Wang, Shaohe et al. (2015) International Worm Meeting "The ninein-related protein NOCA-1 organizes non-centrosomal microtubule arrays in diverse tissues by acting individually and in parallel to Patronin."

Desai, Arshad, Cheerambathur, Dhanya K., Green, Rebecca A., Ochoa, Stacy D., Oegema, Karen, Wang, Shaohe, Quintin, Sophie

[International Worm Meeting, 2015]

In metazoans, diverse non-centrosomal microtubule arrays assemble in differentiated tissues to perform mechanical and transport-based functions. How these arrays form remains poorly understood. Here, we show that C. elegans NOCA-1, which shares homology to ninein, a vertebrate protein implicated in microtubule anchoring, functions as an organizer of non-centrosomal microtubule arrays across different tissues. NOCA-1 has multiple isoforms that share a common ninein-homology domain that we show binds microtubule ends in vitro. Distinct NOCA-1 isoforms organize non-centrosomal microtubule arrays in the germline and the embryonic epidermis. In the larval epidermis, a third NOCA-1 isoform functions redundantly with the minus end protection factor Patronin/PTRN-1 to direct assembly of a circumferential microtubule array required for larval growth and morphogenesis. These results reveal involvement of the ninein-related protein NOCA-1 in the formation of non-centrosomal arrays in diverse C. elegans tissues and highlight functional overlap between the ninein and Patronin families of microtubule cytoskeleton-associated proteins.

Green, Rebecca et al. (2019) International Worm Meeting "4D High Content Imaging and Automated Phenotypic Profiling of C. elegans Embryogenesis."

Green, Rebecca, Ochoa, Stacy, Chow, Tiffany, Hendel, Jeff, Zhao, Zhiling, Khaliullin, Renat, Desai, Arshad, Oegema, Karen, Wang, Shaohe

[International Worm Meeting, 2019]

An important challenge is to functionally classify the ~2000 genes (>1400 conserved) that control cell-fate specification and morphogenesis during embryogenesis. Here, we perform a 4D high-content screen by filming embryogenesis using two custom-engineered C. elegans reporter strains, following individual RNAi-based knockdown (>20,000 individual movies). We monitor (1) changes in cell fate specification, by dynamically tracking fluorescently labeled endoderm, mesoderm and ectoderm nuclei, and (2) morphogenic changes during epithelial and neuronal development by monitoring tissue position and tissue shape. Consistent and timely analysis of 20,000 movies requires automation, however, the range and complexity of 4D developmental phenotypes are not easily captured by existing automated methods. To address this challenge, we manually curated a pilot set of 500 genes (>7000 movies) and used this reference to guide the development of custom automated analysis algorithms; this effort ensured that our final automated analysis method captured observed phenotypes across a spectrum of developmental defects. For each RNAi condition, our automated analysis yields phenotypic signatures consisting of >100 continuous parameters. To evaluate the phenotypic similarity between RNAi conditions, we measure the distance between phenotypes in continuous space. To correct for the fact that a strict measure of Euclidean distance penalizes genes with more severe phenotypes, we measure the angle between the average phenotypes for the two conditions (phenotypic angle of deviation; PAD). Finally, we optimized the set of parameters used for automated comparison by assessing performance of the algorithm on a manually-annotated set of phenotypic groups. Our resulting automated method effectively identifies genes whose knockdown leads to similar phenotypes; this allows partitioning of genes into functional groups that are predicted to reflect developmental pathways and will yield a systems-level view of embryonic development. This work represents the first fully automated high-content screen of an intact developing organism and is the most complex morphological profiling effort to date.

Green, Rebecca et al. (2015) International Worm Meeting "­Decoding Embryonic Developmental Pathways Using 4D-High Content Imaging of C. elegans embryos."

Desai, Arshad, Biggs, Ronald, Oegema, Karen, Zhao, Zhiling, Wang, Shaohe, Ochoa, Stacy, Green, Rebecca, Gerson-Gurwitz, Adina, Khaliullin, Renat

[International Worm Meeting, 2015]

­Embryogenesis is a complex process requiring coordination of cell division, signaling, migration, differentiation, and death. Systematically defining the genetic pathways that drive these morphogenetic events during embryogenesis is an important current challenge. Our goal is to construct a comprehensive functional network map of essential developmental genes for the model metazoan, C. elegans. To this end, we have developed a 4D-high-content screening based approach to functionally classify ~2600 developmental genes, using two-specifically engineered marker strains that readout defects in (1) germ layer specification and positioning and (2) cell shape changes and cell migration during morphogenesis. Following RNAi of targets, we image C. elegans embryos throughout the developmental time course (~10hrs) using a CV1000 spinning disk confocal high-content imaging system, which enables collection of developmental data for 50-100 embryos in a single experiment. To date, we have completed a pilot set of >500 genes. Among these, we have recovered expected phenotypes for well described developmental genes as well as severe developmental phenotypes for many uncharacterized genes, validating our overall experimental approach. This pilot data set is being used to develop custom data management algorithms (cropping, orienting, and indexing embryos) and data analysis protocols, including: manual and automated scoring of phenotypic features (Imaris and custom). Using this approach, each individual embryogenesis movie is scored and genes are clustered according to phenotypic profiles. When complete, this will be the first systems-level view of embryonic development in a complex multicellular organism. We anticipate such an effort will translate to higher organisms and help reveal the genetic basis for congenital defects, such as neural tube, craniofacial, and ventral body wall closure abnormalities.

Yang M et al. (2005) Biochem J "Manganese toxicity and Saccharomyces cerevisiae Mam3p, a member of the ACDP (ancient conserved domain protein) family."

Jensen LT, Culotta VC, Yang M, Gardner AJ

[Biochem J, 2005]

Manganese is an essential, but potentially toxic, trace metal in biological systems. Overexposure to manganese is known to cause neurological deficits in humans, but the pathways that lead to manganese toxicity are largely unknown. We have employed the bakers'' yeast Saccharomyces cerevisiae as a model system to identify genes that contribute to manganese-related damage. In a genetic screen for yeast manganese-resistance mutants, we identified S. cerevisiae MAM3 as a gene which, when deleted, would increase cellular tolerance to toxic levels of manganese and also increased the cell''s resistance towards cobalt and zinc. By sequence analysis, Mam3p shares strong similarity with the mammalian ACDP (ancient conserved domain protein) family of polypeptides. Mutations in human ACDP1 have been associated with urofacial (Ochoa) syndrome. However, the functions of eukaryotic ACDPs remain unknown. We show here that S. cerevisiae MAM3 encodes an integral membrane protein of the yeast vacuole whose expression levels directly correlate with the degree of manganese toxicity. Surprisingly, Mam3p contributes to manganese toxicity without any obvious changes in vacuolar accumulation of metals. Furthermore, through genetic epistasis studies, we demonstrate that MAM3 operates independently of the well-established manganese-trafficking pathways in yeast, involving the manganese transporters Pmr1p, Smf2p and Pho84p. This is the first report of a eukaryotic ACDP family protein involved in metal homoeostasis.