Bruce Bowerman et al. (2006) Development & Evolution Meeting "Anterior-Posterior Asymmetry in Beta-Catenin Localization During Embryonic Cell Divisions in the Spiral-Cleaving Polychaete Platynereis dumerilii"

Stephan Schneider, Bruce Bowerman

[Development & Evolution Meeting, 2006]

Beta-catenin functions in the canonical Wnt-signaling pathway by activating target gene expression in the nucleus. Early roles for beta-catenin signaling are surprisingly different among animal phyla. To gain insight into the ancestral role of beta-catenin, we are using the polychaete Platynereis dumerilii. This annelid's slowly evolving gene sequences, and its phylogenetic position as a lophotrochozoan, make it an attractive model, both for inferring ancestral bilaterian functions and for linking invertebrate and vertebrate model systems. Platynereis embryos deploy a highly stereotypic cell division program called spiral cleavage that generates blastomeres of defined size and cell fate, in a series of asynchronous anterior-posterior (a-p) oriented cell divisions. We observed a-p asymmetries in the levels of detectable beta-catenin during the first 70 embryonic cell divisions, from fertilization, through the spiral cleavage stages, and up to the emergence of bilateral symmetry. In every a-p cell division, beta-catenin accumulated to higher levels in the nuclei of posterior daughters. This asymmetry occurs independent of cell size, cell cycle, cell fate, and cell lineage. Interestingly, the asymmetry is lost only in transverse/bilateral cell divisions, and is reversed only in the germline lineage. These results are strikingly reminiscent of the a-p oriented divisions and Wnt pathway asymmetries that occur throughout embryonic and post-embryonic development in C.elegans. We also find similarities to beta-catenin signaling in early deuterostome and insect embryos. We propose that this a-p asymmetry of beta-catenin in daughter cells constitutes an ancestral and versatile developmental module that has been deployed and modified throughout evolution in metazoan embryos.

Sugioka, Kenji et al. (2015) International Worm Meeting "Asymmetric ubiquitination of the contractile ring by CUL-3 E3 ubiquitin ligase complex regulates asymmetric cytokinesis in P0 cell."

Sugioka, Kenji, Bowerman, Bruce

[International Worm Meeting, 2015]

In the ubiquitin proteasome system, E3 ubiquitin ligases serve to ubiquitylate target substrates and thereby target them for the proteasome dependent degradation. During C. elegans early embryogenesis, the CUL-2 and CUL-3 E3 ligase complexes play essential roles by regulating the degradation of developmental factors. However, the targets of these E3 ligases and their developmental significance are not entirely known. Here we found a new role for the CUL-3 E3 ligase during cytokinesis. Interestingly, C. elegans zygote P0 cell is known to undergo an asymmetric cytokinesis, with the contractile ring on one side closing faster than on the opposite side, such that the midbody is eventually off-centered. We found that cul-3(RNAi) resulted in a loss of this asymmetry during cytokinesis. The CUL-3 binding partner and substrate adaptor MEL-26 also is required for this asymmetric cytokinesis. Using a GFP::CUL-3 fusion we generated by CRISPR/Cas9 genome editing, and an anti-MEL-26 antibody, we found that both CUL-3 and MEL-26 localized to the cleavage furrow during cytokinesis, suggesting that this E3 ligase complex functions at the contractile ring. The CUL-3/MEL-26 complex may be selectively transported to the cleavage furrow, as RNAi knockdown of the early endosomal component RAB-5 specifically diminished CUL-3/MEL-26 localization at the cleavage furrow. How does this E3 ligase on the contractile ring regulate asymmetric cytokinesis? Surprisingly, we found that ubiquitylation at the contractile ring was strongly asymmetric, and this ubiquitylation was CUL-3 dependent. A previous study has reported the asymmetric accumulation of contractile ring components, including the non-muscle myosin NMY-2, to the side where the ring closes faster during asymmetric cytokinesis. We found that in cul-3(RNAi) embryos, NMY-2 localization became symmetric, suggesting that CUL-3 dependent ubiquitylation is required for the formation of an asymmetric contractile ring. Simultaneous staining of ubiquitin and NMY-2 showed that poly-ubiquitin exclusively localized to the fast closing side while NMY-2 localized to both sides with enrichment on the fast side. Moreover, poly-ubiquitin only localized to the inner contractile ring while NMY-2 also localized to the outer ring. In summary, we have found that the CUL-3/MEL-26 E3 ubiquitin ligase regulates the asymmetric ubiquitination of an unknown protein at the inner contractile ring on the fast closing side during this asymmetric cytokinesis. To our knowledge, this is the first example of an asymmetric ubiquitination within a cell. Moreover, this ubiquitylation is required for the asymmetric localization of NMY-2 to the contractile ring and for the asymmetry of this cytokinesis. .

