The vast majority of theses in this collection are open access and freely available. There are a small number of theses that have access restricted to the WWU campus. For off-campus access to a thesis labeled "Campus Only Access," please log in here with your WWU universal ID, or talk to your librarian about requesting the restricted thesis through interlibrary loan.
Date Permissions Signed
5-31-2022
Date of Award
Spring 2022
Document Type
Masters Thesis
Department or Program Affiliation
Biology
Degree Name
Master of Science (MS)
Department
Biology
First Advisor
Galati, Nick
Second Advisor
Serrano-Moreno, José Ramón
Third Advisor
Leaf, David Scott, 1955-
Fourth Advisor
Young, Jeff C. (Jeffery C.)
Abstract
Cilia are microtubule-based organelles that project from the surface of individual cells. When cilia are formed, they are nucleated from centrioles that are known as basal bodies. Microtubules elongating from the basal body give rise to the axoneme of the cilium, which gives cilia their rod-like structure. The axoneme is surrounded by a specialized plasma membrane that is unique, but continuous with the plasma membrane that surrounds the rest of the cell. The ciliary membrane is enriched with ion channels and membrane-bound proteins that are essential for cilia function. Interestingly, the cilium maintains a distinct environment from the rest of the cell, despite there being no membrane separation from the area within the cilium (i.e., ciliary compartment) and the rest of the cell. Cilia function includes generating hydrodynamic force for motility and participating in signal transduction that is necessary for sensation and animal development. Calcium ions (Ca2+) are necessary for cilia to achieve these functions, yet questions remain as to how Ca2+ levels are maintained within cilia (i.e., ciliary Ca2+ levels). The current “outside-in” model suggests that Ca2+ channels fill cilia with Ca2+ from outside of the cell (i.e., extracellular) and that Ca2+ can diffuse freely between the ciliary compartment and inside of the cell (i.e., intracellular; Delling et al. 2016). However, the base of the cilium can be embedded within the Golgi apparatus (i.e., Golgi), which is a rich source of intracellular Ca2+. Since the Golgi modulates Ca2+ levels at other cellular structures (Follit et al. 2006; Micaroni et al. 2012), this raises the question – does the Golgi impact ciliary Ca2+ levels from inside the cell? We first tested this by disrupting the structural integrity of the Golgi with Brefeldin A (BFA) and found that on average cilia from cells treated with BFA show higher Ca2+ than cells treated with the DMSO control. To further test the current “outside-in” model, histamine and thapsigargin were used to stimulate the release of Ca2+ from the Golgi and endoplasmic reticulum (i.e., ER), which are both rich intracellular Ca2+ stores. Unexpectedly, Ca2+ released from the Golgi and ER did not freely diffuse into cilia, as predicted by the “outside-in” model. Rather, cilia displayed a non-uniform response to intracellular Ca2+ release, suggesting that free diffusion is not the underlying mechanism of Ca2+ transfer from intracellular Ca2+ stores into the ciliary compartment. Experiments involving mechanical stimuli and the Ca2+ channel blocker, lanthanum (III) chloride (LaCl3) show that cilia display a more homogeneous response to extracellular Ca2+. This work suggests that cilia display a differential response to Ca2+ dependent on whether Ca2+ is of intracellular or extracellular origin, consequently, expanding the current “outside-in” model of ciliary Ca2+ homeostasis. To continue testing this model in future studies, a custom cell line of NIH 3T3 fibroblast cells (Arl13b-GCaMP6-mCherry) was developed to permanently express the genetically encoded Ca2+ sensor used to quantity Ca2+ in cilia in this study.
Type
Text
Keywords
Cilia, Calcium, Golgi
Publisher
Western Washington University
OCLC Number
1322997619
Subject – LCSH
Cilia and ciliary motion; Calcium; Golgi apparatus
Format
application/pdf
Genre/Form
Academic theses
Language
English
Rights
Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.
Rights Statement
http://rightsstatements.org/vocab/InC/1.0/
Recommended Citation
Fisher, Hannah R., "Primary cilia display a non-uniform response to intracellular calcium release" (2022). WWU Graduate School Collection. 1109.
https://cedar.wwu.edu/wwuet/1109