Mondal, S. et al. (2019) International Worm Meeting "Probabilistic addition of mitochondria in the growing touch neuronal process identified using a long-term growth and imaging microfluidic device."

Koushika, S.P., Awasthi, A., Mondal, S., Sure, G.P.R., Dubey, J.

[International Worm Meeting, 2019]

The long cellular architecture of neurons requires regulation in part through transport and anchoring events to distribute intracellular organelles. Mitochondria are one such organelle that are present at a constant density in touch neurons. As the neuron grows with increase in animal length, mitochondria are added at the rate of ~0.4 per hour to to result in the density of ~5/100 ?m in all adult animals. To identify how mitochondria are added along the neuron we developed a microfluidic device to feed and immobilize C. elegans for high-resolution imaging over a period of several days. Repeated sub-cellular imaging of an identified touch neuron in a given animal, over 36 hours in our microfluidic device, shows no preferential location for mitochondrial addition. New mitochondria are added anywhere along the neuronal process when the average separation between the two pre-existing mitochondria crosses the threshold of 24 micrometers. Mitochondria along the neuronal process show turnover and new mitochondria may arise due to entry from the cell body and from fission of moving mitochondria in the neuronal process. Using our long-term growth and imaging device we have identified a how a slow cell biological process occurs in individual neurons in identified animals without population averaging.

Guruprasada R. Sure et al. (2006) East Asia C. elegans Meeting "Identifying the players involved in the transport of mitochondria in Caenorhabditis elegans neurons"

Guruprasada R. Sure, Shaili N Johri, Swetha Mohan, Sandhya P Koushika

[East Asia C. elegans Meeting, 2006]

The growth, maintenance, and survival of neurons rely critically on organelle transport between the cell body and the synapse. Mitochondria are involved in the ATP production, calcium homeostasis and synaptic vesicles recycling. Mitochondria, unlike other organelles are transported bi-directionally and remain stationary 75% of the time. In neurodegenerative diseases such as Alzheimer""s and Parkinson""s, abnormal mitochondrial distribution has been observed. We are trying to identify the molecular players involved in the transport of mitochondria using classical genetic approaches. To address this question we take a classical genetic approach using a transgenic strain jsIs609 in which GFP is expressed in the mechanosensory neurons and targeted to the mitochondrial matrix. We have performed EMS mutagenesis and screened around 3000 genomes and isolated 17 mutants. These fall into different classes, such as 100% increase in number of mitochondria in axons, 50-75% fewer mitochondria in the axons, abnormal accumulation of mitochondria in minor neurites and large spherical mitochondria in the cell bodies of the neurons. We are currently performing complementation analyses of these mutants. We have also analyzed the distribution of mitochondria in mutants with known roles for the transport of organelles and in the dynamics of mitochondria, such as anc-1, unc-16, unc-116, rab-7 and unc-73. In unc-116 Kinesin-I mutants fewer mitochondria are observed in the axons whereas unc-16 ortholog of Sunday driver have more number of mitochondria in the axons. To analyze the mitochondrial behavior/dynamics with respect to metabolism we have performed starvation analyses. We find that the cell bodies of the neurons are fragmented during starvation but the axons are spared and are normal. We are currently imaging mitochondrial motility and dynamics across the developmental stages. # Guruprasada Reddy Sure and Swetha Mohan contributed equally to this work.

Starr, D. et al. (2015) International Worm Meeting "UC Davis-University of Maryland Eastern Shore Graduate Admissions Pathway: Collaborating to broaden participation in molecular and cellular biology."

Hom, C., Starr, D.

