Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Biology and Biomedical Sciences: Developmental, Regenerative and Stem Cell Biology

Language

English (en)

Date of Award

Spring 4-29-2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Lilianna Solnica-Krezel

Abstract

The PLAC8 gene encodes for a small, cysteine-rich protein conserved in vertebrates that is a member of a large family of PLAC8-motif containing proteins with diverse functions in animals, plants, and algae. Recently, high levels of PLAC8 expression have been detected in aggressive colorectal cancer and invasive breast cancer, and potentially contributing to the cancer pathogenesis. However, the molecular and cellular functions of PLAC8 in vertebrate development, homeostasis, and disease remain unclear. To determine the function of PLAC8 under disease and normal conditions, in this thesis work, I identified plac8.1 as a PLAC8 homolog in zebrafish, a vertebrate model system amenable to various embryologic and genetic approaches. Zebrafish plac8.1 was maternally and ubiquitously expressed until four days post fertilization when its transcript became enriched in the gut. During the process of gastrulation, Plac8.1 protein distribution gradually shifted from the cytosol to the cell membrane. At larval stages, Plac8.1 accumulated at the apical region of epithelial cells of the gut and the kidney. This dynamic gene expression and protein localization patterns suggest that Plac8.1 may have functions during zebrafish embryogenesis and organogenesis.

In the first part of this thesis, I aimed to address questions concerning the effects of high levels of PLAC8 on cell behavior by overexpressing Plac8.1 during early embryogenesis. Embryos overexpressing Plac8.1 manifested morphological defects starting at early gastrulation. Epiboly as well as convergence and extension gastrulation movements were delayed in plac8.1-overexpressing embryos, a spectrum of phenotypes resembling impaired E-cadherin function in zebrafish. Indeed, E- cadherin levels were significantly reduced in plac8.1-overexpressing embryos by a cell-autonomous and post-transcriptional mechanism. Similarly, PLAC8-overexpressing colon cancer cells exhibited reduced cell surface E-cadherin and many features of epithelial-mesenchymal transition: EMT). Furthermore, knockdown of PLAC8 in colon cancer cells resulted in increased level of E-cadherin.

In the second part of this thesis, I aimed to determine the requirement for plac8.1 function during development by employing two loss-of-function approaches. Upon injection of either of two non-overlapping antisense morpholino oligonucleotides: MOs) that effectively reduced Plac8.1 protein levels, embryos displayed an array of defects that phenocopied the class of mutants with defective cilia. Consistently, the motile cilia morphology and motility were impaired in plac8.1 morphants. Moreover, in immunoprecipitation experiments, Plac8.1 bound Cops4, a component involved in ubiquitination regulation. Also Plac8.1 and Cops4 cooperated to regulate motile cilia morphology and motility. As a second loss-of-function approach, I generated loss-of-function allele plac8.1stl33 using Transcription Activator-Like Effector Nucleases: TALENs), a method that utilizes sequence-specific nucleases. Similar to plac8.1 morphants, plac8.1stl33 embryos showed motile cilia morphology and beating function defects. We hypothesize that zebrafish Plac8.1 functions at the ciliary base, possibly by modulating intraflagellar transport through ubiquitination modifications.

Our in vivo studies of zebrafish plac8.1 have uncovered pleiotropic functions of plac8.1 during development. In addition, the overexpression experiments shed light on mechanisms underlying human disease. Based on the above results, I propose that Plac8.1 overexpression interferes with regulation of protein stability, whereas Plac8.1 is required for motile cilia morphogenesis and function. Common denominators of these seemingly unrelated phenotypes include the potential connection with the process of ubiquitination, and the enriched apical localization of Plac8.1. Our work can also inform studies of other members of the PLAC8 family of proteins.

DOI

https://doi.org/10.7936/K7CJ8BGM

Comments

Permanent URL: http://dx.doi.org/10.7936/K7CJ8BGM

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