Assembly and Transport of the Ciliary Inner Dynein Arm, I1 Dynein Open Access

Viswanadha, Rasagnya Madupu (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/79407z05n?locale=en
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Abstract

Motile cilia are essential for the development and for function of many organs in the adult. Large ATPase complexes called dyneins drive motility of cilia. Defects in the assembly of ciliary dyneins result in diseases including Primary Ciliary Dyskinesia (PCD). However, we are only beginning to understand the mechanisms of dynein assembly. To determine how ciliary dyneins are assembled, I focused on the Chlamydomonas inner dynein arm, I1 dynein. I1 dynein precursors in the cytoplasm assemble as a 20S complex similar to the 20S I1 dynein complex isolated from the axoneme. The intermediate chain subunit, IC140 (IDA7), and heavy chains (IDA1, IDA2) are required for 20S I1 dynein precursor assembly in the cytoplasm. Taking advantage of cytoplasmic complementation in zygotes, I determined that the I1 dynein complex is transported to the distal tip of the cilium before incorporating in the axoneme. In addition, cytoplasmic complementation in dikaryons using the conditional kinesin-2 mutant, fla10-1,revealed that transport of the I1 dynein precursor complex is dependent on kinesin-2 activity. Thus, I1 dynein assembly depends upon IFT and at least one additional factor, IDA3, for transport to the ciliary distal tip prior to docking in the axoneme. Together, these data indicate that ciliary axonemal dyneins assemble in a stepwise fashion beginning with precursor complex assembly in the cytoplasm followed by entry to the ciliary compartment and transport by IFT within the cilium before docking in the axoneme.

Table of Contents

TABLE OF CONTENTS
Chapter 1: Introduction

Overview...2
Significance of studying the cilium...5
Structural organization of the Motile Cilia...12
The experimental system: Chlamydomonas reinhardtii and Discovery of IFT...18
I1 dynein as a model to study assembly and Central hypothesis...26
Figures...31

Chapter 2: Preassembled I1 dynein in the cytoplasm cannot bind I1-deficient axonemes

Introduction...52
Results...56

I1 dynein preassembles in the wild-type cytoplasm as a 20S complex...56
Cytoplasmic 20S complex is incompetent to bind axonemes...60
ida3 is not defective in an I1 dynein docking mechanism...61
Cytoplasmic I1 dynein complex fails to enter the ida3 cilium...61

Discussion...62
Figures...69

Chapter 3: I1 is transported to the distal tip by IFT for assembly in the axoneme

Introduction...89
Results...92

I1 is transported to the distal tip of the cilium...92
Transport of I1 dynein requires IFT...94

Discussion...96
Figures...98

Chapter 4: Significance of results and new questions

Summary of results and opportunities...117
Cloning and identification of IDA3...120
Live-cell imaging of I1 dynein...124
Figures...129

Appendix I: Materials and Methods...146
Appendix II: Tables...151
References...152

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