[International Worm Meeting, 2015]

STEM professionals in the 21st century remain predominantly Caucasian/white, in spite of decades of work by professional societies, colleges and universities, and individual scientists to broaden participation. This multifaceted problem includes concerns among students and faculty at minority-serving institutions about the economics of career choice, family pressure to pursue a career in a biomedical field, and limited exposure to natural history. Further, institutional efforts in recruitment by research universities remain rooted in graduate fairs that target senior undergraduates from groups underrepresented in science, whereas connections made via shared research networks provide a more sure means to admission in molecular and cell biology. The UC Davis-University of Maryland Eastern Shore (UMES) Molecular and Cellular Biology Graduate Admissions Pathways (MCBGAP) program addresses this challenge via collaborations between faculty at the two institutions and a research co-mentoring program that brings UMES undergraduates to UC Davis for summer research. The program is funded by a grant from the University of California Office of the President and the UC Davis College of Biological Sciences.MCBGAP supported two cohorts of five UMES students in the summers of 2014 and 2015. The MCBGAP program consists of reciprocal student-faculty visits, close interactions between key UC Davis and UMES faculty, monthly Skype meetings that involve mentors and students, and research, professional development, and field trips in the summer. MCBGAP has catalyzed change both at UMES, where students are given the opportunity to self-identify as researchers at a tier 1 research university, and at UC Davis, where increased numbers of faculty recognize the need to be proactive in graduate recruiting and admissions, and multiple deans have committed time to mentor students and funds to support additional undergraduates from Historically Black Colleges and Universities for summer research and mentoring. The experience has also inspired us to apply for a Postbaccalaureate Research Education Program (PREP) from the NIH.

Yunhee Kim et al. (2006) East Asia C. elegans Meeting "Identification and Characterization of T23C6.3, a Novel gene in Caenorhabditis elegans"

Yunhee Kim, Joohong Ahnn

[East Asia C. elegans Meeting, 2006]

Calcineurin is a calcium/calmodulin-dependent serine/threonine protein phosphatase and is well known to have diverse cellular functions in different cell types and organisms. T23C6.3 has been screened as one of the candidate molecules which interact with calcineurin by Yeast-two-hybrid system. T23C6.3 is a nematode-specific novel gene in C.elegans. To find out the spatial and temporal expression pattern of T23C6.3, 2.2kb 5'upstream region and full-length genomic DNA of T23C6.3 fused with GFP was injected to wild-type animal. T23C6.3 is strongly expressed in the nucleus of intestine, hypodermis, vulva and uterine regions and the expression begins around 60-cell stage and progresses all larval stages and adult. To elucidate the biological function of T23C6.3 in C.elegans, a mutant(jh145) has been isolated by PCR-based TMP-UV mutagenesis method. The mutant has a deletion of 880 base pairs from exon 2 to exon 3 which leads to a premature termination of translation. Since T23C6.3 is a potential calcineurin binding partner, several calcineurine-defective phenotypes such as brood size, body size, serotonin- and levamisole-mediated egg-laying, were tested. The mutant(jh145) showed no significant difference to wild-type animal in those assays. However, the double mutants, tax-6(p675);T23C6.3(jh145) and cnb-1(jh103);T23C6.3(jh145), are more severe in terms of brood size, body size and serotonin- and levamisole-mediated egg-laying compare to tax-6(p675) and cnb-1(jh103) respectively. These results suggest that the mutation of T23C6.3 enhances the calcineurin-mutant phenotypes and thus, T23C6.3 genetically interacts with calcineurin. To make sure the direct interaction of T23C6.3 with calcineurin, we are currently investigating the physical interaction between T23C6.3 and calcineurin by in vitro-binding assay.

Andrew Giles et al. (2008) C.elegans Neuronal Development Meeting "Validating a Multi-Worm Tracker for use in a Genetic Screen for Abnormal Habituation in C. elegans"

Catharine Rankin, Nicholas Swierczek, Andrew Giles, Rex Kerr

[C.elegans Neuronal Development Meeting, 2008]

