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Date of Award

Winter 2023

Document Type

Masters Thesis

Department or Program Affiliation

Biology

Degree Name

Master of Science (MS)

Department

Biology

First Advisor

Galati, Nick

Second Advisor

Amacher, Jeanine

Third Advisor

Emory, Steven R.

Fourth Advisor

Pollard, Dan A.

Abstract

Centrosomes are required for human cells to divide and differentiate into the many different embryonic tissues that form throughout development. Mutations that drastically disrupt centrosomes cause embryonic lethality and cancer. More subtle mutations cause congenital birth defects including blindness, olfactory deficits, and limb, heart, and brain malformations. PCNT is an essential human gene that encodes for the centrosome protein Pericentrin. Pericentrin organizes the structure of the centrosome by serving as a scaffold protein. Pericentrin also interacts with other centrosome proteins, which play a role in centrosome-mediated microtubule formation. Additionally, Pericentrin recruits enzymes that are involved in centrosome duplication and maturation, including the critical centrosome regulating enzyme, Aurora-A kinase. Although Pericentrin recruits Aurora-A, it is not clear whether Pericentrin regulates Aurora-A kinase activity. To test this possibility, single molecule imaging of Aurora-A activity in live cells was accomplished using Förster Resonance Energy Transfer (FRET) quantified with Fluorescence Lifetime Imaging Microscopy (FLIM; FRET-FLIM).

Pericentrin exists in two separate populations within cells. One population resides at the centrosome, and a second population, known as satellites, exist as dynamic granules that traffic to and from the centrosome. Populations of these satellites are found at or near both the centrosome and the Golgi apparatus. Additionally, the satellites mimic the distribution pattern of the Golgi throughout the cell cycle. During interphase, the satellites cluster at both the centrosome and Golgi. However, by mitosis the satellites and the Golgi are both nearly fully dispersed and are spread evenly throughout the entirety of the cell. To observe potential effects of changes in Golgi morphology on satellites, the Golgi was broken apart into small vesicles using the Golgi disruptor drug Brefeldin A. Using this methodology, we determined that Golgi morphology plays a role in the distribution of centriolar satellites by causing Golgi-associated satellites to disperse alongside the Golgi.

Capitalizing on the FLIM capabilities of the Stellaris, we set out to characterize autofluorescence of C. elegansusing FLIM microscopy. The model organism C. elegans has autofluorescence that is problematic for biological imaging assays. The autofluorescence spectrum and fluorescence lifetime characterization of C. elegans autofluorescence is currently unknown. We characterized the autofluorescence emission spectra at four excitation wavelengths, 405, 473, 561 and 647 nm. Of these spectral scans the green species (473 nm excitation) of autofluorescence overlaps with the commonly used fluorescent protein GFP’s spectra. However, by utilizing FLIM, we determined that the green autofluorescence can be easily separated from GFP fluorescence due to their different fluorescence lifetime properties. The separation of lifetime between the autofluorescence and GFP fluorescence improves the ability to detect and quantify GFP fluorescence in C. elegans cilia.

Overall, this work provides a foundation for future experiments to determine effects of Pericentrin overexpression on Aurora-A kinase, a model of regulation of centriolar satellites via the Golgi, as well as a novel method for characterizing autofluorescence using FLIM.

Type

Text

Keywords

Pericentrin, Aurora-A Kinase, FRET-FLIM, FLIM, Centriolar Satellites, Golgi, Centrosome, C. elegans, Autofluorescence, GFP

Publisher

Western Washington University

OCLC Number

1371989662

Subject – LCSH

Centrosomes; Fluorescence microscopy; Energy transfer; Golgi apparatus

Format

application/pdf

Genre/Form

masters 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.

Available for download on Sunday, March 10, 2024

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