Bembenek, Joshua N. (2011) International Worm Meeting "Analysis of Protease-Dead Separase: Development of a New Method to Study Dominant-Negative Transgenes."

Bembenek, Joshua N.

[International Worm Meeting, 2011]

The C. elegans embryo is an ideal system to study several important processes including egg activation and cell division. We are studying the role of separase, a protease required for chromosome segregation, in regulation of RAB-11 vesicle trafficking during anaphase. A key question is whether the protease activity of separase is required for its role in vesicle exocytosis. To address this question we generated a protease-dead separase mutant (GFP::SEP-1PD) and found that it accumulated more strongly at sites of action within the cell during anaphase, suggesting that it traps substrates. GFP::SEP-1PD expression causes high rates of embryonic lethality and severe adult phenotypes, suggesting that it is dominant-negative and interferes with endogenous separase function. Using the generally applicable method described below to propagate strains with dominant-negative transgenes, we plan to investigate the protease-dependent functions of separase in the C. elegans embryo. The most common method for expression of fluorescent fusion proteins in embryos involves generation of integrated transgenes under the control of the pie-1 promoter, using unc-119 as a selection marker. However, this system is not inducible and prevents generation of lines expressing dominant-negative proteins because strains are inviable. Using standard methods, we obtained lines that express GFP::SEP-1PD which could not be propagated more than a few generations. However, gfp(RNAi) feeding eliminated transgene expression and allowed propagation of GFP::SEP-1PD lines for many generations without obvious deleterious defects. Animals removed from gfp(RNAi) for 5 generations recover transgene expression and associated phenotypes. To obtain more severely dominant-negative or over-expressing transgenes, animals can be rescued after bombardment directly on gfp(RNAi) feeding bacteria. We also took advantage of the fact that the pie-1 promoter has little expression in the male germline. Backcrossing GFP::SEP-1PD males to unc-119 females allows propagation of the line without using gfp(RNAi) and circumventing the 5 generation delay to obtain expression. F1 Heterozygous GFP::SEP-1PD hermaphrodites show high levels of embryonic lethality and 50% of the surviving F2 animals are sterile or lay 100% dead embryos. Transgenic males can also be crossed to mutant lines to examine genetic interactions. Bombardment of an unc-119(ed3);him-5(ok1896) mutant (made by Judy Yanowitz) may also facilitate isolation of dominant-negative transgenes. These methods are immediately applicable for studies of dominant-negative transgenes using widely established techniques and should open new lines of investigation in the C. elegans embryo.

Joshua Bembenek et al. (2006) Development & Evolution Meeting "Separase is Required for Cytokinesis in C. elegans"

John White, Joshua Bembenek

[Development & Evolution Meeting, 2006]

Separase is a widely conserved protease that promotes chromosome segregation at the metaphase to anaphase transition by cleaving a subunit of cohesin, a protein complex that tethers sister chromatids together. It was previously shown that the C. elegans homologue of separase, sep-1, is required for chromosome segregation during embryonic divisions. Unexpectedly, abrogating sep-1 function causes cytokinesis failures. Some experiments indicated that these cytokinesis failures were a secondary defect caused by osmotic and mechanical pressures experienced by the embryo during microscopic observation due to abnormal eggshell formation. Separase also acts in the FEAR network in budding yeast, which stimulates the release of Cdc14 from sequestration in the nucleolus. Cdc14 release is required for promoting anaphase spindle dynamics and subsequently promotes mitotic exit and cytokinesis. Whether Cdc14 is the only target of the FEAR pathway is unknown - cdc-14 is not essential in C. elegans and the nucleolus and nuclear envelope are dispersed during mitosis, indicating that anaphase events may be regulated differently. To investigate whether separase might promote anaphase and cytokinesis in C. elegans, we have analyzed sep-1 mutant phenotypes by an in vivo microscopic analysis of embryos under conditions that minimize mechanical and osmotic pressure. Early meiotic embryos are viable under such conditions and display a prominent period of exocytic membrane activity that is reduced in sep-1(RNAi), possibly explaining the eggshell defect. These conditions can also rescue cytokinesis in other mutants that are sensitive to osmotic and mechanical pressure, but not in sep-1 mutants. Abrogation of sep-1 function using two different temperature-sensitive mutants causes abnormal anaphase spindle movements and cytokinesis failures while relatively normal chromosome segregation still occurs. However, sep-1(RNAi) causes severe chromosome non-disjunction, cytokinesis failures, and defects in sperm pronuclear positioning, centrosome duplication and polarity, which may be associated with improper cell cycle regulation during the transition from meiosis II to the first mitosis. These data support our hypothesis that sep-1 plays a part of a signalling pathway, which promotes cytokinesis in the C. elegans embryo.