Lowry, Josh et al. (2015) International Worm Meeting "A genetic screen for mutants with defects in the regulation of gonad membrane dynamics during adult oogenesis."

Lowry, Josh, Bowerman, Bruce

[International Worm Meeting, 2015]

C. elegans hermaphrodites produce oocytes in an assembly line fashion with syncytial nuclei located at the periphery of the distal arm sharing a common cytoplasm called the rachis. As oocyte nuclei move proximally and approach the bend of the gonad they begin to cellularize and then enlarge as they move through the proximal arm toward the spermathecum and uterus. This process results in the production of oocytes equivalent in volume to the entire gonad every six hours1. Thus oogenesis must involve a dynamic regulation of the complex membrane architecture of the gonad as oocyte nuclei move proximally and undergo cellularization. To identify genes that mediate this regulation of membrane dynamics, we have screened for temperature-sensitive mutants that share two phenotypes: osmotically sensitive eggshell-defective (Osm) after up-shifts of L4 larvae to the restrictive temperature, and adult sterility (Ste) after up-shifts of L1 larvae. Because eggshell formation and gonad development both require membrane trafficking, we hypothesized that Osm/Ste mutants might be defective in membrane-trafficking functions important for the dynamic regulation of the adult gonad membrane architecture during oogenesis. From a collection of 102 Osm mutants, we identified a subset of 25 that are recessive and also are highly penetrant Ste mutants. To determine if these Osm/Ste mutants have defects in adult oogenesis, we applied a high-throughput positional cloning approach that uses Illumina-based whole-genome sequencing and SNP mapping2,3. After identifying candidate mutations, we then used genetic complementation tests with available deletion alleles to determine gene identity for 10 Osm/Ste mutants. With one exception, all of the genes we identified are highly conserved and a subset-vps-15 (yeast vacuolar protein sorting), drp-1 (dynamin-related protein), ippk-1 (homolog of vertebrate inositol polyphosphate kinase 1)-have orthologs in other organisms that are known to be involved in membrane trafficking or membrane metabolism. We also identified an allele of sqv-8, which is required for the glycosylation of extracellular matrix proteins, and alleles of genes that are likely to be required for more general processes of gene expression and thus are unlikely to be specifically required for membrane regulation: abtm-1 (ABC-family iron transport protein, mitochondrial), crn-3 (cell death-related nuclease), and rpl-7 (large ribosomal subunit protein). To explore the requirements for vps-15, drp-1, and ippk-1, we have examined gonad membrane architecture after up-shifting mutants to the restrictive temperature at the L1, L4 and early adult stages, using confocal microscopy and a fluorescent marker labeling the plasma membrane.

Chuang, Chien-Hui et al. (2017) International Worm Meeting "ZYG-9 and TAC-1 are involved in early meiotic spindle assembly in C. elegans oocytes."

Bowerman, Bruce, Chuang, Chien-Hui

[International Worm Meeting, 2017]

Oocytes of many animals form bipolar spindles in the absence of centrosomes, and how these acentrosomal spindles establish bipolarity remains unclear. In C. elegans oocyte meiosis I, spindle microtubules first form a cage-like structure peripheral to the chromosomes, with multiple small and peripheral spindle poles appearing as assembly progresses. These small poles later merge together and ultimately form two mature poles on the opposite sites of the aligned chromosomes (Wolff et al., 2016). Previous studies have documented defects in oocyte meiotic spindle structure in ZYG-9 or TAC-1 depleted oocytes (Matthews LR et al., 1998; Bellanger JM and Gonczy P, 2003; Yang et al., 2003), but when these assembly defects first appear is not known. Using live imaging, we are investigating the temporal requirements for ZYG-9 and TAC-1 during meiotic spindle assembly. Here we show that ZYG-9 and TAC-1 are required very early in spindle formation, with defects that then further interfere with the establishment of spindle bipolarity. Using GFP and mCherry fusions to mark microtubules and chromosomes, respectively, in zyg-9 (-) oocytes, we have detected defects very early in meiotic spindle assembly. Rather than forming a cage-like structure with all microtubules restricted to the periphery, we instead observed a cage-like structure with some microtubules extending across the interior, projecting through the volume occupied by chromosomes. This abnormal microtubule scaffold subsequently assembled into a multi-polar spindle network, with individual bivalents sometimes surrounded by discrete small bipolar spindles. Ultimately chromosomes often segregated toward more than two poles. Similarly, using GFP fused to ASPM-1 to mark spindle poles, we found that wild-type oocytes had two stable ASPM-1 foci established by metaphase, while zyg-9 (-) oocytes had multiple ASPM-1 foci that dynamically coalesced and dispersed throughout most of meiosis I. ZYG-9 and TAC-1 form a complex that promotes microtubule assembly (Bellanger JM and Gonczy P, 2003; Le Bot N et al., 2003; Srayko M et al., 2003), and TAC-1 depleted oocytes showed similar phenotypes to the zyg-9 (-) oocytes. We are currently investigating the localization of the ZYG-9/TAC-1 complex during early meiotic spindle assembly to further advance our understanding of how these proteins contribute to the acentrosomal assembly of bipolar spindles during oocyte meiotic cell division. References: Wolff et al., Mol Biol Cell, 2016; Matthews LR et al., J Cell Biol., 1998; Bellanger JM and Gonczy P, J Cell Biol., 2003; Yang et al., Dev Biol., 2003; Le Bot N et al., Curr Biol., 2003; Srayko M et al., Curr Biol., 2003.

