Dissertation
Centrosomal Regulation of the C. Elegans β-Catenin SYS-1 is Mediated by Microtubule Motors
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2021
DOI: 10.17077/etd.005743
Abstract
Asymmetric cell division (ACD), the unequal distribution of cell fate determinants between daughter cells, is a critical system underlying the development and maintenance of varied tissue types in a multicellular organism. Despite widespread utilization of such divisions, the mechanisms responsible for induction of asymmetry are not well understood due to the pleiotropic, and often functionally redundant, signaling systems involved. The Wnt/β-catenin asymmetry pathway, most well characterized in C. elegans, induces asymmetric distribution of Wnt signaling components to polarize a mother cell and differentially regulate Wnt target genes in its daughters. SYS-1/-catenin, the transcriptional activator of this signaling pathway, exhibits a conserved and symmetrical localization to mitotic centrosomes. Recent data indicate that centrosomal localization of SYS-1 serves as a clearance mechanism to increase the robustness of Wnt-mediated polarity. However, the method of SYS-1 accumulation at the centrosome is unknown. In the early C. elegans embryo, centrosomal localization of SYS-1 is dependent on the centrosomal scaffolding protein RSA-2. Depletion of RSA-2 by RNAi prevents SYS-1 centrosomal loading and results in accumulation in the region of kinetochore microtubules, consistent with a role for dynein-based trafficking of SYS-1. Therefore, I hypothesize that minus-end directed microtubule trafficking facilitates SYS-1 centrosomal localization. Here I demonstrate via RNAi screen that depleting microtubule transport proteins demonstrates several dynein subunits required for proper centrosomal localization of both embryonic SYS-1 and its negative regulators. Depletion of some components of the dynein complex, e.g. DLC-1 and DYLT-1, rarely but completely uncoupled SYS-1 from centrosomes. Similarly, I demonstrate that a temperature sensitive allele of the dynein heavy chain, dhc-1(or195), shows a disproportionate effect on SYS-1 localization at all temperatures. At the permissive temperature, SYS-1 centrosomal localization is severely hypomorphic, and at the restrictive temperature SYS-1 is nearly indistinguishably enriched on centrosomes. Consistent with impaired SYS-1 degradation, we conclude that loss of these SYS-1 trafficking components can induce SYS-1 mediated cell fate changes. Interestingly, knockdown of ECPS-1, the C. elegans homolog for mammalian dynein/proteasome adaptor Ecm29, instead stabilizes centrosomal SYS-1 and enhances its centrosomal enrichment. Microtubule depletion via nocodazole treatment also demonstrates an enhanced enrichment of centrosomal SYS-1, strongly suggesting a role for microtubule-mediated trafficking in SYS-1 negative regulation as well. Collectively, these data suggest that retrograde trafficking of both SYS-1 and its negative regulators via the dynein motor is necessary for appropriate centrosomal clearance of SYS-1. Disruptions to this system, while sufficient only rarely for the induction of cell fate changes per se, are sufficient to limit the robustness of SYS-1 dependent asymmetric cell divisions, rendering them significantly more vulnerable to ectopic perturbation.
Details
- Title: Subtitle
- Centrosomal Regulation of the C. Elegans β-Catenin SYS-1 is Mediated by Microtubule Motors
- Creators
- Joshua W Thompson
- Contributors
- Bryan T Phillips (Advisor)Douglas W Houston (Committee Member)Veena Prahlad (Committee Member)Christopher S Stipp (Committee Member)Tina L Tootle (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Integrated Biology
- Date degree season
- Spring 2021
- DOI
- 10.17077/etd.005743
- Publisher
- University of Iowa
- Number of pages
- ix, 92 pages
- Copyright
- Copyright 2021 Joshua W Thompson
- Language
- English
- Description illustrations
- illustrations (some color)
- Description bibliographic
- Includes bibliographical references (pages 81-92).
- Public Abstract (ETD)
Asymmetric cell division, unequal distribution of cell fate determinants between daughter cells, underlies the development and maintenance of varied tissue types. The Wnt/β-catenin asymmetry pathway studied mostly in the nematode Caenorhanditis elegans organizes dividing cells asymmetrically, distributing proteins that positively and negatively regulate a specific set of genes to opposing sides of the dividing cell. This enables these daughter cells to differentially regulate a set of Wnt-specific target genes after division by adjusting the regulatory proteins they inherit. Despite this effort to establish asymmetry by the dividing cell, the main protein activating these genes, β-catenin, localizes symmetrically to the two hubs of the cytoskeleton called centrosomes. Each of the daughter cells then symmetrically inherits one of these centrosomes. My lab has shown that this enables cells to clear their SYS-1, leaving just enough that it may be appropriately regulated by the daughter cells that inherit it. However, the C. elegans β-catenin, SYS-1, gets to these centrosomes very quickly, only during specific times in the life of a cell, and requires help from another protein, RSA-2. Therefore, we think the cell actively moves SYS-1 to these centrosomes. By preventing the activity of molecular motors that walk along the cytoskeleton, I will demonstrate that one family of motors, called dynein, does have an effect on the regulation of SYS-1. In fact, I present evidence that suggests the dynein motor seems to traffic both SYS-1 and the proteins that negatively regulate it to centrosomes for more precise regulation of SYS-1 target genes.
- Academic Unit
- Biology
- Record Identifier
- 9984097276202771
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