Joshua N. Bembenek et al. (2007) International Worm Meeting "Secretion of the Eggshell through Cortical Granule Exocytosis Occurs During Anaphase I in C. elegans."

Jay M. Campbell, Christopher Richie, Jayne M. Squirrell, Joshua N. Bembenek, John G. White, Kevin Eliceiri, Andy Golden

[International Worm Meeting, 2007]

Fertilization triggers events such as the resumption of the cell cycle and cortical granule exocytosis (CGE) in oocytes of several species. The proper execution of these egg activation events is required for the transition from an arrested oocyte to a dividing embryo. Osmotic integrity defective (OID) mutants are characterized by abnormal eggshell formation in C. elegans. Interestingly, separase, a conserved protease required for chromosome separation during anaphase, as well as other cell cycle regulators cause OID when depleted from the embryo. We sought to understand the basis of OID and why cell cycle regulators display this phenotype, by investigating the process of eggshell formation. Live cell imaging revealed a wave of secretion which initiated near the meiotic spindle and spread across the cortex in young embryos undergoing anaphase I. Utilizing lectin staining, expression of a Golgi protein fused to GFP and TEM, we found a large population of vesicles in oocytes and at the cortex of young embryos that had not completed anaphase I. The Golgi labeling colocalized with lectin-stained vesicles. Live cell imaging demonstrated that these vesicles underwent exocytosis during anaphase I. In older embryos, these vesicles were not observed but the eggshell stained strongly with lectins. We conclude from these data that CGE releases eggshell components during anaphase I. A number of OID mutants did not properly form cortical granules in oocytes. Separase and other cell cycle mutants caused the retention of cortical granules in older embryos and inhibited CGE. However, secretion was not inhibited in either chs-1, a putative cargo of cortical granules or cks-1, a cell cycle mutant that demonstrates anaphase and cytokinesis failures but not OID. This suggests that not all OID mutants are defective in secretion and that cell division failures do not necessarily prevent CGE. These data demonstrate that disruption of cortical granule function is a basis of the OID phenotype. Furthermore, our observations of the dynamics of separase localization suggest that a specific pathway involving separase may regulate CGE during anaphase in C. elegans. During prometaphase I separase colocalized with outer kinetochore components in cortically-localized filaments and accumulated on both chromosomes and the meiotic spindle. As embryos proceeded through metaphase, separase transferred from these filaments to cortical granules and was lost during the exocytic wave in anaphase. These observations suggest that separase may act to coordinate CGE with chromosome segregation during egg activation.

