Regulation of ciliary motility Öffentlichkeit

Abstract

The overall goal of this work is to determine the mechanisms that regulate dynein motors involved in ciliary/flagellar motility. Cilia are highly conserved organelles that play essential motile and sensory roles required for normal development and function of most organs in the adult; cilia are found on nearly every differentiated cell where they play vital motility and cell signaling functions: failure in assembly or function can result in a wide range of developmental disorders or diseases called "ciliopathies". Ciliary/flagellar motility is regulated by conserved kinases and phosphatases that are localized in the axoneme. I focused on the heterotrimeric phosphatase PP2A, and tested the hypothesis that the axoneme contains a specialized B-type subunit required for targeting the PP2A C-(catalytic) and A-(scaffold) subunits. I identified an axonemal PP2A B-subunit (PR55 B subfamily). The Chlamydomonas PP2A B-subunit gene maps to LG1 at the PF4 locus. The pf4 cells display a slow swimming phenotype and are defective in phototaxis. This motility phenotype is similar to the phenotype of I1 dynein mutants. However, I1 dynein is fully assembled in pf4 axonemes. The B-subunit gene is mutated in pf4, and immunoblots confirmed that the B-subunit is absent in pf4 cells. The PP2A C-subunit, while expressed in pf4 cells, fails to assemble in the axoneme. Two independent, UV-induced pf4 intragenic revertants (pf4rV5, pf4rV11) were recovered from a genetic screen for suppressors. Both revertants contain a seven-base pair insertion which alters several amino acids in the fifth WD-repeat of the B-subunit protein. The reading frame is restored and the new B-subunit is expressed in both revertants. Notably, PP2A is assembled in the revertant axonemes, phototaxis is restored and swimming is partially restored. The revertant cells confirm that the pf4 phenotype is a consequence of the mutation in the PP2A B-subunit, and, consistent with my hypothesis, the results reveal that the PP2A B-subunit is required for assembly of the PP2A holoenzyme in the axoneme. The results reveal that PP2A is required for normal motility.

Table of Contents

Table of contents:

Distribution Agreement
Approval Sheet
Abstract Cover Page
Abstract
Cover Page
Acknowledgments
Table of Contents
Overview and Significance






1
Figures









5-16


Chapter 1: Introduction







17

Prelude








18



An overview of Cilia and Flagella





18


Experimental model system





21
The Axoneme







24
Axonemal dyneins







27
Sliding-switching model for ciliary bending



30
Dynein regulation by phosphorylation




31
Protein kinases-PKA and CK1-located in the axoneme


33
Axonemal Phosphorylation





35
The protein phosphatase PP2A





36
The PR55/B/B55 Family of PP2A B-subunits



40
Summary and design of the hypothesis




42

Figures








45-64
Chapter 2: The foundation for the study of PP2A and the identification
65
of the axonemal PP2A B-subunit gene







Foundation for study of the protein phosphatase PP2A


66
Results and Discussion:






70
Identification of the Chlamydomonas axonemal PP2A B-subunit

70
gene and protein
Figures








75-96
Chapter III: The mutant pf4 reveals that assembly of axonemal PP2A
97
requires the B-subunit and that this B-subunit is required for normal ciliary
motility
Results and Discussion:






98
The flagellar mutant, pf4, is defective in the B-subunit gene


98
The B-subunit is required for targeting of PP2A to the axoneme

100
The pf4 mutant has defective motility and fails to perform phototaxis
101
Intragenic pf4 revertants restore PP2A assembly, rescue phototaxis and near 102
wild-type motility
The pf4 double mutants display more severe phenotypes


104
Figures









107-136
Chapter IV: Discussion and new questions




137

Summary and opportunities






138
Role of PP2A in the regulation of ciliary motility



140
Where does PP2A localize in the axoneme?




141

About this Dissertation

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Cell
Stichwort	Flagella Dynein Cilia PP2A Axoneme
Committee Chair / Thesis Advisor	Sale, Winfield S, Emory University
Committee Members	Hepler, John R, Emory University Powers, Maureen, Emory University Caspary, Tamara, Emory University Pallas, David C, Emory University Faundez, Victor, Emory University

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