Lowry, Josh et al. (2013) International Worm Meeting "Using Next-Generation Sequencing to Determine Gene Identity in Temperature-Sensitive, Embryonic Lethal Mutants."

Connolly, Amy, Lowry, Josh, Bowerman, Bruce

[International Worm Meeting, 2013]

While ~2500 genes in C. elegans are essential, temperature-sensitive alleles are available for only 100-200 of these genes. A major hurdle following any genetic screen has been the positional cloning of causal mutations. This process can be difficult and time consuming, and in the past most research has focused on narrow sub-classes of mutant phenotypes. To more rapidly determine gene identity in conditionally lethal mutants, we are employing simultaneous whole-genome sequencing and SNP mapping (Doitsidou et al, 2010). Briefly, the mutant of interest is outcrossed to the polymorphic strain CB4856.Then populations from ~50 F2 homozygous mutant progeny are pooled and genomic DNA extracted and sequenced on an Illumina HiSeq 2000. SNP mapping data is extracted from the resulting sequencing data using the CloudMap tool (Minevich et al, 2012), defining a chromosomal region in which the mutation lies. The same sequencing data is then used to generate a list of candidate mutations in that region. Our goal is to map and identify the causal mutations in hundreds of conditionally lethal mutants, sampling a wide range of mutant phenotypes in an effort to explore new areas of essential gene function research. One class of mutants we are now exploring exhibit lethal defects at multiple stages of development. Mutants shifted to the restrictive temperature at the L4 stage produce embryos with eggshell defects. Shifting up at the L1 stage produces sterile adults with gonad development defects. We hypothesize that this class of mutants, the Osm/Ste class, may prove useful for studying the role of membrane trafficking during gonad morphogenesis. To date, we have obtained WGS-SNP mapping data for 16 mutants, with another set of 8 soon to follow. A short list of candidate mutations has been determined for each of them, with complementation tests and transgenic rescue to follow where appropriate. Current candidates include rpl-7, gip-2, atx-2, aco-2, abtm-1 and drp-1. This high-throughput approach will allow us to clone more mutants far more quickly than has previously been possible.

Chuang, Chien-Hui et al. (2013) International Worm Meeting "Investigating loss-of-function suppressors of C. elegans centrosomal defective mutants."

O'Rourke, Sean, Chuang, Chien-Hui, Bowerman, Bruce

[International Worm Meeting, 2013]