Turpin, Christopher et al. (2021) International Worm Meeting "Elucidating the Role of Securin in Regulating Separase during Cortical Granule Exocytosis"

Turpin, Christopher, Bembenek, Joshua

[International Worm Meeting, 2021]

Meiosis is a tightly regulated series of events leading to gamete formation. A key player in this process is the protease Separase. Known for its role in chromosome segregation, we have defined a role for Separase in vesicular trafficking during cell division. Securin is an inhibitory chaperone of Separase that is degraded at anaphase onset after ubiquitination by the Anaphase Promoting Complex/Cyclosome (APC/C). After Securin degradation, activated Separase cleaves a subunit of cohesin to allow chromosome segregation. Separase also shows dynamic localization during the cell cycle. In prometaphase, Separase localizes to kinetochores and mysterious cortical filaments. During anaphase I, Separase transfers to the midbivalent between homologous chromosomes where cohesin localizes, and concurrently appears on vesicles, called cortical granules, in the cortex. How the dynamic localization of Separase is regulated is currently unknown. Here, we investigated how APC/C and Securin regulate Separase during meiosis I. First, we observed that Securin is degraded by anaphase I and is not present on vesicles. Second, APC/C activity is required for Separase to localize to vesicles. apc/c RNAi results in arrested embryos with Separase and Securin remaining localized to cortical filaments. Third, partial securin RNAi causes precocious Separase vesicle localization. These observations suggest that Securin degradation is required for Separase to localize to vesicles during anaphase. To further test this, we made Non-Degradable Securin fused to GFP (SecurinDM::GFP). SecurinDM::GFP is stably expressed during anaphase I and causes embryonic lethality. SecurinDM::GFP causes chromosome segregation defects, polar body extrusion failure and inhibits cortical granule exocytosis. Importantly, expression of SecurinDM::GFP causes a reduced and delayed localization of Separase to vesicles during anaphase I. We conclude that degradation of Securin regulates the localization of Separase to vesicles to coordinate exocytosis with chromosome segregation. Our findings suggest a novel role for the APC/C and Securin in controlling the localization of Separase, in addition to regulating its protease activity for chromosome segregation and cortical granule exocytosis during anaphase I.

Mitchell, Diana et al. (2013) International Worm Meeting "Methods to Study Toxic Transgenes: Analysis of Protease-Dead Separase in Membrane Trafficking."

Uehlein, Lindsey, Mitchell, Diana, Bembenek, Joshua

[International Worm Meeting, 2013]

The C. elegans embryo provides an ideal system to study the processes of egg activation and cell division. We have demonstrated a novel role for separase, a conserved protease that facilitates chromosome segregation, in the regulation of membrane trafficking during cortical granule exocytosis and cytokinesis. We are interested in determining the role of separase's protease function in the regulation of membrane trafficking. To address this, we generated protease-dead separase mutant tagged with GFP (GFP::SEP-1-PD). We find that GFP::SEP-1-PD accumulates more strongly than wt GFP::SEP-1 at the furrow and midbody during anaphase and cytokinesis, suggesting it could be substrate trapping and have a role in membrane trafficking. The expression of GFP::SEP-1-PD causes embryonic lethality, suggesting that it is a dominant-negative transgene that interferes with endogenous separase function. To enable further study of GFP::SEP-1-PD, we developed methods that allow propagation of worm lines containing toxic transgenes. We are using these methods to enable the study of the protease function of separase in membrane trafficking in the C. elegans embryo. The first method involves feeding worms gfp(RNAi) bacteria to silence the transgene. Transgene expression takes on average 5 generations after removal from gfp(RNAi). Transgene re-expression is also temperature sensitive, allowing further control over its re-expression. We are using this method to obtain large numbers of embryos expressing GFP::SEP-1-PD for biochemical and proteomics analysis, which may allow us to identify novel separase substrates. The second method takes advantage of the pie-1 promotor, which drives transgene expression, and is not expressed in the male germline. Male worms containing GFP::SEP-1-PD can used to propagate the transgene and circumvent the generation delay in the gfp(RNAi) feeding method. We are employing this method to examine genetic interactions of PD-separase with genes in the membrane trafficking pathway known to be involved in cortical granule exocytosis and/or cytokinesis. The general application of these methods facilitates the study of dominant-negative/toxic transgenes using standard techniques in the C. elegans model system.

