The long-term goal of this project is to characterize the mechanisms that distinguish germ cells from somatic cells, a fundamental problem in developmental biology. In C. elegans, soma- germline asymmetries are established in the zygote before the first division, through the asymmetric partitioning of proteins and protein/RNA complexes in the cytoplasm. Cytoplasmic partitioning is regulated by conserved polarity regulators (PAR proteins), which localize asymmetrically in the zygote cortex. The goal of this proposal is to uncover how PAR activity at the cortex regulates polarity in the cytoplasm.
Specific Aim I (SA1) will focus on the mechanisms that segregate the RNA-binding protein MEX-5 to the anterior cytoplasm. Preliminary data suggest that MEX-5 asymmetry depends on phosphorylation by PAR-1, a kinase on the posterior cortex, which increases MEX-5 diffusion locally in the posterior cytoplasm. SA2 will focus on the germline determinant PIE-1, which segregate to the posterior, opposite MEX-5 and in a steeper gradient. We will investigate how PIE-1 integrates both cortical and cytoplasmic cues to form a distinct gradient. Finally, SA3 will focus on the mechanisms that localize P granules to the posterior. P granules are evolutionarily conserved RNA-protein complexes specific to the germline. We have discovered a new P granule component PPTR-1 for P granule integrity during mitosis;our initial findings suggest that the germline specificity of P granules depends on regulated assembly and does not require cytoplasmic partitioning. We will use a combination of biochemical, genetic, and live microscopy approaches to test each of these hypotheses, and identify the genes and biochemical interactions involved. Unlike other well-studied embryonic polarity models, our system does not rely on pre-localized RNAs to generate asymmetry, and thus gives us the unique opportunity to explore the mechanisms that directly segregate proteins in the cytoplasm.

Public Health Relevance

Cell polarity is essential to generate cell diversity during development, to prevent uncontrolled cell growth, and for the every-day functioning of many polarized cell types (epithelial cells, neurons, and lymphocytes). By taking advantage of a simple model system, our studies will illuminate the mechanisms that build and maintain the architecture of many cell types and prevent tumor formation.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD037047-13
Application #
8284208
Study Section
Development - 1 Study Section (DEV1)
Program Officer
Ravindranath, Neelakanta
Project Start
1999-08-18
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
13
Fiscal Year
2012
Total Cost
$261,350
Indirect Cost
$101,990
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
McEwen, Tamara J; Yao, Qiuming; Yun, Sijung et al. (2016) Small RNA in situ hybridization in Caenorhabditis elegans, combined with RNA-seq, identifies germline-enriched microRNAs. Dev Biol 418:248-57
Paix, Alexandre; Folkmann, Andrew; Rasoloson, Dominique et al. (2015) High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes. Genetics 201:47-54
Wang, Jennifer T; Seydoux, Geraldine (2014) P granules. Curr Biol 24:R637-8
Chen, Bi-Chang; Legant, Wesley R; Wang, Kai et al. (2014) Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346:1257998
Paix, Alexandre; Wang, Yuemeng; Smith, Harold E et al. (2014) Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Genetics 198:1347-56
Wang, Jennifer T; Smith, Jarrett; Chen, Bi-Chang et al. (2014) Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans. Elife 3:e04591
Wang, Jennifer T; Seydoux, Geraldine (2013) Germ cell specification. Adv Exp Med Biol 757:17-39
Voronina, Ekaterina; Paix, Alexandre; Seydoux, Geraldine (2012) The P granule component PGL-1 promotes the localization and silencing activity of the PUF protein FBF-2 in germline stem cells. Development 139:3732-40
Voronina, Ekaterina; Seydoux, Geraldine; Sassone-Corsi, Paolo et al. (2011) RNA granules in germ cells. Cold Spring Harb Perspect Biol 3:
Motegi, Fumio; Zonies, Seth; Hao, Yingsong et al. (2011) Microtubules induce self-organization of polarized PAR domains in Caenorhabditis elegans zygotes. Nat Cell Biol 13:1361-7

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