Dissertation/Thesis Abstract

The biophysics of germline determination in the Caenorhabditis elegans early embryo
by Daniels, Brian R., Ph.D., The Johns Hopkins University, 2009, 182; 3357181
Abstract (Summary)

In order to generate cellular diversity in developing organisms while simultaneously maintaining the developmental potential of the germline, germ cells must have the ability to preferentially endow germline daughter cells with a portion of the cytoplasm containing specialized cell-fate determinants (collectively referred to as the ‘germplasm’) that are not inherited by somatic cells. The asymmetric division of the Caenorhabditis elegans (C. elegans) early embryo is marked by the eccentric positioning of the mitotic spindle and asymmetric partitioning of cell-fate determinants, such as the protein PIE-1 (Pharynx In Excess) and ribonucleoproteinous subcellular organelles known as P granules, both of which are required for proper germline specification.

To investigate the physical mechanisms that drive asymmetric partitioning and eccentric spindle displacement, we introduce a novel in vivo assay to probe the spatial and temporal variations in the viscoelastic properties of the cytoplasm of developing C. elegans embryos. Our results indicate that unlike highly differentiated cells, the cytoplasm of C. elegans zygotes is purely viscous with no measurable elasticity. The shear viscosity of the cytoplasm, which is about 3 orders of magnitude greater than that of water, does not vary along the anterior-posterior axis or with time throughout the first cell division. These results support the hypothesis that the eccentric positioning of mitotic spindles stems from the asymmetric distribution of elementary force generators as opposed to a heterogeneous distribution of viscoelastic properties, as well as eliminate “viscous trapping” as a possible mechanism of P granules partitioning to posterior. Moreover, we demonstrate that cytoplasmic streaming alone is not sufficient to induce asymmetric partitioning of organelles. Our measurements also provide the first measurements of the elementary forces that control spindle positioning in early C. elegans embryo.

To test the hypothesis that intracellular elasticity correlates with the level of cellular differentiation, we use a novel method of Biolistic Intracellular Nanorhology to compare the intracellular rheology of de-differentiated induced ploripotent stem (iPS) cells and the differentiated parental fibroblasts from which they were derived. Our results demonstrate that highly elastic IMR90 parental cells lose elasticity when induced to become pluripotent stem cells, supporting a correlative relationship between intracellular elasticity and cellular differentiation.

The germline inheritance of the protein PIE-1 is accomplished by first asymmetrically localizing the protein to the germplasm prior to cleavage, and subsequently degrading residual levels of the protein in the somatic cytoplasm after cleavage. However, despite its critical role in cell fate determination, the enrichment of germline determinants in the germplasm remains poorly understood. Here, combining live-cell fluorescence methods and kinetic modeling, we demonstrate that the enrichment process does not involve previously proposed protein immobilization, intracellular compartmentalization, or localized protein degradation. Alternatively, our results support a novel heterogeneous reaction/diffusion model for PIE-1 enrichment in which the diffusion coefficient of PIE-1 is reversibly reduced in the posterior, resulting in an overall protein gradient across the zygote at steady-state.

Indexing (document details)
Advisor: Wirtz, Denis
School: The Johns Hopkins University
School Location: United States -- Maryland
Source: DAI-B 70/04, Dissertation Abstracts International
Subjects: Cellular biology, Chemical engineering, Biophysics
Keywords: Asymmetric cell division, Cell fate, Germline
Publication Number: 3357181
ISBN: 9781109138658