Verbrugghe, Koen J.C. et al. (2011) International Worm Meeting "Condensin I and the spindle midzone prevent furrow regression induced by chromosome mis-segregation in C. elegans embryos."

Chan, Raymond C., Verbrugghe, Koen J.C., Csankovszki, Gyorgyi, Bembenek, Joshua N.

[International Worm Meeting, 2011]

During cell division, chromosomes invariably segregate before the cleavage furrow ingresses to partition the cell. Chromosomes that obstruct the furrow could block cytokinesis leading to altered cell ploidy, a defect common in cancer. Most cellular checkpoints minimize chromosome mis-segregation by delaying cell cycle progression until the chromosomes are replicated and bi-oriented to the spindle poles. However, this strategy may not be compatible with embryonic development, during which the timing of cell divisions must be closely coordinated. In C. elegans embryos, somatic cells with chromosome or spindle abnormalities can initiate cytokinesis (1-3). However, the cleavage furrow did not regress in embryos containing chromatin bridges induced by topoisomerase II (top-2) RNAi (4). To address whether this phenomenon of sustained furrow integrity is seen in other chromomsome mis-segregation events, we inactivated several genes essential for segregation. While most conditions that generate chromatin bridges do not trigger cleavage furrow regression, the loss of the condensin complexes does. Condensin I and II contain the Structural Maintenance of Chromosome 2 and 4 homologs, MIX-1 and SMC-4, and additional subunits unique to each complex. The disruption of a shared subunit of condensin I and II was sufficient to induce both chromosome mis-segregation and furrow regression. We found that disruption specific to each condensin complex could functionally separate these two defects. Loss of condensin II caused chromosome mis-segregation, while mutations in condensin I did not. Furrow regression is only seen when chromatin bridges occur in the condensin I mutants, suggesting that condensin I may be required to sustain furrow integrity. Condensin I is highly enriched on chromatin bridges. Surprisingly, condensin I normally localizes to the spindle midzone, and disruption of the spindle midzone via spd-1 deficiency recapitulates this condensin I mutant phenotype. Together our data support the presence of a pathway, resembling the abscission checkpoint (5), which prevents furrow regression in the presence of chromatin bridges in C. elegans embryos. 1. M Brauchle et al. Curr Biol 13, 819 (2003). 2. AH Holway et al. J Cell Biol 172, 999 (2006). 3. SE Encalada et al. Mol Biol Cell 16, 1056 (2005). 4. JN Bembenek et al. Curr Biol 20, 259 (2010). 5. P Steigemann et al. Cell 136, 473 (2009).

Bai, Xiaofei et al. (2015) International Worm Meeting "The Protease Activity of Separase Is Required for Both of Chromosome Segregation and Regulation of Membrane Trafficking During Cytokinesis."

Klebanow, Lindsey, Mitchell, Diana, Bembenek, Joshua, Bai, Xiaofei

[International Worm Meeting, 2015]