Habituation is the gradual decrease in the behavioral response to an irrelevant stimulus. It is a fundamental form of learning in which organisms stop responding to stimuli that do not have significant consequences; this allows them to reserve their energy to respond to stimuli that predict biologically relevant consequences. The cellular and molecular mechanisms that mediate this form of learning are not well-understood. C. elegans can be used as a model to study this because their response to a non-localized tap (called the tap withdrawal response) has been shown to habituate (Rankin et al. 1990). Conducting a large scale genetic screen would be beneficial in order to identify a number of novel genes that play a role in habituation. Until now this has been a daunting task because it takes a long time to analyze an individual strain (approx. 40 hours by manual behavioural analysis; 6 hours using an automated system that tracks individual worms). The Kerr lab has developed a system that is capable of simultaneously analyzing the habituation of multiple worms (as many as 50 reliably) in real-time. This lowers the time needed to assess an individual strain to 10 minutes, making a genetic screen for habituation feasible. Prior to conducting a screen it is important to validate the system to be sure it will properly identify habituation abnormalities. We have validated the system by demonstrating a number of essential habituation characteristics, such as the interstimulus interval dependence on both the rate and asymptotic level of habituation in wild-type worms (Rankin and Broster 1992). We have also tested several previously identified mutants, such as dop-1 and cat-2, which show altered short-term habituation (Sanyal et al. 2004) and found that the system is capable of reproducing the same phenotypes as previously reported. We are currently optimizing the system for use in a genetic screen to identify genes that affect different characteristics of habituation.

Olson, S.K. (2019) International Worm Meeting "Incorporation of CRISPR/Cas9 into a semester-long investigative lab on the cell biology of C. elegans."

Olson, S.K.

[International Worm Meeting, 2019]

Course-based undergraduate research experiences (CUREs) aim to provide authentic research experiences for all students, and are particularly transformative for students from underserved backgrounds. Since 2012, I have incorporated a semester-long CURE centered on my lab's research program into the lab section of an Advanced Cell Biology course that enrolls 24 students. Students explore localization patterns, depletion phenotypes, and putative interactions of uncharacterized proteins previously identified in an RNAi screen for eggshell defects in C. elegans, and the findings feed directly into my research program. While the Advanced Cell lab was transformative for students, the model became unsustainable due to the large amount of work needed to generate 4-5 new fluorescently-tagged worm strains each year. Thus, I recently redesigned the Advanced Cell lab so the students themselves generated the worm strains to be studied using CRISPR/Cas9 gene editing. Students selected a protein from a list relevant to our research goals, spent 2 weeks designing guide RNA and homology-directed repair constructs to introduce localization tags, affinity tags, and degrons into their proteins, and assembled the vectors using the streamlined and modular SapTrap assembly method. Following injection of constructs into worms, students screened for genomic edits and spent the rest of the semester conducting localization, phenotypic, and/or co-IP studies to explore the role of their protein in eggshell formation. Students were also exposed to the ethics of CRISPR technology through dedicated discussion time. In the first trial of the CRISPR lab, 6/8 groups successfully cloned their constructs, 4/8 had evidence of genome edits, and 1/8 completed their proposed experiments. Current efforts aim to improve the cloning process to better streamline the experiment and improve success rates. Assessment of the 2012-2017 labs showed that Advanced Cell students scored higher than national CURE and SURE averages on metrics including engagement, learning gains, and confidence, which have been shown to encourage more students to pursue careers in science.

Lisa R Girard et al. (2005) International Worm Meeting "WormBook: An Online Review of C. elegans Biology and Resource for Experimental Methods"

Martin Chalfie, Tristan J Fiedler, Lincoln D Stein, Paul W Sternberg, Lisa R Girard, Todd W Harris

[International Worm Meeting, 2005]

