. (2001) International C. elegans Meeting "The C. elegans ptc and ptr genes: A cholesterol connection?"

Tim Schedl, Patricia E Kuwabara, Min-Ho Lee, Gregory Jefferis, Olivier Zugasti

[International C. elegans Meeting, 2001]

Patched (PTC) is a multipass membrane protein controlling cell fate and proliferation; in humans, PTCH functions as a tumour suppressor. Biochemical analyses have shown that PTC is a receptor for Hedgehog (Hh). It was postulated that PTC and the serpentine membrane protein Smoothened (SMO) form a complex whereby Patched inhibits SMO; Hh relieves this inhibition by binding to PTC. In turn, SMO activates the transcriptional regulator Ci to express TGF-beta and Wnt family members. In C. elegans , BLAST and Clustal W analyses indicate that there are 3 ptc genes and 26 ptc-related ( ptr ) genes. Moreover, the hydropathy plots of ptc and ptr genes are very similar; these proteins are predicted to encode 12-pass membrane proteins with topologies similar to those of Patched proteins identified in other organisms. The PTC and PTR proteins also belong to a larger family of proteins containing sterol sensing domains (SSD); members of the SSD family include PTC, Dispatched (CHE-14), HMG CoA reductase, SCAP, SREBP and NPC1. We have searched the C. elegans genome for other components of the Hh/PTC pathway. There are no obvious Hedgehog or Smoothened homologues encoded by the genome although a large family of Hedgehog-like proteins have been identified (Aspock et al.1999. Genome Res. 9: 909), some of which may have signalling properties. Moreover, the activity of TRA-1, the single C. elegans homologue of Ci, appears to have been usurped by the sex determination pathway. The apparent absence of many of the components of the Hedgehog/Patched signalling pathway in C. elegans has led us to examine the role of Ce-ptc-1 in the development of the worm. From an evolutionary standpoint, a study of the Ce-ptc genes might shed light on the ancestral roles of Patched proteins or perhaps uncover new functions. Results of RNAi and mutational deletion studies will be presented indicating that ptc-1 is an essential germline gene (Kuwabara et al. 2000, Genes Dev.14:1933). Animals lacking ptc-1 activity are essentially sterile with multinucleate germ cells arising from a probable defect in germline cytokinesis. The membranes normally separating individual germ cell nuclei are absent in ptc-1 mutants; the loss of these membranes allow multiple nuclei to cycle synchronously through mitosis. We conclude that these membranes maintain autonomous domains within the germline syncytium. It is unclear whether ptc-1 mutants display cytokinesis defects because the cleavage furrow is not formed or because it is not stabilized. Anti-PTC-1 polyclonal antibodies indicate that PTC-1 protein is normally enriched at the apices of the membranes separating individual germ cells. One interpretation of the ptc-1 mutant phenotype is that ptc-1 normally plays a germline-specific role in membrane trafficking. In addition to our studies on the ptc genes, we are undertaking a global analysis of the 26 C. elegans ptr genes. Preliminary results of these studies will be presented. Taken together, our analyses of the ptc-1 and ptr genes combined with studies of other C. elegans SSD protein encoding genes, such as che-14 (Michaux et al. 2000. Curr. Biol. 10:1098) and npc-1 and npc-2 (Sym et al. 2000. Curr. Biol. 10:527) indicate that the SSD proteins are involved in a diverse range of developmental processes, which may all share a common link to cholesterol.

Patricia E Kuwabara (1999) International C. elegans Meeting "The C. elegans ptc genes play essential, distinct and apparently Hh-independent developmental roles"

Patricia E Kuwabara

[International C. elegans Meeting, 1999]