Centrosomes act as microtubule organizing centers, and are critical for cell division and cell polarity. Identifying the regulators and components of centrosomes is an important step in understanding cell division. To this end, our lab has completed genome-wide modifier screens to identify genes that, when reduced in function using feeding RNAi, can suppress lethality in three conditional centrosome-defective mutants: spd-2(or183ts), spd-5(or213ts) and zyg-1(or278ts). The spd-2 gene encodes a coiled-coil protein essential for centrosome duplicaion and maturation; zyg-1 encodes a kinase required for daughter centriole formation; and spd-5 encodes a coiled-coil protein required for centrosome maturation. We have isolated 9 non-essential genes and one essential gene that specifically suppress the embryonic-lethality associated with one or more of the three mutants. We are currently investigating these 10 genes for their roles in regulating microtubule dynamics and mitotic spindle assembly and positioning. One gene, H37N21.1/hpo-11, strongly and specifically suppresses the lethality of spd-5 after RNAi knockdown. When we use RNAi to knock down H37N21.1/hpo-11 in wild-type embryos, we observe a substantial delay in rotation of the first mitotic spindle and a highly penetrant loss of spindle rocking during anaphase at the one-cell stage. These results suggest there may be abnormalities in microtubule dynamics in embryos lacking H37N21.1. Interestingly, H37N21.1 is conserved, with a mammalian ortholog called MADM (Mlf-1 adaptor molecule). MADM encodes a kinase-like protein localizes to centrosomes, and physically associates with the Mlf1 oncoprotein, which is involved in acute myeloid leukemia. However, the role of MADM at centrosome and in leukemia is little understood. We are investigating the cellular localization and function of H37N21.1, and of the other suppressors, to understand how they influence centrosome fuction and suppress lethality.

Canman, Julie C. et al. (2009) International Worm Meeting "The cloning of three conditional fast-inactivating icp-1 (INCENP) alleles."

Bowerman, Bruce, Canman, Julie C., Oegema, Karen

[International Worm Meeting, 2009]

Cell division is a dynamic process controlled by a number of proteins, among which some are multifunctional and are required at multiple steps. Loss-of-function of these master regulators, by classical techniques of reverse genetics, leads to a complex pleiotropic phenotype due to the combination of defects at these various steps. The role of these master regulators in a specific aspect of cell division is thus challenging to address. One such important master regulatory complex is the Chromosome Passenger Complex (CPC), which participates throughout meiosis, mitosis, and during cytokinesis. Indeed, disrupting function of the CPC by traditional forward or reverse genetics results in massive chromosome segregation defects as well as a failure to divide. To examine the role of the CPC during cytokinesis specifically, we have taken a forward genetics approach in C. elegans to isolate conditional alleles which phenocopy loss-of-function of the CPC showing both massive chromosome segregation and cytokinesis defects. Using this approach we found three mutant lines that are all completely impaired in chromosome segregation and cytokinesis. These mutants all failed to complement with each other indicating they are alleles of the same gene. Using traditional visible marker mapping, we mapped these mutants to the left arm of chromosome I, which contains the C. elegans INCENP homolog, ICP-1INCENP, the core scaffolding protein of the CPC. We sequenced the icp-1 locus in these mutants and found that all three mutants contain single mis-sense mutations in icp-1 resulting in single amino acid changes. All three alleles are fast-inactivating, showing a fully penetrant loss-of-function phenotype within seconds of shifting to the restrictive temperature. Thus, they will be powerful tools for studying the role of the CPC in cytokinesis independent of chromosome segregation during meiosis and mitosis.

Connolly, Amy et al. (2011) International Worm Meeting "A screen for C. elegans oocyte meiotic spindle-defective mutants."

Connolly, Amy, Yochem, John, Bowerman, Bruce, Lowry, Josh

[International Worm Meeting, 2011]

We are interested in identifying and investigating essential genes involved in oocyte meiotic spindle assembly, as part of a large-scale screen for temperature-sensitive, embryonic lethal-mutants. We initially identify mutants based on temperature-sensitive embryonic lethality, and then examine their early embryonic cell divisions using time-lapse DIC imaging. In a small number of these mutants, we detect an abnormal number of maternal pronuclei in the one-cell zygote, suggestive of oocyte meiotic spindle assembly or function defects. This far we have found several mutants with either multiple or no maternal pronuclei. One new mutant, or1129ts, has multiple maternal pronuclei, while another or974ts sometimes lacks a maternal pronucleus. We have narrowed the locations of or1129ts and or974ts to linkage group IV and V, respectively, by using Restricted-site Associated DNA polymorphism (RAD) mapping. RAD mapping relies on crossing the mutant into the polymorphic Hawaiian strain (CB4856), and then isolating and pooling F2s that are again homozygous for the mutation. The genomic DNA is purified then cut with a restriction enzyme (EcoRI) and ligated to barcoded adaptors. Finally, Illumina DNA sequencing is used to sample SNPs throughout the genome and narrow the mutation to a 1-3 Mb region. The or1129ts and or974ts mutations complement the kinesin mutants klp-18(or447ts) IV and bmk-1(or627ts) V, respectively, the only other meiotic spindle mutants we know of that map to these linkage groups (see abstract by Osterberg et al), suggesting that these mutations are alleles of other genes. We are now using whole genome sequencing to determine the identity of the affected gene in each mutant. We will characterize their mutant phenotypes using GFP and mCherry fusions to microtubules, histones, and other proteins for live cell imaging, as part of an ongoing effort to define the genetic pathways and networks that control C. elegans meiotic spindle assembly.