*Both the first and second authors contributed equally to this project. WormBook (to be located at http://www.wormbook.org)is a new, online book containing original reviews on all aspects of C. elegans biology. It will provide a text companion to WormBase, the model organism database (MOD) for C. elegans. WormBase provides the data; WormBook will provide the narrative that interprets and synthesizes that data. The WormBook concept capitalizes on the World Wide Web to link in-text references (e.g. genes, alleles, proteins, and literature citations) with primary biological databases (e.g. WormBase, PubMed and Wormatlas). Researchers using these linked primary databases will have simultaneous access to current review articles in WormBook that will facilitate the exchange of ideas and promote further research. WormBook, as the first of what is sure to be many text companions to the growing number of model organism databases, represents an online database and publishing model that can be easily incorporated by other biological disciplines where the rapid proliferation of primary data must be managed in the context of biological knowledge. The goals of the WormBook project are to: 1) Develop a publicly available database, and surrounding infrastructure that will allow users to take maximal advantage of the resources in WormBook and provide a paradigm for other community efforts facilitated by WormBooks freely available software for rapid develop of similar MOD companion websites 2) Commission, edit and annotate reviews on a wide variety of topics in C. elegans biology for inclusion in WormBook 3) Offer a repository (WormMethods) for experimental techniques and methods pertinent to nematode research 4) Integrate the contents of WormBook and WormBase Utilizing an online format for WormBook will provide numerous advantages over print. Some of these advantages include: support for a wider range and more extensive use of media such as figures and movies, integration by means of reciprocal hyperlinks with other websites, easier and more regular updates and retention of control over content. The initial version of WormBook is slated for release in June 2005 and will eventually contain over 100 chapters.

Akiko Miyara et al. (2006) East Asia C. elegans Meeting "Analysis of ttx-8: novel thermotaxis gene conserved among various organisms"

Akane Ohta, Ikue Mori, Masatoshi Okumura, Yoshifumi Okochi, Akiko Miyara

[East Asia C. elegans Meeting, 2006]

C. elegans can associate the food condition with the cultivation temperature and migrate to the cultivation temperature when looking for the food. This response to temperature is called thermotaxis. Several neurons and genes required for thermotaxis have been identified, but molecular mechanism of thermotaxis is still poorly understood. The ttx-8(nj21) and ttx-8(nj34) mutants are defective in thermotaxis and partially defective in chemotaxis. We conducted the rescue experiments and found that ttx-8 encodes novel protein. The predicted protein structure of ttx-8 gene consists of five transmembrane domains in the amino terminus and four coiled-coil domains in the carboxyl terminus. RIC-3, identified in C. elegans at first and conserved among several species has transmembrane domains in the amino terminus and coiled-coil domains in the carboxyl terminus as well (Halevi et al., 2002; Halevi et al., 2003). RIC-3 is thought to be required for the maturation of acetylcoline receptor (Halevi et al., 2002), so TTX-8 may play a similar role such as folding, assembly, transmission or anchoring of some kind of membrane protein. To determine the cells where ttx-8 gene is expressed, we constructed ttx-8 promoter-gfp fusion gene and injected in N2 strain. GFP expression driven by ttx-8 promoter was observed in neurons. To identify the cells where TTX-8 functions, ttx-8 cDNA was expressed in various neurons of ttx-8(nj34) mutants. When ttx-8 cDNA was expressed in AFD, AIY and AIZ neuron of ttx-8(nj34) mutants, about 50% of transgenic animals showed normal thermotaxis phenotype, suggesting that TTX-8 functions in AFD, AIY and AIZ neuron. We found that TTX-8 may interact with Y11D7A.12, a novel protein with a FLYWCH zinc finger domain, by Yeast two-hybrid screening. Y11D7A.12 has been reported to interact with MEC-4, a mechanically-gated ion channel (see abstract by Everett et al., IWM 2001). We are in the process of making sure that TTX-8 interacts with Y11D7A.12 in detail and analyzing the subcellular localization of TTX-8. We believe that the further analysis of ttx-8 in C. elegans would give us new important information about the function of this protein, conserved from nematode to mammals.

Powell, J.R. (2017) International Worm Meeting "A course-based undergraduate research experience to identify novel regulators of innate immune signaling in C. elegans."

Powell, J.R.