Patched-dependent cell signalling is widespread in the animal kingdom, playing roles in developmental patterning, cell proliferation, and carcinogenesis. The first patched (ptc) gene was identified in Drosophila because of its role in establishing segment polarity. In humans, Ptc is a tumour-suppressor protein in basal cell carcinomas, and mutations in PTCH are implicated in Gorlin's syndrome. Biochemical evidence indicates that Ptc and the serpentine protein Smoothened (Smo) form a receptor complex for the morphogen Hedgehog (Hh). Current models suggest that Ptc inhibits Smo, and that Hh relieves this inhibition by binding to Ptc. In turn, Smo promotes the transcription of TGF- b and Wnt family members. In C. elegans , three genes encode proteins with strong sequence similarity to Drosophila and vertebrate PTC proteins. In addition, a number of other predicted C. elegans proteins comprise a subfamily of distant PTC relatives. Here it is reported that ptc-1 and ptc-3 are the C. elegans genes most closely related to the ptc genes so far identified in other phyla; ptc-2 appears to be a pseudogene that arose through a recent duplication of ptc-1 . Genomic and cDNA sequence analyses have been performed for both ptc-1 and ptc-3 . Similar to other Ptc proteins, the predicted PTC-1 and PTC-3 proteins each have 12 potential membrane spanning domains and a cholesterol sensing domain. PTC-1 and PTC-3 share approximately 33% identity with Ptc proteins from other phyla. Inactivation of ptc-1 or -3 by RNAi reveals that each gene is essential for development: ptc-1 is needed for cellularisation during gametogenesis, whereas ptc-3 is required for late embryogenesis. To corroborate the ptc-1(RNAi) and ptc-3(RNAi) phenocopies, mutants have been obtained that delete regions of either ptc-1 or ptc-3 (thanks to B. Barstead and L. Liu). Each of the deletion mutants displays phenotypic properties similar to those observed by RNAi. Surprisingly, both Hh and Smo appear to be absent from the C. elegans genome. Therefore, these findings suggest that the control of ptc activity may have diverged during evolution, and further raise the possibility that PTC may have functions that are both Hh- and Smo-independent.

Mehra A et al. (1994) Worm Breeder's Gazette "Direct Interaction Between FEM-3 and a Carboxy-Terminal Fragment of TRA-2A"

Spence AM, Heck L, Kuwabara PE, Mehra A

[Worm Breeder's Gazette, 1994]

Direct Interaction between FEM-3 and a Carboxy-Terminal Fragment Or TRA-2A Arun Mehra, Linda Heck, Patricia Kuwabara*, and Andrew Spence Dept. of Medical Genetics, University of Toronto, 1 King's College Circle, Toronto, Canada, and *MRC Laboratory of Molecular Biology, Hills Rd., Cambridge, UK

Soloviev, Alexander et al. (2009) International Worm Meeting "The Patched-related protein PTR-2 is required for somatic cytokinesis in C. elegans."

Soloviev, Alexander, Kuwabara, Patricia, Zugasti, Olivier

[International Worm Meeting, 2009]

C. elegans is positioned at an evolutionary cusp with regard to the Hedgehog (Hh) /Patched (Ptc) signalling pathway. The worm lacks an obvious Hh orthologue, but encodes a large number of Ptc-related proteins (Zugasti et al., 2005). Moreover, the Ptc family of proteins has undergone a significant expansion in the worm (Burglin and Kuwabara, 2006). It remains unclear whether the Ptc proteins of C. elegans participate in cell-cell signalling; however, we have shown that ptc-1 plays an essential role in the development of the germ line. Mutants lacking ptc-1 are sterile because of a defect in cytokinesis (Kuwabara et al., 2000). In order to determine whether the PTC proteins play more global role in cytokinesis, we sought to determine whether the absence of any member of this family disrupted somatic cytokinesis. To this end, we identified the ptr-2 (ptc-related) gene. We have shown that ptr-2(RNAi) causes embryos to arrest due to a cytokinesis defect that results in the formation of multinucleated blastomeres. In the most severe cases, the embryos arrest at the 1-cell stage of development. Time-lapse recordings of ptr-2(RNAi) embryos reveal that a knockdown of ptr-2 activity affects multiple aspects of cell division, including chromosome condensation/segregation, spindle positioning and cleavage furrow formation (Zugasti et al., 2005). Given that there are a large number of ptc and ptr genes in the worm, we felt that it was important to establish that the ptr-2(RNAi) defects were not due to off target effects. We show that a mutation in ptr-2 gives rise to an embryonic cytokinesis defect, although the mutant phenotype is not identical to that observed by RNAi. To understand the basis of the multiple defects caused by the knockdown of ptr-2, we have also examined the intracellular localisation of PTR-2 protein using an anti-PTR antibody. We will show that PTR-2 is dynamically distributed in vesicles throughout the cell-cycle and discuss the role of the cytoskeleton in PTR-2 localisation.