Chromosomal segregation and cytokinesis are tightly regulated processes during cell division. Separase is a cysteine protease that is involved in the cleavage of cohesin, which allows chromosome segregation from the onset of mitotic anaphase. Besides the canonical proteolytic function of separase in chromosome segregation, we also found that separase is localized to vesicles and is required for exocytosis during cytokinesis. To better understand the functions of the separase protease activity in chromosome segregation and cytokinesis, we generated a protease-dead separase mutant with a point mutation fused to green fluorescent protein GFP (SEP-1PD::GFP). We recently demonstrated that SEP-1PD::GFP expression is dominant negative. In order to better understand this phenotype, we investigated the SEP-1PD::GFP cellular phenotypes using live-cell imaging. Consistent with the dominant negative phenotype, we found that SEP-1PD::GFP expression causes chromosome nondisjunction, possibly due to impaired cohesin cleavage by the inactive protease. To test this, we used RNAi to partially knock down the cohesin scc-1, the subunit cleaved by separase, which significantly rescues embryo lethality and chromosome segregation defects. Additionally, SEP-1PD::GFP also shows enhanced accumulation to the ingressing furrow and the midbody during cytokinesis compared to wild-type protein (SEP-1WT::GFP). Previously, we have shown that RNAi depletion of separase causes an accumulation of RAB-11 positive vesicle at the cleavage furrow and midbody. To test whether SEP-1PD::GFP impairs RAB-11 trafficking during cytokinesis, we generated a strain with RAB-11::mCherry and SEP-1PD::GFP. Interestingly, we found that the expression of SEP-1PD::GFP also causes the abnormal accumulation of RAB-11 vesicles at the cleavage furrow. This result suggests that the protease activity of separase affects exocytosis of RAB-11 vesicles with the plasma membrane. Collectively, we favor the possibility that protease-dead separase traps substrates that should be cleaved by endogenous separase which impairs cohesin cleavage and RAB-11-vesicle exocytosis. In chromosome segregation, the protease activity of separase will cleave the cohesion SCC-1, whereas during cytokinesis, the protease activity of separase may cleave a putative substrate allowing RAB-11-vesicles to undergo exocytosis during cytokinesis. In the future, we hope to identify the putative substrates of separase required for exocytosis using GFP trap beads to purify both SEP-1WT::GFP and SEP-1PD::GFP from C. elegans..

Bai, Xiaofei et al. (2017) International Worm Meeting "A potential role for midbodies in developing tissues of C. elegans."

Simmons, Ryan, Chen, Bi-Chang, Turpin, Chris, Bembenek, Joshua, Klebanow, Lindsey, Bai, Xiaofei, Betzig, Eric, Mitchell, Diana

[International Worm Meeting, 2017]

The midbody forms at the end of cytokinesis and facilitates abscission, the final separation of the daughter cells. Recently, the midbody has been implicated in several developmental processes, including cell fate specification, dorsoventral axis formation, neurite growth, cilium formation, and apical polarity during epithelial lumen formation. To investigate developmental roles of the midbody, we examined midbody fate in the invariant lineage of the C. elegans embryo. Cytokinesis occurs in stereotypical patterns that are unique and tissue specific with a defined pattern of midbody inheritance. In the first mitosis, a relatively symmetric furrow results in a centrally positioned midbody that is always internalized by the P1 daughter cell. In the next AB cell division, a highly asymmetric furrow positions the midbody next to EMS, which internalizes it instead of either AB daughter cell. A dramatic shift in midbody behavior is observed in several tissues at around the 300-cell stage when the embryo undergoes global morphogenetic changes. In two lumen-forming tissues, the intestine and the pharynx, midbodies form after symmetric furrowing and migrate across the cell to the future apical midline. This coordinated midbody migration event coincides with polarization events in intestinal epithelia. At the apical midline, the midbody ring is internalized and disappears. However, Aurora B kinase (AIR-2) remains on the apical surface for over an hour after cytokinesis and polarization. A similar apical localization pattern is observed for AIR-2 in the pharyngeal primordium. Finally, in cells that form the inner labial sensilla (ILS), we observe symmetrical cytokinesis followed by a midbody migration event that leads to a focal aggregation of AIR-2. AIR-2 persists along the leading edge of developing dendrites (which also may be apical) as they migrate towards the anterior end of the embryo, anchor at the tip of the embryo and elongate. Other midbody markers are either internalized or maintained with AIR-2 in a tissue-specific manner. Inactivating several fast-inactivating temperature sensitive cytokinesis mutants late in embryogenesis causes severe defects in positioning, continuity and shaping of the intestinal and pharyngeal lumen. Many animals fail to hatch, but among those that do, a high percentage show defects in neuronal DiI staining. These data suggest that the proper execution of cytokinesis, which shows surprising flexibility during development, and specific cytokinesis regulators such as AIR-2, may regulate different aspects of development including the final interphase architecture of a terminally dividing cell.