[International Worm Meeting, 2017]

The positive impact of participation in undergraduate research has been well established. Apprentice-style research experience rewards students with a close mentoring experience; however, these positions are limited by faculty time and resources. Additionally, although students underrepresented in the sciences show even greater benefits from research, majority students often disproportionately seek out and receive apprentice positions. We have converted the laboratory component of Bio 211 Genetics at Gettysburg College into a semester-long intensive research experience. Because Genetics is a required course in three majors, this course-based research experience creates a more equitable entry point into research that extends the benefits of research to a broader group of students in a cost-effective manner. The students in Bio 211 Genetics perform an RNAi screen to look for regulators of immune signaling in C. elegans. With about 32 students enrolled in the course, the class can collectively screen approximately 500 RNAi clones in one semester, with built-in redundancy to ensure high data quality. After a primary screen, the students repeat the experiment on a subset of hits, image and quantify target gene expression using a compound epifluorescence microscope, and sequence the RNAi clones to confirm their identity. Through a series of progressive experiences over the semester, we encourage students to develop their scientific communication abilities by reading and discussing the primary literature, presenting their projects orally in group meetings and formal symposia, and maintaining a lab notebook. Assessment via the Grinnell CURE survey indicated that this course had a dramatically positive impact on student learning. For example, in the first semester, student intent to pursue graduate degrees in the life sciences rose from 26% at the beginning of the semester to 52% by the end. Genetics student rated their learning gains in this course equivalent or even higher than students who took a parallel survey after a summer apprentice-style research experience (the SURE survey) on elements such as ability to read and understand primary literature, tolerance for obstacles faced in the research process, understanding of the research process, and readiness for more demanding research.

Matyas Gorjanacz et al. (2008) European Worm Meeting "Screening temperature sensitive embryonic lethal mutants to identify genes in nuclear envelope assembly"

Ulrike Bauer, Iain W Mattaj, Geraldine Seydoux, Matyas Gorjanacz

[European Worm Meeting, 2008]

The nuclear envelope (NE) of eukaryotic cells provides an essential. barrier that separates the genome from the cytoplasm. During mitotic. prophase the NE disassembles to enable chromosome segregation and in. anaphase and telophase it reassembles around the chromatin. Little is known. about the factors or the mechanisms by which they act during NE re-. formation at the end of mitosis.. The genome wide RNAi screens have identified only a very small number. of genes required for NE assembly. However, given the complexity of NE. assembly, the finding of only a small number of genes whose depletion. affects this process strongly suggested that the RNAi screens carried out. until now have failed to identify many genes whose products may function in. during NE assembly. Furthermore, many of the identified factors not only. affect NE formation, but also cause defects in other, earlier mitotic. events like chromosome condensation and de-condensation, chromosome. segregation, etc. This makes it impossible to be sure whether NE assembly. defects are primary events or secondary consequences of the earlier. problems. Recently we demonstrated that this disadvantage could be overcome. in our studies of the chromatin-associated BAF-1 protein (Gorjanacz et al.,. 2007; EMBO J). We now wish to extend our use of this method by screening a. collection of temperature sensitive embryonic lethal Caenorhabditis elegans. mutations for those that affect NE assembly. We are performing this screen. in three major steps. First, by using DIC microscopy, we screen for strains. showing nuclear appearance defects after a short temperature up-shift from. the permissive temperature (15degreesC) to the restrictive temperature (25degreesC).. Second, to further select the candidates and to prove their specific NE. defects after the temperature up-shift, we immunostain these mutant embryos. with antibodies against inner nuclear membrane proteins and nucleoporins.. Finally, to select the specific genes that are directly involved in NE. assembly we analyze them with our newly developed fast response temperature. controller device in a series of temperature up-shift experiments during. different cell-cycle stages. Screening the collection of 850 temperature. sensitive embryonic lethal mutations we identified 30 candidates, which by. DIC microscopy and by immunostainig showed reproducible strong NE defects.. 5 out of these 30 candidates seem to have a direct and specific effect on. NE assembly. These 5 "NE-specific" candidates will be SNP mapped and the. role of identified genes in NE assembly will be characterized in vivo and. in vitro.