Regulation of ciliary dynein by the axonemal protein kinase CK1 公开

Abstract

Regulation of ciliary dynein by the axonemal protein kinase CK1

By Avanti Gokhale

Cilia are highly conserved organelles that play essential motile and sensory roles
required for normal development and function of most organs in the adult. The goal of
my work is to study mechanisms that regulate dynein motor activity and control
ciliary/flagellar motility. Genetic and functional studies revealed ciliary dynein is
regulated by phosphorylation, and that a CK1-like kinase is located in the axoneme and
thought to phosphorylate the intermediate chain IC138 of the inner dynein arm isoform
I1. I postulated that CK1 is targeted and anchored on the outer doublet microtubules, near
I1 dynein, to regulate dynein-driven microtubule sliding and control ciliary movement.
To test this, I cloned and characterized the axonemal CK1 in Chlamydomonas, and
confirmed it is a highly conserved member of the CK1 family of kinases. Analysis of
ciliary structural mutants and immunofluorescence analysis revealed that CK1 is
anchored on the outer doublet microtubules, along the entire length of the axonemes.
Consistent with this localization, chemical crosslinking in axonemes revealed that CK1
directly interacts with tubulin.
To study the physiologocial role of axonemal CK1, I took advantage of an in vitro
microtubule sliding assay to measure dynein activity in mutant axonemes lacking the
radial spoke structures ( pf17). Importantly, in absence of the radial spokes, axonemal
CK1 activity is misregulated resulting in global inhibition of dynein-driven microtubule
sliding. Consistent with this conclusion, CK1 inhibitors rescued dynein activity in pf17
axonemes. Using a novel approach, to produce CK1-depleted axonemes, I tested the idea
that removal of CK1 would restore dynein activity. As predicted, CK1 depletion restored
dynein-driven microtubule sliding, mimicking the effect of CK1 inhibitors. Using a
purified, recombinant CK1, I reconstituted the CK1-depleted pf17 axonemes and restored
inhibition of dynein activity, in a CK1 kinase-inhibitor sensitive manner. Furthermore,
reconstitution of CK1-depleted axonemes with a purified, recombinant "kinase-dead"
CK1 did not restore the inhibition of dynein activity. These results demonstrated that
CK1 kinase activity is required for regulation of dynein-driven microtubule sliding.
This work addresses fundamental aspects of signal transduction and regulation of
dynein motors in cilia. The data are consistent with the model that CK1 is a structural
component of the axoneme, localized on the outer doublet microtubules in position to
regulate phosphorylation of IC138 and control microtubule sliding. Additional
challenges include identifying the precise location of CK1 in the axoneme and identifying
interacting proteins that anchor and localize CK1. One prediction is that CK1 is located
adjacent to I1 dynein, anchored by a novel class of CK1-anchoring proteins (CKAPs).
Further tests also include identification of key phosphorylated residues in IC138 to
directly test how IC138 contributes to regulation of dynein activity and ciliary motility.

Table of Contents

Table of contents: Chapter 1: Introduction 1 I. Overview and Significance 2 Significance 3 The experimental system 5 II. Structural and mechanism of motile cilia 9 Central pair apparatus 14 Radial spokes 15 Dynein regulatory complex 17 The dynein motors: each has a place; each has a purpose 18 Inner dynein arm 22 I1 dynein 23 A sliding microtubule, "switching" model for ciliary motility 28 III. Focus of this thesis 33 Figures and tables 40-66 Chapter 2: Cloning and characterization of the Chlamydomonas protein 67 kinase CK1 Introduction 68 Materials and methods 72 Results 80 Identification of axonemal CK1 80 Biochemical characterization of Chlamydomonas CK1 81 Localization of CK1 82 The Chlamydomonas pf27 is not defective in the CK1 gene and CK1 is fully assembled in pf27 axonemes 84 Discussion 86 Figures and tables 93-119 Chapter 3: Identification of CK1-interacting proteins in the axoneme 120 Introduction 121 Materials and methods 127 Results 132 CK1 directly interacts with tubulin 132 CK1 interacts with I1 proteins 134 Discussion 137 Figures 147-178 Chapter 4: Regulation of dynein-driven microtubule sliding by axonemal 179 kinase CK1 Introduction 180 Materials and methods 184 Results 187 Depletion of CK1 rescued microtubule sliding in RS mutant axonemes; rescue of microtubule sliding requires I1 dynein 187 Exogenous CK1 restores inhibition of microtubule sliding in a DRB/CK1-7 sensitive manner 188 CK1 kinase activity is required for inhibition of microtubule sliding 189 Discussion 190 Figures 194-207 Chapter 5: Significance of results and new questions 208 Summary and opportunities 209 Role of CK1 in normal, wild type ciliary motility 211 pf27 reveals a new regulatory pathway 215 Figures 218-229 References 230

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Cell
关键词	Dynein Cilia Casein Kinase 1
Committee Chair / Thesis Advisor	Sale, Winfield S, Emory University
Committee Members	Powers, Maureen, Emory University Saxe, Charles, American Cancer Society Faundez, Victor, Emory University L'Hernault, Steven, Emory University

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