Qiao, Mengmeng et al. (2015) International Worm Meeting "C. elegans WD40 domain containing protein LAWD-1 possibly mediates protein-protein interactions during epithelial morphogenesis."

Kuwabara, Patricia, Qiao, Mengmeng

[International Worm Meeting, 2015]

Morphogenesis in C. elegans is a highly dynamic process in which a bean shaped embryo is transformed into its final adult form. During this process, epithelial cells extend to form sheets and tubes that shape the body and form organs. Disrupted epithelial morphogenesis often leads to embryonic arrest and defects, such as those displayed by the vab-1 and vab-2 mutants, which encode the ephrin and eph receptor, respectively(1,2). Here, we describe the role of the lawd-1 (lumen associated with WD40 domain) gene in epithelial morphogenesis. The lawd-1 mutants cr7 and tm4605 each display a highly penetrant viable Vab (Variable abnormal) phenotype and a less penetrant embryonic lethality. Genetic analyses indicate that neither allele is likely to be null, hence a new knockout allele is being generated using CRISPR/Cas9. Both lawd-1 mutants are rescued by a recombineered fosmid containing lawd-1::mCherry (C-terminal). The LAWD-1::mCh reporter is expressed throughout development in all epithelial tissues except the hypodermis. Given that lawd-1 mutants have a Vab phenotype, the absence of hypodermal expression, except in seam cells, is surprising although this may simply be due to a lack of germline expression despite the ability of the reporter to rescue lawd-1 mutants. We are making a germline reporter to test this possibility. When lawd-1::mCh is co-expressed in embryos with the epithelial markers hmp-1::gfp, dlg-1::gfp or lifeact::gfp, we find that LAWD-1::mCh is apically enriched in epithelial cells. The lawd-1 gene is predicted to encode multiple isoforms, a number of which have been detected by western analysis. Some of these isoforms have the potential to form an N-terminal double-propeller with 2x7 WD40 repeats. The presence of a LAWD-1 WD40 domain has led us to hypothesise that LAWD-1 mediates protein-protein interactions with other proteins involved in epithelial morphogenesis. To test this hypothesis we have co-immunoprecipitated LAWD-1::mCh and identified potential interacting partners by mass spectrometry. Several of the candidates isolated have known roles in epithelial development. One such candidate is VAB-10B, a spectraplakin isoform that interacts with actin during elongation3. Interestingly, a synthetic lethal interaction has also been detected between lawd-1 and sma-1, which encodes the actin interacting protein beta-H spectrin4. We are currently validating the candidate LAWD-1 interacting proteins identified in our screen.Ref. 1Chin-Sang ID et al. (1999) Cell. 99;781. 2George SE et al. (1998) Cell. 92;633. 3Bosher JM et al. (2003) J. Cell Biol. 161;757. 4McKeown C et al. (1998) Development. 125(11);2087.

Kuwabara PE et al. (2000) Parasitol Today "RNAi--prospects for a general technique for determining gene function."

Kuwabara PE, Coulson AR

[Parasitol Today, 2000]

Gene discovery programs centred around expressed sequence tag (EST) and genome sequencing projects have predictably led to an exponential surge in the number of parasite gene sequences deposited in public databases. To take advantage of this wealth of sequence information, it is essential to develop rapid methods for elucidating the biological function or mode of action of individual genes. Here, Patricia Kuwabara and Alan Coulson discuss the virtues of a powerful epigenetic gene disruption technique, RNA-mediated interference (RNAi), which was originally developed for the nematode Caenorhabditis elegans. It is anticipated that this technique will not only provide insights into gene function, but also help investigators to mine the genome for candidate drug intervention or vaccine development targets, some of which may not be readily apparent on the basis of sequence information alone.