Michael Dybbs et al. (2001) International C. elegans Meeting "A screen for new Hic mutants"

Josh Kaplan, Michael Dybbs

[International C. elegans Meeting, 2001]

ABSTRACT International Worm Meeting 2001 A screen for new Hic mutants Michael Dybbs and Joshua Kaplan 361 LSA, Dept. of Mol. and Cell Biology, University of California, Berkeley, CA 94720 Previous studies by our lab and others have shown that release of acetylcholine at the C. elegans neuromuscular junction (NMJ) is regulated by a G-protein signaling network. These studies have shown that serotoninergic signaling through goa-1 G a o and dgk-1 DAG kinase inhibits acetylcholine release. An opposing muscarinic signaling pathway through egl-30 G a q and egl-8 PLC b enhances acetylcholine release via the production of diacylglycerol ( DAG). Two RGS proteins, egl-10 RGS and eat-16 RGS, further regulate this pathway by stimulating the GTPase activity of goa- 1 G a o ( eat-16 RGS) and egl-30 G a q ( egl-10 RGS). Mutations in the inhibitory pathway ( goa-1 , dgk-1 ) make animals hypersensitive to paralysis by the acetylcholine esterase inhibitor, aldicarb (Hic) Mutations in the muscarinic pathway ( egl-30 , egl-8 ) make animals resistant to aldicarb (Ric). In order to identify additional molecules which negatively regulate release, we are conducting a broad screen for Hic mutants. Aldicarb sensitivity could be a result of either mutations that effect presynaptic neurotransmitter release or postsynaptic response to neurotransmitter in the muscle. We are distinguishing these effects using levamisole, a cholinergic agonist that acts directly on the acetylcholine receptors in the muscle. We are focusing on mutants that display a wild-type muscle sensitivity and are thus acting presynaptically. Mutants isolated in our pilots screens will be described.

Dasgupta, Krishnakali et al. (2009) International Worm Meeting "Understanding the role of PKC-1 signaling pathway in regulating Dense Core Vesicle secretion."

Sieburth, Derek, Dasgupta, Krishnakali

[International Worm Meeting, 2009]

Synaptic vesicles (SV) and dense core vesicles (DCV) both exhibit calcium dependent neurotransmitter release. However, differences in their biogenesis, release properties, and recycling suggest that each type of organelle may use some unique proteins to carry out their functions. PKC-1, a protein kinase C ortholog in worms, is one such candidate for a unique DCV regulating molecule, since it has been shown to regulate neuropeptide secretion, while possibly not affecting SV release.(1) PKC-1 is most similar to the human eta and epsilon isoforms which are activated by the second messenger DAG via their C1 domains. In order to identify additional components that act in the PKC-1 pathway to regulate DCV secretion, we have used a constitutively active PKC-1 mutant which lacks the autoinhibitory domain and is predicted to be active in the absence of DAG. Expression of this transgene specifically in motor neurons causes hypersensitivity to an acetylcholinesterase inhibitor, aldicarb (Hic) and loopy locomotion. We screened for suppressors of either the loopy or Hic phenotype of animals expressing the constitutive PCK-1, and identified ~30 suppressors that restore movement or aldicarb response. We are in the process of mapping them and characterizing their effects on neuropeptide secretion. Presently, we have identified one aldciarb resistant candidate allele, vj3, which shows decreased uptake of NLP-21::YFP by coelomocytes and increased accumulation of DCVs in axons. However, vj3 mutants do not cause an accumulation of the SV marker at synapses, suggesting that vj3 mutants specifically reduce DCV secretion. vj3 maps to a small interval on LG I that does not contain any known neurotransmitter secretion genes. We are currently in the process of fine mapping, and tesing candidate genes for aldicarb resistant phenotypes by RNAi. 1.PKC-1 regulates secretion of neuropeptides; Derek Sieburth1, Jon M Madison & Joshua M Kaplan; Nature Neuroscience 10, 49 - 57 (2007).