O'Rourke, Sean et al. (2009) International Worm Meeting "EFA-6 induces microtubule catastrophe in early C. elegans embryos."

O'Rourke, Sean, Bowerman, Bruce, Kwong, Ronald, Christensen, Sara

[International Worm Meeting, 2009]

During mitotic spindle assembly, microtubules polymerize outwards from centrosomes and reach the cell cortex. Microtubule-cortical contacts are important for spindle positioning and function. In early C. elegans embryos, microtubules contact the cortex briefly before undergoing catastrophe. Cortical molecules that regulate the switch from growing to shrinking microtubules have not been described. Here we show that a cortically-localized, putative GTPase exchange factor called EFA-6 promotes microtubule catastrophe, as embryos lacking this protein display long microtubules at the cell cortex. We have found that microtubule plus ends reside at the cortex greater than 5 times longer in efa-6 mutant embryos. However, astral microtubules in embryos lacking EFA-6 are nucleated and grow at identical rates, when compared to wild-type embryos. The abnormally long microtubules cause centrosomes to detach from the male pronucleus, abolish anaphase spindle oscillations, and can suppress the embryonic lethality associated with partial loss of cytoplasmic dynein. EFA-6 is widely conserved and is known to exchange GDP for GTP on ARF6 GTPases. However, ARF-6 and EFA-6 exchange factor activity are not required for this regulation of microtubule dynamics. Instead, the N-terminus of EFA-6 is essential for this activity, as is a pleckstrin homology domain that presumably mediates cortical localization. Thus, EFA-6 has novel, exchange factor-independent function(s) during early embryonic cell divisions. We are currently testing whether the EFA-6 N-terminal domain is sufficient to promote microtubule catastrophe when it is targeted to the cortex through a CAAX motif, independent of its own PH domain.

Keikhaee, Reza et al. (2011) International Worm Meeting "repo-1(or430ts): a C. elegans mutant with a reversed anterior-posterior body axis."

Keikhaee, Reza, Nash, Bruce, Bowerman, Bruce, Yochem, John

[International Worm Meeting, 2011]

Over the course of one hour, following fertilization of an oocyte, the apolar one-cell C. elegans zygote is transformed into a highly polarized cell that divides asymmetrically to produce anterior and posterior daughters with different sizes, contents, cell cycle times and developmental potentials. While many of the molecules required for anterior-posterior (AP) cell polarity are conserved across animals, the mechanisms that establish it remain unclear. We have identified a dominant, temperature-sensitive, embryonic-lethal allele (or430ts) of the C. elegans ortholog of SF3a66 called or430ts. SF3a66 is a subunit of human SF3a (splicing factor 3a) that is involved in the processing of pre-mRNA, but recent studies have shown that SF3a66 may also act as a microtubule binding and bundling protein independent of its RNA splicing role, and the C. elegans ortholog is a synthetic multivulva (synMuv) protein. We found that at the restrictive temperature (26 deg C), embryos produced by or430ts/or430ts worms (hereafter called mutant zygotes or embryos) exhibit a remarkable reversal of AP cell polarity at the one-cell stage. Differential interference contrast microscopy (DIC) showed a decrease in cortical ruffling and pseudocleavage, and the sperm and egg pronuclei meet centrally in this mutant, instead of meeting toward the posterior pole as in wild type, suggesting a loss of AP polarity in repo-1(or430ts) mutant zygotes. Spinning disk confocal microscopy with GFP transgenic strains showed that the posterior polarity proteins PAR-2 and PIE-1 are localized to the anterior pole in one-cell stage mutant zygotes, confirming that polarity is reversed; hence the gene name repo-1, for reversed polarity. We also have observed microtubule defects in repo-1(or430ts) one-cell zygotes, including defective rotation of the centrosome/pronuclear complex, a lack of P0 mitotic spindle elongation, and chromosome segregation defects following the first embryonic mitosis. Since depletion of microtubules has been shown to delay AP polarity induction, we are interested in the possibility that the dominant or430ts mutation results in reversed polarity by affecting microtubule dynamics. Because arrested meiotic spindles also have been shown to cause a partial reversal of AP polarity, we currently are investigating meiotic spindle dynamics in repo-1(or430ts) mutant to determine if abnormalities in meiotic spindle assembly or dis-assembly might be responsible for the reversed polarity phenotype.